ALBERT

Notes: Anabaena strain 90 produces three hepatotoxic heptapeptides (microcystins), two seven-residue depsipeptides called anabaenopeptilide 90A and 90B, and three six-residue peptides called anabaenopeptins. The anabaenopeptilides belong to a group of cyanobacterial depsipeptides that share the structure of a six-amino-acid ring with a side-chain. Despite their similarity to known cyclic peptide toxins, no function has been assigned to the anabaenopeptilides. Degenerate oligonucleotide primers based on the conserved amino acid sequences of other peptide synthetases were used to amplify DNA from Anabaena 90, and the resulting polymerase chain reaction (PCR) products were used to identify a peptide synthetase gene cluster. Four genes encoding putative anabaenopeptilide synthetase domains were characterized. Three genes, apdA, apdB and apdD, contain two, four and one module, respectively, encoding a total of seven modules for activation and peptide bond formation of seven l-amino acids. Modules five and six also carry methyltransferase-like domains. Before the first module, there is a region similar in amino acid sequence to formyltransferases. A fourth gene (apdC), between modules six and seven, is similar in sequence to halogenase genes. Thus, the order of domains is co-linear with the positions of amino acid residues in the finished peptide. A mutant of Anabaena 90 was made by inserting a chloramphenicol resistance gene into the apdA gene. DNA amplification by PCR confirmed the insertion. Mass spectrometry analysis showed that anabaenopeptilides are not made in the mutant strain, but other peptides, such as microcystins and anabaenopeptins, are still produced by the mutant.

Notes: Salmonella pathogenesis is a complex and multifactorial phenomenon. Many genes required for full virulence in mice have been identified, but only a few of these have been shown to be necessary for the induction of enteritis. Likewise, at least some of the Salmonella virulence factors affecting enteritis do not appear to be required for infection of systemic sites in mice. This suggests that subsets of virulence genes influence distinct aspects of Salmonella pathogenesis. Recently, considerable progress has been made in characterizing the virulence mechanisms influencing enteritis caused by non-typhoid Salmonella spp. The Salmonella pathogenicity island-1-encoded type III secretion system mediates the translocation of secreted effector proteins into target epithelial cells. These effector proteins are key virulence factors required for Salmonella intestinal invasion and the induction of fluid secretion and inflammatory responses.

Notes: The recent emergence of food-borne pathogens, such as Salmonella enterica serotype Enteritidis (S. enteritidis) and Escherichia coli O157:H7, has generated increasing interest in how infectious diseases can invade, persist and spread within new host populations. To alter their host range pathogens require adaptations, which ensure their circulation in a new animal population. Adaptations for circulation in different populations of vertebrate hosts seem to have been acquired multiple times within the genus Salmonella because extant Salmonella serotypes differ greatly with regard to host range. In this article, mechanisms involved in host adaptation are deduced by considering the influence of the host immune response on circulation of Salmonella serotypes within populations of vertebrate animals. This approach contributes to the identification of genes involved in host adaptation and provides new insights into the emergence of food-borne pathogens.

Notes: Transcription from the bop promoter in the haloarchaeon Halobacterium NRC-1, is highly induced under oxygen-limiting conditions. A DNA gyrase inhibitor, novobiocin, was previously shown to block bop gene induction and suggested that DNA supercoiling mediates transcriptional induction. A region of non-B structure was found 3′ to the TATA box within an 11 bp alternating purine–pyrimidine sequence (RY box), which correlated to both increased DNA supercoiling and transcriptional induction. Here, saturation mutagenesis of the RY box region has been used to show that single-base substitutions of A(r)G either 23 or 19 bp 5′ to the transcription start site temper the effect of DNA supercoiling based on novobiocin insensitivity of transcription. Mutagenesis of the region 5′ to the TATA box showed its involvement in DNA supercoiling modulation of transcription, defined the 3′ end of the upstream activator sequence (UAS) regulatory element, and ruled out the requirement for a TFB (TFIIB) Recognition Element. Spacing between the TATA box and UAS was found to be critical for promoter activity because insertion of partial or whole helical turns between the two elements completely inhibited transcription indicating that the UAS element does not function as a transcriptional enhancer. The results are discussed in the context of DNA melting and flexibility around the TATA box region and the involvement of multiple regulatory and transcription factors in bop promoter activity.

Notes: We investigated the transcription of the urease gene cluster ureABIEFGH in Helicobacter pylori to determine the regulation of gene expression of the highly produced enzyme urease. Northern blot hybridization analysis demonstrated that cells of the wild-type strain grown in an ordinary broth had transcripts of ureAB, ureABI, ureI, ureIE′ and ure′FGH, but cells of a ureI-disrupted mutant had only the ureAB transcript. When the wild-type cells were exposed to pH 8 for 30 min, very little mRNA was detected. However, when exposed to pH 6, a large amount of the ureIE′′ transcript, which was longer than the ureIE′ transcript, together with the additional transcripts ureABIEFGH and ure′EFGH were detected. Rifampicin addition experiments demonstrated that urease mRNAs, and the ureIE′ transcripts in particular, are more stable at pH 5.5 than at pH 7. In accord with these results, urease activity in the crude cell extract of the pH 5.5 culture was twice as much as that of the pH 7 culture, although the amounts of UreA and UreB detected by immunoblot analysis were similar. The transcription start point of ureI was identified by primer extension using a ureA promoter-deleted mutant, and a consensus sequence of RpoD-RNA polymerase was found in the ureI promoter. The 3′ end of the ureIE′′ mRNA, determined using S1 nuclease mapping, revealed that the transcript is able to cover the majority of the ureE open reading frame (ORF) that might be sufficient for UreE activity. Based on the above results, we conclude that the urease gene cluster of H. pylori consists of two operons, ureAB and ureIEFGH, and that primary transcripts of the latter as well as the read-through transcript, ureABIEFGH, are cleaved to produce several species of mRNA. It has been suggested that the ureIEFGH operon is regulated post-transcriptionally by mRNA decay in response to environmental pH. We are tempted to speculate that the ureE′′ transcript present in acidic pH may contribute to produce an active product that can proceed the nickel incorporation to the active centre, the final step of urease biosynthesis.

Notes: Systemic infections by Salmonella enterica, such as typhoid fever, are a significant threat to human health. Recent studies indicate that the function of a type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) is central for the ability of S. enterica to cause systemic infections and for intracellular pathogenesis. This review summarizes approaches leading to the identification of SPI2, the molecular genetics and evolution of SPI2, and the current understanding of the regulation of gene expression. Recent studies have indicated that SPI2 is used by intracellular Salmonella to actively modify functions of the host cells. The role of SPI2 during pathogenesis of salmonellosis and current models regarding function will be discussed.

Notes: Natural sequence variation was investigated among serogroup A subgroup IV-1 Neisseria meningitidis isolated from diseased patients and healthy carriers in The Gambia, West Africa. The frequencies of DNA import were analysed by sequencing fragments of four linked genes encoding the immunogenic outer membrane proteins TbpB (transferrin binding protein B) and OpaA (an adhesin) plus two housekeeping enzymes. Seventeen foreign tbpB alleles were independently imported into the 98 strains tested, apparently due to immune selection. The median size of the imported DNA fragments was 5 kb, resulting in the occasional concurrent import of linked housekeeping genes by hitchhiking. Sequences of tbpB from other strains of N. meningitidis as well as commensal Neisseria lactamica and Neisseria spp. isolated from the same geographical area revealed that these species share a common tbpB gene pool and identified several examples of interspecific genetic exchange. These observations indicate that recombination can be more frequent between related species than within a species and indicate that effective vaccination against serogroup B meningococcal disease may be difficult to achieve.

Notes: Antirepressors have been identified as proteins interacting with transcriptional repressors leading to expression of the repressed genes. The defective satellite phage/plasmid P4 has the capacity to derepress the unrelated prophage P2 after infection, thereby getting access to the late functions of the helper that are required for P4 lytic growth. The derepression of prophage P2 is mediated by the P4 E protein that function as an antirepressor by binding to the P2 immunity repressor C. A P2 mutant, sos, has been isolated that is insensitive to the action of the P4 E protein. In the present study, we show that sos is a point mutation in the P2 immunity repressor gene C and that it makes P4 E unable to turn the transcriptional switch of P2 from the lysogenic state to the lytic mode in a two plasmid reporter system. Furthermore, the interaction between C and E, when analysed in the yeast two-hybrid system, is blocked by the sos mutation. An analysis of C mutants indicates that the dimerization function of C is located in the C-terminal part of the protein and the dimerization defective mutants are unable to bind to their operator DNA. The sos mutation does not affect the capacity of the protein to dimerize. Using the yeast two-hybrid system, compensatory E mutants have been isolated that can interact with Sos, but they are unable to turn the transcriptional switch controlled by the Sos repressor. However, one point mutation in the E protein is shown to be unable to turn the transcriptional switch controlled by the wild-type C repressor.

Notes: CAP1 encodes a basic region-leucine zipper (bZip) transcriptional regulatory protein that is required for oxidative stress tolerance in Candida albicans. Cap1p is a homologue of a Saccharomyces cerevisiae bZip transcription factor designated Yap1p that is both required for oxidative stress tolerance and localized to the nucleus in response to the presence of oxidants. Oxidant-regulated localization of Yap1p to the nucleus requires the presence of a carboxy-terminal cysteine residue (C629) that is conserved in Cap1p as C477. To examine the role of this conserved cysteine residue, C477 was replaced with an alanine residue. This mutant protein, C477A Cap1p, was analysed for its behaviour both in S. cerevisiae and C. albicans. Wild type and C477A Cap1p were able to complement the oxidant hypersensitivity of a Δyap1 S. cerevisiae strain. Whereas a Yap1p-responsive lacZ fusion gene was oxidant inducible in the presence of YAP1, the C. albicans Cap1p derivatives were not oxidant responsive in S. cerevisiae. Introduction of wild type and C477A Cap1p-expressing plasmids into C. albicans produced differential resistance to oxidants. Glutathione reductase activity was found to be inducible by oxidants in the presence of Cap1p but was constitutively elevated in the presence of C477A Cap1p. Western blot assays indicate Cap1p is post-translationally regulated by oxidants. Green fluorescent protein fusions to CAP1 showed that this protein is localized to the nucleus only in the presence of oxidants while C477A Cap1p is constitutively nuclear localized. Directly analogous to S. cerevisiae Yap1p, regulated nuclear localization of C. albicans Cap1p is crucial for its normal function.

Notes: The DNA-binding domain of the Escherichia coli DnaA protein is represented by the 94 C-terminal amino acids (domain 4, aa 374–467). The isolated DNA-binding domain acts as a functional repressor in vivo, as monitored with a mioC::lacZ translational fusion integrated into the chromosome of the indicator strain. In order to identify residues required for specific DNA binding, site-directed and random PCR mutagenesis were performed, using the mioC::lacZ construct for selection. Mutations defective in DNA binding were found all over the DNA-binding domain with some clustering in the basic loop region, within presumptive helix B and in a highly conserved region at the N-terminus of presumptive helix C. Surface plasmon resonance (SPR) analysis revealed different binding classes of mutant proteins. No or severely reduced binding activity was demonstrated for amino acid substitutions at positions R399, R407, Q408, H434, T435, T436 and A440. Altered binding specificity was found for mutations in a 12 residue region close to the N-terminus of helix C. The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temperatures. dnaX suppressors dnaA71 and dnaA721 map to the region close to helix C and bind DNA non-specifically.

Notes: The integrase (Int) proteins encoded by bacteriophages HK022 and λ catalyse similar site-specific integration and excision reactions between specific DNA regions known as attachment (att) sites. However, the Int proteins of HK022 and λ are unable to catalyse recombination between non-cognate att sites. The att sites of both phages contain weak binding sites for Int, known as ‘core-type’ sites. Negatively acting nucleotide determinants associated with specific core sites (λ B′, HK022 B′, HK022 C) are responsible for the barrier to non-cognate recombination. In this study, we used challenge phages to demonstrate that the λ and HK022 Ints cannot bind to core sites containing non-cognate specificity determinants in vivo. We isolated mutants of the HK022 Int, which bind the λ B′ core site. Two mutants, D99N and D99A, have changed a residue in the core-binding (CB) domain, which may be directly contacting the core site DNA. We suggest that binding to the λ B′ site was accomplished by removing the negatively charged aspartate residue, which normally participates in a conflicting interaction with the G4 nucleotide of the λ B′ site. We showed that, although our mutants retain the ability to recombine their cognate att sites, they are unable to recombine λatt sites.

Notes: The fim switch of Escherichia coli is responsible for phase-variable expression of type 1 fimbriae. Switching in the ON-to-OFF and OFF-to-ON directions is promoted by the FimB recombinase, while the FimE recombinase directs switching predominantly in the ON-to-OFF direction. The effects of local promoter activity and the H-NS nucleoid-associated protein on inversion of the switch were assessed. In contrast to FimB-mediated inversion, inversion of the switch by the FimE recombinase was unaffected by the H-NS status of the cell. Transcription towards the switch from within a translationally inactivated fimE gene was found to bias the switch strongly in the OFF direction, creating a FimE+-like phenotype in the absence of the FimE protein. This biasing was H-NS dependent and was also contingent on transcription from within the switch. These data show that local transcription and a nucleoid-associated protein both contribute to the modulation of a site-specific recombination event on the bacterial chromosome.

Notes: The fnrN gene from Rhizobium leguminosarum UPM791 controls microaerobic expression of both nitrogen fixation and hydrogenase activities in symbiotic cells. Two copies of fnrN are present in this strain, one chromosomal (fnrN1) and the other located in the symbiotic plasmid (fnrN2). Their expression was studied by cloning the regulatory regions in lacZ promoter-probe vectors. The fnrN genes were found to be autoregulated: they are expressed only at basal levels under aerobic conditions; they are highly expressed under microaerobic conditions; and they are expressed at basal levels in the double mutant DG2 (fnrN1 fnrN2) under any condition. The promoters of both genes contain two FnrN-binding sequences (anaeroboxes), centred at positions −12.5 (proximal anaerobox) and −44.5 (distal anaerobox). Expression analysis and gel retardation experiments with fnrN1-derivative promoter mutants altered in key bases of the anaerobox sequences demonstrated that binding of FnrN1 to the distal anaerobox is necessary for microaerobic activation of transcription, and that binding of FnrN1 to the proximal anaerobox results in transcriptional repression. The apparent affinity of FnrN1 for the proximal anaerobox was fivefold lower than for the distal anaerobox, resulting in repression of transcription of fnrN1 only at high-FnrN1 concentrations. This positive and negative autoregulation mechanism ensures an equilibrated expression of fnrN in response to microaerobic conditions.

Notes: Listeria monocytogenes is capable of growth within the cytoplasm of infected host cells. Escape from the host cell phagosome is mediated primarily through secretion of listeriolysin, a haemolytic factor which functions to actively lyse the phagosomal membrane. Listeriolysin negative mutants of L. monocytogenes are non-haemolytic on blood agar plates and demonstrate a significant reduction of virulence in the mouse model of infection. We have developed a system for the identification of in vivo induced genes in L. monocytogenes which utilizes the listeriolysin gene, hly, as both a reporter of gene expression and as a means of selection of promoter elements expressed in vivo. The system is analogous to in vivo expression technology (IVET) first reported for Salmonella, however, as listeriolysin functions in the environment of the host phagosome the loci identified in this study are most likely expressed during residence in the phagosome. The system was successfully tested using the promoter of the inducible virulence gene plcA. A bank was created by fusing a promoterless copy of hly to random promoter elements in a listeriolysin negative IVET host. Sequential inoculations of mice with this bank resulted in the isolation of clones with increased survival potential in the mouse model relative to a negative control, but which remained haemolysin negative on blood agar plates. Nine in vivo induced loci were identified including genes encoding a DNA topoisomerase III, a cellobiose transporter and a fumarase. Two isolates represented fusions to proteins of unknown function and three isolates contained no significant homologues in the database. A mutant in the fumarase gene demonstrated reduced virulence for mice and an inability to grow in cultured mouse phagocytes.

Notes: Saccharomyces cerevisiae Ste12p plays a key role in coupling signal transduction through MAP kinase modules to cell-specific or morphogenesis-specific gene expression required for mating and pseudohyphal (PH)/filamentous growth (FG). Ste12p homologues in the pathogenic yeasts Candida albicans and Filobasidiela neoformans apparently play similar roles during dimorphic transitions. Here we report the isolation and characterization of the first Ste12 protein from a true filamentous fungus. Aspergillus nidulans steA encodes a protein with a homeodomain 63–75% identical to those of other Ste12 proteins, with greatest similarity to FnSte12αp. SteAp and Ste12αp lack the pheromone induction domain found in budding yeast Ste12p, but have C-terminal C2/H2-Zn+2 finger domains not present in the other Ste12 proteins. A ΔsteA strain is sterile and differentiates neither ascogenous tissue nor fruiting bodies (cleistothecia). However, the development of sexual cycle-specific Hülle cells is unaffected. Filamentous growth, conidiation and the differentiation of PH-like asexual reproductive cells (metulae and phialides) are normal in the deletion strain. Northern analysis of key regulators of the asexual and sexual reproductive cycles support the observation that although SteAp function is restricted to the sexual cycle, cross regulation between the two developmental pathways exists. Our results further suggest that while several classes of related proteins control similar morphogenetic events in A. nidulans and the dimorphic yeasts, significant differences must exist in the regulatory circuitry.

Notes: The two glyceraldehyde-3-phosphate dehydrogenase-encoding genes (gap) of Synechocystis were shown to be expressed as monocistronic transcripts. Whereas gap1 expression is slow and weak, gap2 gene induction is rapid and strong. Transcription of the gap2 gene was shown to depend on functional photosynthetic electron transport and on active carbon metabolism. The basal promoter of gap2 (P, −45 to +34, relative to the transcription start site) is controlled by three cis-acting elements designated A (−443 to −45), B (+34 to +50, in the untranslated leader region) and C (+50 to +167, in the coding region) that, together, promote a 100-fold stimulation of P activity. Element B was found to behave as a transcriptional enhancer, in that it was active regardless of its position, orientation and distance relative to P. All three cis-acting stimulatory elements exhibit a common 5′-agaTYAACg-3′ nucleotide motif that appears to be conserved in cyanobacteria and may be the target for a transcriptional enhancer. We also report that gap2 transcription depends on a Gram-positive-like −16 promoter box (5′-TRTG-3′) that was obviously conserved throughout the evolution of chloroplasts. This is the first report on the occurrence of a −16 promoter element in photoautotrophic organisms.

Notes: Apicomplexan protozoa possess a family of micronemal and cell surface-associated proteins, each comprised a combination of cell-adhesive vertebrate von Willebrand factor (vWF)-like A domains and thrombospondin (TSP) type 1-like domains. The human malaria parasite Plasmodium falciparum has in the extracellular portion of the CS protein TRAP-related protein (CTRP) six tandemly arrayed A domains followed by seven TSP type 1-like domains, whereas a second member of this family, thrombospondin-related anonymous protein (TRAP), contains a single vWF-like A domain and a single TSP type 1-like domain. Here we show that CTRP transcripts are present within the infected mosquito midgut and that CTRP protein is expressed with a punctate distribution and a predominance at the apical end of mosquito midgut-stage ookinetes. This expression pattern is analogous to micronemal expression of TRAP in Plasmodium sporozoites. Disruption of the CTRP gene by homologous recombination in cultures of the human malaria parasite P. falciparum demonstrates that CTRP is essential for mosquito midgut development. Oocyst formation was never observed following membrane feeds of CTRP disruptant lines to Anopheline mosquitoes, despite the development of mature ookinetes. We propose that CTRP is involved in essential recognition or motility processes at the ookinete cell surface within the mosquito midgut.

Notes: The class B M1-V577 penicillin-binding protein (PBP) 3 of Escherichia coli consists of a M1–L39 membrane anchor (bearing a cytosolic tail) that is linked via a G40–S70 intervening peptide to an R71–I236 non-catalytic module (containing the conserved motifs 1–3) itself linked via motif 4 to a D237–V577 catalytic module (containing the conserved motifs 5–7 of the penicilloyl serine transferases superfamily). It has been proposed that during cell septation the peptidoglycan crosslinking activity of the acyl transferase module of PBP3 is regulated by the associated M1–I236 polypeptide itself in interaction with other components of the divisome. The fold adopted by the R71–V577 polypeptide of PBP3 has been modelled by reference to the corresponding R76–S634 polypeptide of the class B Streptococcus pneumoniae PBP2x. Based on these data and the results of site-directed mutagenesis of motifs 1–3 and of peptide segments of high amphiphilicity (identified from hydrophobic moment plots), the M1–I236 polypeptide of PBP3 appears to be precisely designed to work in the way proposed. The membrane anchor and the G40–S70 sequence (containing the G57–Q66 peptide segment) upstream from the non-catalytic module have the information ensuring that PBP3 undergoes proper insertion within the divisome at the cell septation site. Motif 1 and the I74–L82 overlapping peptide segment, motif 2 and the H160–G172 overlapping peptide segment, and the G188–D197 motif 3 are located at or close to the intermodule junction. They contain the information ensuring that PBP3 folds correctly and the acyl transferase catalytic centre adopts the active configuration. The E206–V217 peptide segment is exposed at the surface of the non-catalytic module. It has the information ensuring that PBP3 fulfils its cell septation activity within the fully complemented divisome.

Notes: Helicobacter pylori cells are naturally competent for the uptake of both plasmid and chromosomal DNA. However, we demonstrate that there are strong barriers to transformation of H. pylori strains by plasmids derived from unrelated strains. We sought to determine the molecular mechanisms underlying these barriers. Transformation efficiency was assessed using pHP1, an Escherichia coli–H. pylori shuttle vector conferring kanamycin resistance. Transformation of 33 H. pylori strains was attempted with pHP1 purified from either E. coli or H. pylori, and was successfully introduced into only 11 strains. Digestion of H. pylori chromosomes with different restriction endonucleases (REs) showed that DNA methylation patterns vary substantially among strains. The strain most easily transformed, JP26, was found to have extremely low endogenous RE activity and to lack a restriction–modification (R–M) system, homologous to MboI, which is highly conserved among H. pylori strains. When we introduced this system to JP26, pHP1 from MboI.M+ JP26, but not from wild-type JP26, transformed MboI R−M+ JP26 and heterologous MboI R−M+ wild-type H. pylori strains. Parallel studies with pHP1 from dam+ and dam−E. coli strains confirmed these findings. These data indicate that the endogenous REs of H. pylori strains represent a critical barrier to interstrain plasmid transfer among H. pylori.

Notes: The pilus of Neisseria gonorrhoeae (the gonococcus Gc), the causative agent of gonorrhoea, promotes attachment of the gonococcus to the host epithelium and is essential for the establishment of disease. The ability of N. gonorrhoeae to infect previously exposed individuals is partially due to pilus antigenic variation. In addition, variation of the pilus has been proposed to function in the adaptation of the gonococcus to host environments. Previously, we described the development of a competitive reverse transcriptase (RT)-PCR assay that quantifies the frequency of pilin antigenic variation within a gonococcal population. Using this assay, the effect of different biologically relevant environmental conditions on the frequency of pilin antigenic variation was tested. Of the environmental conditions examined in vitro, only limited iron affected a significant change in the frequency of antigenic variation. Further investigation revealed that an observed increase in pilin antigenic variation reflected an increase in other DNA recombination and DNA repair processes within iron-starved cultures. In addition, this low iron-induced increase was determined to be independent of changes in RecA expression and was observed in a Fur mutant strain. As gonococci encounter conditions of low iron during infection, these data suggest that iron-limitation signals for increased recombinational events that are important for gonococcal pathogenesis.

Notes: IHF (integration host factor) mutants exhibit asynchronous initiation of chromosome replication from oriC as determined from flow cytometric analysis of cultures where RNA synthesis was inhibited with rifampicin. However, the run-out kinetics of chromosome replication in ihf mutants shows that they continue to produce oriCs for some time in the absence of RNA synthesis resulting in a twofold increase in the oriC per mass ratio. An ihf dnaA double mutant did not exhibit this continued increase of the oriC per mass ratio. This indicates that ihf mutants can initiate replication from oriC in a rifampicin-resistant initiation mode but requires fully functional DnaA protein. The origin per mass ratio, determined by a quantitative Southern blotting technique, showed that the ihf mutants had an origin per mass ratio that was 60% of the wild type although it had a normal DnaA protein concentration. This shows that the initiation mass was substantially higher in the ihf mutants. The oriC per terminus ratio, which was also determined by Southern blotting, was very low in the ihf mutant, although it grew with the same doubling times as the wild-type strain. This indicates that cells lacking IHF replicate their chromosome(s) very fast.

Notes: Several strains of the human opportunistic pathogen Pseudomonas aeruginosa infect plants, nematodes and insects. Our laboratory has developed a multihost pathogenesis system based on the P. aeruginosa clinical isolate PA14, in which non-mammalian hosts are used to screen directly for virulence-attenuated mutants. The majority of PA14 mutants isolated using non-mammalian hosts also displayed reduced virulence in a burned mouse model. Surprisingly, only a few host-specific virulence factors were identified, and many of the P. aeruginosa mutants were attenuated in virulence in all the hosts. These studies illustrate the extensive conservation in the virulence mechanisms used by P. aeruginosa to infect evolutionarily diverged hosts, and validate the multihost method of screening for virulence factors relevant to mammalian pathogenesis. Through the use of genetically tractable hosts, the multihost pathogenesis model also provides tools for elucidating host responses and dissecting the fundamental molecular interactions that underlie bacterial pathogenesis.

Notes: A meningococcal genomic expression library was screened for potent CD4+ T-cell antigens, using patients' peripheral blood lymphocytes (PBLs). One of the most promising positive clones was fully characterized. The recombinant meningococcal DNA contained a single, incomplete, open reading frame (ORF), which was fully reconstructed with reference to available genomic sequence data. The gene was designated autA (auto-transporter A) as its peptide sequence shares molecular characteristics of the auto-transporter family of proteins. Only a single copy of this gene was detected in the meningococcal, and none in the gonococcal, genomic sequence databases. The complete autA gene, when cloned into an expression vector, expressed a protein of approximately 68 kDa. Purified rAutA recalled strong secondary T-cell responses in PBLs of patients and some healthy donors, and induced strong primary T-cell responses in healthy donors. The human B-cell immunogenicity and cross-reactivity of AutA, purified under native conditions, was confirmed in dot immunoblot experiments. Immunoblots with rabbit polyclonal antibodies to rAutA demonstrated the conserved nature, antigenicity and cross-reactivity of AutA amongst meningococci of different serogroups and strains representing different hypervirulent lineages. AutA showed homology with another meningococcal and gonococcal ORF (designated AutB). AutB was cloned and expressed and used to raise an autB-specific antiserum. Immunoblot experiments indicated that AutB is not expressed in meningococci and does not cross-react with AutA. Thus, AutA, being a potent CD4+ T-cell and B-cell-stimulating antigen, which is highly conserved, deserves further investigation as a potential vaccine candidate.

Notes: A number of well-known bacterial toxins ADP-ribosylate and thereby inactivate target proteins in their animal hosts. Recently, several vertebrate ecto-enzymes (ART1–ART7) with activities similar to bacterial toxins have also been cloned. We show here that psiblast, a position-specific-iterative database search program, faithfully connects all known vertebrate ecto-mono(ADP-ribosyl)transferases (mADPRTs) with most of the known bacterial mADPRTs. Intriguingly, no matches were found in the available public genome sequences of archaeabacteria, the yeast Saccharomyces cerevisiae or the nematode Caenorhabditis elegans. Significant new matches detected by psiblast from the public sequence data bases included only one open reading frame (ORF) of previously unknown function: the spvB gene contained in the virulence plasmids of Salmonella enterica. Structure predictions of SpvB indicated that it is composed of a C-terminal ADP-ribosyltransferase domain fused via a poly proline stretch to a N-domain resembling the N-domain of the secretory toxin TcaC from nematode-infecting enterobacteria. We produced the predicted catalytic domain of SpvB as a recombinant fusion protein and demonstrate that it, indeed, acts as an ADP-ribosyltransferase. Our findings underscore the power of the psiblast program for the discovery of new family members in genome databases. Moreover, they open a new avenue of investigation regarding salmonella pathogenesis.

Notes: Expression of the Saccharomyces cerevisiae nuclear gene CYB2 encoding the mitochondrial enzyme l-(+)-lactate–cytochrome c oxidoreductase (EC 1.2.2.3) is subject to several strict metabolic controls at the transcriptional level: repression due to glucose fermentation, derepression by ethanol, induction by lactate and inhibition under anaerobic conditions or in response to deficiency of haem biosynthesis. In this respect, the data obtained from the transcriptional analysis of the CYB2 gene contribute to a better understanding of the control of mitochondrial biogenesis. In this study, we show that Hap1p is the main transcriptional activator involved in the control of CYB2 transcription. We found that Hap1p activity, known to be oxygen dependent, is effected by DNA–protein interaction with two binding sites present in the CYB2 promoter. Control is moreover dependent on carbon sources. This regulation by the carbon substrates is subordinate to the activity of the complex Hap2/3/4/5p, which counteracts the negative effect of the URS1 element. Finally, our results suggest that the Adr1p transcriptional activator is also required in CYB2 transcription control. This work provides new data which allows a better understanding of the molecular mechanisms implicated in the co-regulation at the transcriptional level of the genes encoding proteins involved in various aspects of oxidative metabolism.

Notes: Salmonella species translocate effector proteins into the host cell cytoplasm using a type III secretion system (TTSS). The translocation machinery probably contacts the eukaryotic cell plasma membrane to effect protein transfer. Data presented here demonstrate that both SspB and SspC, components of the translocation apparatus, are inserted into the epithelial cell plasma membrane 15 min after Salmonella typhimurium infection. In addition, a yeast two-hybrid interaction between SspC and an eukaryotic intermediate filament protein was identified. Three individual carboxyl-terminal point mutations within SspC that disrupt the yeast two-hybrid interaction were isolated. Strains expressing the mutant SspC alleles were defective for invasion, translocation of effector molecules and membrane localization of SspC. These data indicate that insertion of SspC into the plasma membrane of target cells is required for invasion and effector molecule translocation and that the carboxyl terminus of SspC is essential for these functions.

Notes: The pilus antigenic variation (Av) system of Neisseria gonorrhoeae is one of several high-frequency variation systems that utilize gene conversion to switch between numerous forms of an antigen on the cell surface. We have tested three predictions of the first models that explain the movement of DNA during pilin Av: (i) Av requires two recombinations at short regions of identity, (ii) circular intermediates exist that carry pilE/pilS hybrid loci and (iii) these pilE/pilS hybrid loci target the pilS sequences to a recipient pilE gene. We confirm that normal pilin Av utilizes recombination at very short regions of DNA sequence identity and that these recombination events can occur independent of homologous recombination functions. We have isolated covalently closed circular DNA molecules carrying hybrid pilin loci, but propose that an alternative hybrid molecule is the intermediate of pilin Av. Our most striking finding is that transformation of isolated pilE/pilS hybrid loci targets the pilS sequences of the hybrid to a recipient pilE at frequencies much higher than normal recombination frequencies. These results show that the different steps of a model that explains pilin Av can be separately tested to support the validity of these novel models that account for the high-frequency gene conversions that mediate pilin Av.

Notes: In Bacillus subtilis, the ComQXPA quorum-sensing system controls cell density-dependent phenotypes such as the production of degradative enzymes and antibiotics and the development of genetic competence. Bacillus subtilis (natto) NAF12, a mutant defective in poly-γ-glutamate (γ-PGA) production, was derived from B. subtilis (natto) NAF4 by Tn917-LTV1 insertional mutagenesis. Determination of the mutant DNA sequences flanking the Tn917-LTV1 insert revealed that the insertion had inactivated comP in this mutant, indicating that γ-PGA synthesis in B. subtilis (natto) is under the control of the ComP–ComA signal transduction system. A comparison of the amino acid sequences revealed striking variation in the primary structures of ComQ (44% identity), ComX (26%) and the sensor domain of ComP (36%) between B. subtilis (natto) NAF4 and B. subtilis 168. In contrast, the amino acid and nucleotide sequences of the kinase domains of ComP and of the ComA response regulator share 95% and 100% identity respectively. The comP genes of NAF4 and 168 restored the impaired competence of B. subtilis BD1658 (comP::cat) and γ-PGA production of B. subtilis (natto) NAF12 (comP::Tn917-LTV1) to only 15% of the level achieved by the respective parent comP genes. However, when introduced together with the cognate comQ and comX genes, the comP genes restored the relevant defect of the heterologous comP mutants nearly to wild-type levels. Analogous to the comCDE system of Streptococcus strains and the agrBCDE system of Staphylococcus aureus, the concerted variation in the comQXP genes appears to establish specific intercellular communication between B. subtilis strains sharing the same pheromone system.

Notes: The spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized. The C-terminal DNA-binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). A 12-amino-acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target. Insertional inactivation of spo0A in C. beijerinckii blocked the formation of solvents (as well as spores and granulose). Sequences resembling Spo0A-binding motifs (TGNCGAA) are found in the promoter regions of several of the genes whose expression is modulated at the onset of solventogenesis in Clostridium acetobutylicum and C. beijerinckii. These include the upregulated adc gene, encoding acetoacetate decarboxylase (EC 4.1.1.4), and the downregulated ptb gene, encoding phosphotransbutyrylase (EC 2.3.1.c). In vitro gel retardation experiments using C. acetobutylicum adc and C. beijerinckii ptb promoter fragments and recombinant Bacillus subtilis and C. beijerinckii Spo0A suggested that adc and ptb are directly controlled by Spo0A. The binding affinity was reduced when the 0A boxes were destroyed, and enhanced when they were modified to conform precisely to the consensus sequence. In vivo analysis of wild-type and mutagenized promoters transcriptionally fused to the gusA reporter gene in C. beijerinckii validated this hypothesis. Post-exponential phase expression from the mutagenized adc promoter was substantially reduced, whereas expression from the mutagenized ptb promoter was not shut down at the end of exponential growth.

Notes: The Cpx envelope stress response of Escherichia coli is controlled by a two-component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors. We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tethering an MBP–CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment. Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy. Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon. Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activation of the σE stress response. Because the σE response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process.

Notes: The two-component regulatory proteins ResD and ResE are required for anaerobic nitrate respiration in Bacillus subtilis. ResD, when it undergoes ResE-dependent phosphorylation, is thought to activate transcriptionally anaerobically induced genes such as fnr, hmp and nasD. In this report, deletion analysis of the fnr, hmp and nasD promoter regions was carried out to identify cis-acting sequences required for ResDE-dependent transcription. The results suggest that the hmp and nasD promoters have multiple target sequences for ResDE-dependent regulation and that fnr has a single target site. Gel mobility shift assays and DNase I footprinting analyses were performed to determine whether ResD interacts directly with the regulatory regions of the three genes. Our results indicate that ResD specifically binds to sequences residing upstream of the hmp and nasD promoters and that phosphorylation of ResD significantly stimulates this binding. In contrast, a higher concentration of ResD is required for binding to the fnr promoter region and no stimulation of the binding by ResD phosphorylation was observed. Taken together, these results suggest that ResD activates transcription of fnr, hmp and nasD by interacting with DNA upstream of these promoters. Our results suggest that phosphorylation of ResD stimulates binding to multiple ResD binding sites, but is much less stimulatory if only a single binding site exists.

Notes: A two-component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, encoded by resD and resE genes of the res operon (resABCDE), has a regulatory role in both aerobic and anaerobic respiration. In terms of aerobic respiration, resD functions upstream of ctaA, a gene required for haem A biogenesis and hence for the synthesis of haem A-containing cytochrome terminal oxidases. Although ResD is probably a transcription factor, there was no direct evidence that ResD protein, either phosphorylated or unphosphorylated, interacts directly with regulatory regions of ResD-controlled genes. Here, we report the overexpression and purification of ResD and ResE and their role in gene activation. ResD can be phosphorylated by ResE in vitro and is a monomer in solution in either the phosphorylated or unphosphorylated state. The binding activity of ResD to the ctaA promoter was examined by gel shift assays and DNase I footprinting assays. DNase I footprinting showed both unphosphorylated and phosphorylated ResD binding to the ctaA promoter and showed that there are three binding sites (A1, A2 and A3), two (A1 and A2) upstream of the −35 promoter region and one (A3) downstream of the −10 of the promoter. The role of each site in ctaA promoter activity and ResD binding was characterized using deletion analysis, followed by the DNase I footprinting and in vivo transcription assays of promoter–lacZ fusions. Our results showed that the concentration of ResD required to bind at each site is different and that ResD binding at the A1 site is independent of the other two ResD binding sites, but that the concentration of ResD∼P required to protect site A2 is reduced when site A3 is present. In vivo transcription assays from promoter–lacZ fusion constructs showed that DNA containing ResD-binding site A2 was essential for promoter activity and that promoter constructs containing both binding sites A2 and A3 were sufficient for full promoter activity.

Notes: The flhDC operon of Salmonella typhimurium is the master control operon required for the expression of the entire flagellar regulon. The flagellar master operon was placed under the tetracycline-inducible promoter PtetA using the T-POP transposon. Cells containing this construct are motile in the presence of tetracycline and non-motile without inducer present. No flagella were visible under the electron microscope when cells were grown without inducer. The class 1, class 2 and class 3 promoters of the flagellar regulon are temporally regulated. After addition of tetracycline, the class 1 flhDC operon was transcribed immediately. Transcription of flgM (which is transcribed from both class 2 and class 3 promoters) began 15 min after induction. At 20 min after induction, the class 2 fliA promoter became active and intracellular FliA protein levels increased; at 30 min after induction, the class 3 fliC promoter was activated. Induction of fliC gene expression coincides with the appearance of FlgM anti-sigma factor in the growth medium. This also coincides with the completion of hook–basal body structures. Rolling cells first appeared 35 min after induction, and excess hook protein (FlgE) was also found in the growth medium at this time. At 45 min after induction, nascent flagellar filaments became visible in electron micrographs and over 40% of the cells exhibited some swimming behaviour. Multiple flagella assemble and grow on individual cells after induction of the master operon. These results confirm that the flagellar regulatory hierarchy of S. typhimurium is temporally regulated after induction. Both FlgM secretion and class 3 gene expression occur upon completion of the hook–basal body structure.

Notes: Nitrogen metabolism in Aspergillus nidulans is regulated by AREA, a member of the GATA family of transcription factors. One mechanism that modulates AREA activity involves the rapid degradation of the areA transcript when sufficient NH4+ or Gln are available. This signalling mechanism has been shown to require a region of 218 nucleotides within the 3′ untranslated region of areA mRNA. We demonstrate that this region functions independently in a heterologous transcript and acts to accelerate degradation of the poly(A) tail, which in turn leads to rapid transcript degradation in response to the addition of NH4+ or Gln to the growth medium. areA transcript degradation is inhibited by cycloheximide, but this is not a general consequence of translational inhibition. We believe that this is the first reported example in which specific physiological signals, acting through a defined sequence within a transcript, have been shown to promote accelerated poly(A) degradation, which in turn triggers transcript degradation.

Notes: Many strains of the important human pathogen Streptococcus pyogenes form aggregates when grown in vitro in liquid medium. The present studies demonstrate that this property is crucial for the adherence, the resistance to phagocytosis and the virulence of S. pyogenes. A conserved sequence of 19 amino acid residues (designated AHP) was identified in surface proteins of common S. pyogenes serotypes. This sequence was found to promote bacterial aggregation through homophilic protein–protein interactions between AHP-containing surface proteins of neighbouring bacteria. A synthetic AHP peptide inhibited S. pyogenes aggregation, reduced the survival of S. pyogenes in human blood and attenuated its virulence in mice. In contrast, mutant bacteria devoid of surface proteins containing AHP-related sequences did not aggregate or adhere to epithelial cells. These bacteria are also rapidly killed in human blood and show reduced virulence in mice, underlining the pathogenic significance of the AHP sequence and S. pyogenes aggregation.

Notes: Transcription of the type IV pilus subunit gene of Pseudomonas aeruginosa is controlled by a two-component signal transduction system. PilS, the histidine kinase, is membrane bound and PilR, its cognate response regulator, is cytoplasmic. The signal that activates PilS is unknown. PilS has three domains: (i) The N-terminus, predicted to form six transmembrane (TM) helices; (ii) a central linker domain; and (iii) the C-terminal transmitter domain containing all the conserved residues of sensor kinases. A translational fusion of the gfp gene (green fluorescent protein) to the 3′ end of pilS was used to determine the position of PilS in the bacterial cell. Epifluorescence microscopy revealed that PilS is retained to the poles of P. aeruginosa but is distributed evenly about the membrane of Escherichia coli. Deletions of the PilS–GFP fusion revealed that the TM domain was sufficient and necessary to bring GFP to the membrane of P. aeruginosa and E. coli but was not sufficient to confine GFP to the poles. Retention to the poles of P. aeruginosa required both the TM and linker domains. Replacement of the PilS TM domain with an E. coli membrane protein, MalG, still allowed polar localization. Therefore, the PilS TM domain positions the linker domain close to the membrane allowing it to interact with the putative polar anchor which is specific to P. aeruginosa.

Notes: NC2 (Dr1/DRAP1) and Mot1p are global repressors of transcription that have been isolated in both Saccharomyces cerevisiae and humans. NC2 is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TBP and inhibition of TFIIA and TFIIB. Mot1p is an ATPase that removes DNA-bound TBP upon ATP hydrolysis. In this work, we studied the core promoter specificity of NC2 in vivo using a strain that carries mutated NC2β activity. We show that NC2, like Mot1p, is required for transcription of the HIS3 and HIS4 TATA-less core promoters. Furthermore, whereas neither Mot1p nor NC2 appear to function as repressors of the HIS3 gene in cells growing exponentially in glucose, we find that both are required for repression of the HIS3 TATA promoter when cells go through the diauxic shift. Thus, the activity of these factors is similarly regulated depending upon the physiological conditions, and it appears that core promoters activated or repressed by them in vivo might be distinguishable by whether or not they contain a canonical TATA sequence. Finally, although NC2 is an essential factor for yeast viability, we isolated a mutation in a non-essential component of the holoenzyme, Sin4p, that bypasses the requirement for NC2.

Notes: Screening of an Aspergillus niger differential cDNA library, constructed by subtracting cDNA fragments of a xlnR loss-of-function mutant from wild-type cDNA fragments, resulted in the cloning of the gene encoding d-xylose reductase (xyrA). Northern blot analysis using an A. niger wild-type strain, a xlnR multiple-copy strain and a xlnR loss-of-function mutant confirmed that the xyrA gene is regulated by XlnR, the transcriptional activator of the xylanolytic enzyme system in A. niger. d-xylose reductase catalyses the NADPH-dependent reduction of d-xylose to xylitol, which is the first step in d-xylose catabolism in fungi. Until now, XlnR was shown to control the transcription of genes encoding extracellular hydrolytic enzymes involved in cellulose and xylan degradation. In the present study, we show that A. niger is able to harmonize its sugar metabolism and extracellular xylan degradation via XlnR by regulating the expression of XyrA.

Notes: The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter. MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point. In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and −10 region. Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression. Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter. The two MelR binding sites required for this optimal repression are separated by 177 bp. We suggest that, in the absence of melibiose, MelR forms a loop between these two sites. We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated. However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and −10 region. Parallels with the Escherichia coli araC–araBAD regulatory region are discussed.

Notes: The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle-docking process requires that Ypt proteins are located in the vesicle membrane. C-terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6-2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild-type or mrs6-2 cells incubated at 25°C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6-2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.

Notes: Using a combined in vivo and in vitro approach, we demonstrated that the transposition products generated by IS911 from a dimeric donor plasmid are different from those generated from a plasmid monomer. When carried by a monomeric plasmid donor, free IS911 transposon circles are generated by intra-IS recombination in which one IS end undergoes attack by the other. These represent transposition intermediates that undergo integration using the abutted left (IRL) and right (IRR) ends of the element, the active IRR–IRL junction, to generate simple insertions. In contrast, the two IS911 copies carried by a dimeric donor plasmid not only underwent intra-IS recombination to generate transposon circles but additionally participated in inter-IS recombination. This also creates an active IRR–IRL junction by generating a head-to-tail IS tandem dimer ([IS]2) in which one of the original plasmid backbone copies is eliminated in the formation of the junction. Both transposon circles and IS tandem dimers are generated from an intermediate in which two transposon ends are retained by a single strand joint to generate a figure 8 molecule. Inter-IS figure 8 molecules generated in vitro could be resolved into the [IS]2 form following introduction into a host strain by transformation. Resolution did not require IS911 transposase. The [IS]2 structure was stable in the absence of transposase but was highly unstable in its presence both in vivo and in vitro. Previous studies had demonstrated that the IRR–IRL junction promotes efficient intermolecular integration and intramolecular deletions both in vivo and in vitro. Integration of the [IS]2 derivative would result in a product that resembles a co-integrate structure. It is also shown here that the IRR–IRL junction of the [IS]2 form and derivative structures can specifically target one of the other ends in an intramolecular transposition reaction to generate transposon circles in vitro. These results not only demonstrate that IS911 (and presumably other members of the IS3 family) is capable of generating a range of transposition products, it also provides a mechanistic framework which explains the formation and activity of such structures previously observed for several other unrelated IS elements. This behaviour is probably characteristic of a large number of IS elements.

Notes: Lateral gene transfer (LGT) is a major force in microbial genome evolution. Here, we present an overview of lateral transfers affecting genes involved in isopentenyl diphosphate (IPP) synthesis. Two alternative metabolic pathways can synthesize this universal precursor of isoprenoids, the 1-deoxy-d-xylulose 5-phosphate (DOXP) pathway and the mevalonate (MVA) pathway. We have surveyed recent genomic data and the biochemical literature to determine the distribution of the genes composing these pathways within the bacterial domain. The scattered distribution observed is incompatible with a simple scheme of vertical transmission. LGT (among and between bacteria, archaea and eukaryotes) more parsimoniously explains many features of this pattern. This alternative scenario is supported by phylogenetic analyses, which unambiguously confirm several cases of lateral transfer. Available biochemical data allow the formulation of hypotheses about selective pressures favouring transfer. The phylogenetic diversity of the organisms involved and the range of possible causes and effects of these transfer events make the IPP biosynthetic pathways an ideal system for studying the evolutionary role of LGT.

Notes: The commercially important bacterium Lactococcus lactis contains two FNR-like proteins (FlpA and FlpB) which have a high degree of identity to each other and to the FLP of Lactobacillus casei. FlpA was isolated from a GST–FlpA fusion protein produced in Escherichia coli. Like FLP, isolated FlpA is a homodimeric protein containing both Zn and Cu. However, the properties of FlpA were more like those of the E. coli oxygen-responsive transcription factor FNR than the FLP of L. casei. As prepared FlpA recognized an FNR site (TTGAT-N4-ATCAA) but not an FLP site (CCTGA-N4-TCAGG) in band-shift assays. In contrast to FLP, DNA binding by FlpA did not require the formation of an intramolecular disulphide bond. However, despite containing only two cysteine residues per monomer, FlpA was able to acquire an FNR-like, oxygen-labile [4Fe 4S] cluster. But, whereas the incorporation of a [4Fe 4S] cluster into FNR enhances interaction with target DNA, it abolished DNA binding by FlpA. An FlpA variant (FlpA′) with an N-terminal region designed to be more FLP-like failed to incorporate an iron–sulphur cluster but could now form an intramolecular disulphide. This simple example of protein engineering, converting an oxygen-labile [4Fe 4S] containing FNR-like protein into a dithiol–disulphide FLP-like redox sensor demonstrates the versatility of the basic CRP structure. Attempts to demonstrate an FlpA-based aerobic–anaerobic switch in the heterologous host E. coli were unsuccessful. However, studies with a series of FNR-dependent lac reporter fusions in strains of E. coli expressing flpA or flpB revealed that both homologues were able to activate expression of FNR-dependent promoters in vivo but only when positioned 61 base pairs upstream of the transcription start.

Notes: MarR, the negative regulator of the Escherichia coli multiple antibiotic resistance (marRAB) operon, is a member of a newly recognized family of regulatory proteins. The amino acid sequences of these proteins do not display any apparent homologies to the DNA binding domains of prokaryotic transcription regulators and a DNA binding motif for any one of the MarR homologues is currently unknown. In order to define regions of MarR required for DNA binding, mutant repressors, selected based on their ability to interfere with (negatively complement) the activity of wild-type MarR, were isolated. As determined using gel mobility shift assays, 13 out of 14 negative complementing mutants tested were unable to bind DNA in vitro. Three negative complementing alleles presumably specify truncated repressors and one of these proteins, a 120 residue MarR, can bind DNA in vitro. Most of the negative complementing mutations were clustered within two areas of MarR with features related to a helix–turn–helix DNA binding motif. These regions are presumed to be required for the DNA binding activity of the repressor.

Notes: Saccharomyces cerevisiae PAU genes constitute the largest multigene family in yeast, with 23 members located mainly in subtelomeric regions. The role and regulation of these genes were previously unknown. We detected PAU gene expression during alcoholic fermentation. An analysis of PAU gene regulation using PAU–lacZ fusions and Northern analyses revealed that they were regulated by anaerobiosis. PAU genes display, however, different abilities to be induced by anaerobiosis and this appears to be related to their chromosomal localization; two subtelomeric copies are more weakly inducible than an interstitial one. We show that PAU genes are negatively regulated by oxygen and repressed by haem. Examination of PAU gene expression in rox1Δ and tup1Δ strains indicates that PAU repression by oxygen is mediated by an unknown, haem-dependent pathway, which does not involve the Rox1p anaerobic repressor but requires Tup1p. Given the size of the gene family, PAU genes could be expected to be important during yeast life and some of them probably help the yeast to cope with anaerobiosis.

Notes: Agrobacterium rhizogenes transfers DNA (T-DNA) from its Ri plasmid to plant cells. All T-DNA genes are expressed in plant cells. The rolA gene is the only T-DNA gene that contains an intron in the untranslated leader region of its mRNA. This paper shows that (i) the rolA gene is also transcribed in bacteria; (ii) the 85 bp corresponding to the spliceosomal intron drives prokaryotic gene expression in agrobacteria, in free-living rhizobia and in bacteroids within root nodules; and (iii) promoter activity is abolished by the deletion of 63 bp from its 5′ end and is reduced by mutations changing its sequence near the putative −10 region. The expression pattern of a chimeric reporter gene shows that, in free-living bacteria, gene expression takes place during the exponential phase of growth and increases at the onset of the stationary phase. Within root nodules, reporter gene expression occurs in the invasion, nitrogen fixing and senescent zones.

Notes: Antisense RNAs regulate plasmid replication by several different mechanisms. One of these mechanisms, transcriptional attenuation, was first described for the staphylococcal plasmid pT181, and later for the streptococcal plasmids pIP501 and pAMβ1. Previously, we performed detailed in vitro and in vivo analyses of the pIP501 system. Here, we present an in vitro analysis of the antisense system of plasmid pT181. The secondary structures of antisense and sense RNA species of different lengths were determined. Binding rate constants for sense/antisense RNA pairs were measured, and functional segments required for complex formation were determined. A single-round transcription assay was used for in vitro analysis of transcriptional attenuation. A comparison between pT181 and pIP501 revealed several differences; whereas a truncated derivative of pIP501 antisense RNA was sufficient for stable complex formation, both stem–loop structures of pT181-RNAI were required. In contrast to the sense RNA of pIP501, which showed an intrinsic propensity to terminate (30–50% in the absence of antisense RNA), the sense RNA of pT181 required antisense RNA for induced termination. Rate constants of formation of pT181 sense–antisense RNA complexes were similar to inhibition rate constants, in striking contrast to pIP501, in which inhibition occurred at least 10-fold faster than stable binding.

Notes: Haemophilus influenzae haemagglutinating pili are surface appendages that promote attachment to host cells and facilitate respiratory tract colonization, an essential step in the pathogenesis of disease. In contrast to other well-characterized forms of pili, H. influenzae haemagglutinating pili are two-stranded helical structures. Nevertheless, haemagglutinating pili are assembled by a pathway that involves a periplasmic chaperone and an outer membrane usher, analogous to the prototype pathway involved in the biogenesis of Escherichia coli P pili. In this study, we performed site-directed mutagenesis of the H. influenzae HifB chaperone and HifA major pilus subunit at positions homologous to sites important for chaperone–subunit interactions and subunit oligomerization in P pili. Mutations at putative subunit binding pocket residues in HifB or at the penultimate tyrosine in HifA abolished formation of HifB–HifA periplasmic complexes, whereas mutations at the −14 glycine in HifA had no effect on HifB–HifA interactions but abrogated HifA oligomerization. To define further the constraints of the interaction between HifA and HifB, we examined the interchangeability of pilus gene cluster components from H. influenzae type b strain Eagan (hifA-hifEEag) and the related H. influenzae biogroup aegyptius strain F3031 (hifA-hifEF3031). Functional pili were assembled both with HifAEag and the strain F3031 gene cluster and with HifAF3031 and the strain Eagan gene cluster, underscoring the flexibility of the H. influenzae chaperone/usher pathway in incorporating HifA subunits with significant sequence diversity. To gain additional insight into the interactive surfaces of HifA and HifB, we aligned HifA sequences from 20 different strains and then modelled the HifA structure based on the recently crystallized PapD–PapK complex. Analysis of the resulting structure revealed high levels of sequence conservation in regions predicted to interact with HifB, and maximal sequence diversity in regions potentially exposed on the surface of assembled pili. These results suggest broad applicability of structure–function relationships identified in studies of P pili, including the concepts of donor strand complementation and donor strand exchange. In addition, they provide insight into the structure of HifA and suggest a basis for antigenic variation in H. influenzae haemagglutinating pili.

Notes: Helicobacter pylori NCTC11637 expresses a lipopolysaccharide (LPS) that comprises an O antigen side-chain with structural homology to the human blood group antigen Lewis X (Lex). The role of this molecule in adhesion of H. pylori to gastric epithelial cells was investigated. Mutants expressing truncated LPS structures were generated through insertional mutagenesis of rfbM and galE; genes encode GDP mannose pyrophosphorylase and galactose epimerase respectively. Compositional and structural analysis revealed that the galE mutant expressed a rough LPS that lacked an O antigen side-chain. In contrast, an O antigen side-chain was still synthesized by the rfbM mutant, but it lacked fucose and no longer reacted with anti-Lex monoclonal antibodies (Mabs). The ability of these mutants to bind to paraffin-embedded sections from the antrum region of a human stomach was assessed. Adhesion of the wild type was characterized by tropic binding to the apical surface of mucosal epithelial cells and cells lining gastric pits. In contrast, both the rfbM and galE mutants failed to demonstrate tropic binding and adhered to the tissue surface in a haphazard manner. These results indicate that LPS and, more specifically, LeX structures in the O antigen side-chain play an important role in targeting H. pylori to specific cell lineages within the gastric mucosa. The role of LeX in this interaction was confirmed by the tropic binding of synthetic Lex, conjugated to latex beads, to gastric tissue. The observed pattern of adhesion was indistinguishable from that of wild-type H. pylori.

Notes: Exposure of Escherichia coli to a variety of DNA-damaging agents results in the induction of the global ‘SOS response’. Expression of many of the genes in the SOS regulon are controlled by the LexA protein. LexA acts as a transcriptional repressor of these unlinked genes by binding to specific sequences (LexA boxes) located within the promoter region of each LexA-regulated gene. Alignment of 20 LexA binding sites found in the E. coli chromosome reveals a consensus of 5′-TACTG(TA)5CAGTA-3′. DNA sequences that exhibit a close match to the consensus are said to have a low heterology index and bind LexA tightly, whereas those that are more diverged have a high heterology index and are not expected to bind LexA. By using this heterology index, together with other search criteria, such as the location of the putative LexA box relative to a gene or to promoter elements, we have performed computational searches of the entire E. coli genome to identify novel LexA-regulated genes. These searches identified a total of 69 potential LexA-regulated genes/operons with a heterology index of 〈 15 and included all previously characterized LexA-regulated genes. Probes were made to the remaining genes, and these were screened by Northern analysis for damage-inducible gene expression in a wild-type lexA+ cell, constitutive expression in a lexA(Def) cell and basal expression in a non-inducible lexA(Ind−) cell. These experiments have allowed us to identify seven new LexA-regulated genes, thus bringing the present number of genes in the E. coli LexA regulon to 31. The potential function of each newly identified LexA-regulated gene is discussed.

Notes: We identified an exported protease in Lactococcus lactis ssp. lactis strain IL1403 belonging to the HtrA/DegP family. Inactivation of the chromosomal gene (htrALl) encoding this protease (HtrALl) results in growth thermo-sensitivity at very high temperatures (above 37°C for L. lactis). The role of htrALl in extracellular proteolysis under normal growth conditions was examined by testing the stability of different exported proteins (i.e. fusions, a heterologous pre-pro-protein or a native protein containing repeats), having different locations. In the wild-type (wt) strain, degradation products, including the C-terminal protein ends, were present in the medium, indicating that proteolysis occurs during or after export to the cell surface; in one case, degradation was nearly total. In contrast, proteolysis was totally abolished in the htrA strain for all five proteins tested, and the yield of full-length products was significantly increased. These results suggest that HtrALl is the sole extracellular protease that degrades abnormal exported proteins. In addition, our results reveal that HtrALl is needed for the pro-peptide processing of a natural pro-protein and for maturation of a native protein. We propose that in lactococci, and possibly in other Gram-positive organisms with small sized-genomes, a single surface protease, HtrA, is totally responsible for the housekeeping of exported proteins.

Notes: In the disease course of bacillary dysentery, pathogenic Shigella flexneri invade colonic epithelial cells and spread both within and between host cells. The ability to spread intercellularly allows the organism to infect an entire epithelial layer without significant contact with the extracellular milieu. Using fluorescence activated cell sorter (FACS)-based technology, we developed a rapid and powerful selection strategy for the isolation of S. flexneri mutants that are unable to spread from cell to cell. The majority of mutants identified using this strategy harbour mutations that affect the structure of their lipopolysaccharide or the ability of the bacteria to move intracellularly via actin-based motility; both factors have previously been shown to be essential for cell-to-cell spread. However, using a modified strategy that eliminated both of these types of mutants, we identified several mutants that provide us with evidence that bacterial proteins of the type III secretion system, which are essential for bacterial entry into host cells, also play a role in cell-to-cell spread.

Notes: Using PCR, reverse transcription-PCR (RT-PCR) and colony hybridization in a genomic library, we isolated six genes which encode type II P-type ATPases in Neurospora crassa. The six full-length cDNAs were cloned in a yeast expression vector and transformed into Saccharomyces cerevisiae null Ca2+- or Na+-ATPase mutants. Three cDNAs suppressed the defect of the Ca2+ mutant and two of these protected from Mn2+ toxicity. One cDNA suppressed the defect of the Na+ mutant and two cDNAs were not functional in S. cerevisiae. The expression of the transcripts of the six genes in the presence of Ca2+, Na+, high pH or supporting an osmotic shock indicated that, with the exception of one of the Ca2+-ATPases, the main function of the cloned ATPases is the adaptation to stress conditions. The relationship between the cloned fungal Ca2+- and Na+-ATPases and plant type II P-ATPases is discussed.

Notes: The chromosomal DpnII gene cassette of Streptococcus pneumoniae encodes two methyltransferases and an endonuclease. One methyltransferase acts on double-stranded and the other on single-stranded DNA. Two mRNAs are transcribed from the cassette. One, a SigA promoter transcript, includes all three genes; the other includes a truncated form of the second methyltransferase gene (dpnA) and the endonuclease gene. The truncated dpnA, which is translated from the second start codon in the full gene, was shown to produce active enzyme. A promoter reporter plasmid for S. pneumoniae was devised to characterize the promoter for the second mRNA. This transcript was found to depend on a promoter that responded to the induction of competence for genetic transformation. The promoter contains the combox sequence recognized by a SigH-containing RNA polymerase. As part of the competence regulon, the dpnA gene makes a product able to methylate incoming plasmid strands to protect them from the endonuclease and allow plasmid establishment. Its function differs from most genes in the regulon, which are involved in DNA uptake. Comparison of R6 and Rx strains of S. pneumoniae showed the temperature dependence of transformation in R6 to result from temperature sensitivity of the uptake apparatus and not the development of competence.

Notes: The Saccharomyces cerevisiae nuclear gene OXA1, which is conserved from prokaryotes to human, was shown to be essential for cytochrome c oxidase and F1F0–ATP synthase biogenesis. We have searched for an orthologue of OXA1 in Schizosaccharomyces pombe, another yeast that is highly diverged from S. cerevisiae and which could more closely model higher eukaryotes. In particular, S. pombe exhibits a limited growth under anaerobic conditions and is petite negative, that is it does not tolerate large deletions of its mitochondrial DNA. Surprisingly, two S. pombe cDNAs able to complement an S. cerevisiae oxa1 mutation were isolated. The corresponding genes have different chromosomal locations and intron contents. They encode distinct proteins, both sharing a weak sequence identity one with the other and with Oxa1p. A phenotypic analysis of both single inactivations demonstrates that only one gene is essential for respiration in S. pombe. However, the double inactivation is lethal. This work gives new insight into the dependence of S. pombe viability upon oxa1 function, providing evidence of a connection between petite negativity, a functional respiratory chain and F1F0–ATP synthase complex in S. pombe.

Notes: The outer membrane protects Gram-negative bacteria against a harsh environment. At the same time, the embedded proteins fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction. Unlike membrane proteins from all other sources, integral outer membrane proteins do not consist of transmembrane α-helices, but instead fold into antiparallel β-barrels. Over recent years, the atomic structures of several outer membrane proteins, belonging to six families, have been determined. They include the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate-specific porins (LamB, ScrY) and the TonB-dependent iron siderophore transporters FhuA and FepA. These crystallographic studies have yielded invaluable insight into and decisively advanced the understanding of the functions of these intriguing proteins. Our review is aimed at discussing their common principles and peculiarities as well as open questions associated with them.

Notes: ExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways. The N-terminus (residues 1–234) is a GTPase activating protein (GAP) for RhoGTPases, while the C-terminus (residues 232–453) encodes an ADP-ribosyltransferase. Utilizing a series of N-terminal deletion peptides of ExoS and an epitope-tagged full-length ExoS, two independent domains have been identified within the N-terminus of ExoS that are involved in intracellular localization and expression of GAP activity. N-terminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS. The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90–234 of ExoS, which showed that membrane localization was not required to elicit actin reorganization. ExoS was present in CHO cells as a full-length form, which fractionated with membranes, and as an N-terminally processed fragment, which localized to the cytosol. Thus, ExoS localizes in eukaryotic cells to the perinuclear region and is processed to a soluble fragment, which possesses both the GAP and ADP-ribosyltransferase activities.

Notes: The genome sequence of Helicobacter pylori suggests that this bacterium possesses several Fe acquisition systems, including both Fe2+- and Fe3+-citrate transporters. The role of these transporters was investigated by generating insertion mutants in feoB, tonB, fecA1 and fecDE. Fe transport in the feoB mutant was ≈ 10-fold lower than in the wild type (with 0.5 μM Fe), irrespective of whether Fe was supplied in the Fe2+ or Fe3+ form. In contrast, transport rates were unaffected by the other mutations. Complementation of the feoB mutation fully restored both Fe2+ and Fe3+ transport. The growth inhibition exhibited by the feoB mutant in Fe-deficient media was relieved by human holo-transferrin, holo-lactoferrin and Fe3+-dicitrate, but not by FeSO4. The feoB mutant had less cellular Fe and was more sensitive to growth inhibition by transition metals in comparison with the wild type. Biphasic kinetics of Fe2+ transport in the wild type suggested the presence of high- and low-affinity uptake systems. The high-affinity system (apparent Ks = 0.54 μM) is absent in the feoB mutant. Transport via FeoB is highly specific for Fe2+ and was inhibited by FCCP, DCCD and vanadate, indicating an active process energized by ATP. Ferrozine inhibition of Fe2+ and Fe3+ uptake implied the concerted involvement of both an Fe3+ reductase and FeoB in the uptake of Fe supplied as Fe3+. Taken together, the results are consistent with FeoB-mediated Fe2+ uptake being a major pathway for H. pylori Fe acquisition. feoB mutants were unable to colonize the gastric mucosa of mice, indicating that FeoB makes an important contribution to Fe acquisition by H. pylori in the low-pH, low-O2 environment of the stomach.

Notes: The location of the cis-acting regulatory region for histidine-dependent antitermination of the Bacillus subtilis hut operon was determined. A secondary structure, whose sequences partially overlap with the downstream terminator, was found in the regulatory region of the hut transcript. Mutational analysis of the regulatory region showed that the secondary structure was required for histidine-dependent antitermination. An electrophoretic mobility-shift assay demonstrated that, in response to the presence of histidine and Mg2+, purified HutP bound hut RNA bearing putative secondary structure but not RNA lacking the potential to form putative secondary structure. Native gel electrophoresis showed that HutP existed as a hexamer. A filter-binding assay revealed that the concentration of histidine required for half-maximal binding of HutP to RNA was 3.1 mM and that the Kd for binding of HutP to RNA was ≈ 0.56 µM in the presence of histidine. These results suggested that putative secondary structure in the regulatory region of hut mRNA could function as an antiterminator to inhibit the formation of the terminator structure and that HutP causes expression of the hut structural genes by binding to the putative antiterminator structure in response to the presence of histidine.

Notes: Rad6p plays important roles in post-replication DNA repair, chromatin organization, gene silencing and meiosis. In this study, we show that Rad6p also regulates yeast-hypha morphogenesis in the human pathogen Candida albicans. CaRAD6 gene and cDNAs were isolated and characterized revealing that the gene carries two 5′-proximal introns. CaRad6p shows a high degree of sequence similarity to Rad6 proteins from fungi to man (60–83% identity), and it suppresses the UV sensitivity and lack of induced mutagenesis displayed by a Saccharomyces cerevisiae rad6 mutant. In C. albicans, CaRAD6 expression is induced in response to UV, and CaRad6p depletion confers UV sensitivity, confirming that Rad6p serves a role in protecting this fungus against UV damage. CaRAD6 overexpression inhibits hyphal development, whereas CaRad6p depletion enhances hyphal growth. Also, CaRAD6 mRNA levels decrease during the yeast-hypha transition. These effects are dependent on Efg1p, but not Cph1p, indicating that CaRad6p acts specifically through the Efg1p morphogenetic signalling pathway to repress yeast-hypha morphogenesis.

Notes: Control of transcription in prokaryotes often involves direct contact of regulatory proteins with RNA polymerase. For the σ54 RNA polymerase, regulatory proteins bound to distally located enhancers engage the polymerase via DNA looping. The σ54-dependent nifA promoter of Herbaspirillum seropedicae (Hs) is activated under nitrogen-limiting growth conditions. Potential enhancers for the nitrogen control activators NTRC and NIFA and binding sites for integration host factor (IHF) and σ54-holoenzyme were identified. DNA footprinting experiments showed that these sites functioned for protein binding. Their involvement in the promoter regulation was explored. In vitro, activation of the Hs nifA promoter by NTRC is stimulated by the DNA bending protein IHF. In marked contrast, activation by NIFA is greatly reduced by IHF, thus diminishing potentially destabilizing autoactivation of the nifA promoter by NIFA. Additionally, high levels of NIFA appear to limit NTRC-dependent activation. This inhibition is IHF dependent. Therefore, IHF acts positively and negatively at the nifA promoter to restrict transcription activation to NTRC and one signal transduction pathway.

Notes: LamB of Escherichia coli K12, also called maltoporin, is an outer membrane protein, which specifically facilitates the diffusion of maltose and maltodextrin through the bacterial outer membrane. Each monomer is composed of an 18-stranded antiparallel β-barrel. In the present work, on the basis of the known X-ray structure of LamB, the effects of modifications of the β-barrel domain of maltoporin were studied in vivo and in vitro. We show that: (i) the substitution of the pair of strands β13–β14 of the E. coli maltoporin with the corresponding pair of strands from the functionally related maltoporin of Salmonella typhimurium yielded a protein active in vivo and in vitro; and (ii) the removal of one pair of β-strands (deletion β13–β14) from the E. coli maltoporin, or its replacement by a pair of strands from the general porin OmpF of E. coli, leads to recombinant proteins that lost in vivo maltoporin activities but still kept channel formation and carbohydrate binding in vitro. We also inserted into deletion β13–β14 the portion of the E. coli LamB protein comprising strands β13 to β16. This resulted in a protein expected to have 20 β-strands and which completely lost all LamB-specific activities in vivo and in vitro.

Notes: In response to starvation, Myxococcus xanthus undergoes a multicellular developmental process that produces a dome-shaped fruiting body structure filled with differentiated cells called myxospores. Two insertion mutants that block the final stages of fruiting body morphogenesis and reduce sporulation efficiency were isolated and characterized. DNA sequence analysis revealed that the chromosomal insertions are located in open reading frames ORF2 and asgE, which are separated by 68 bp. The sporulation defect of cells carrying the asgE insertion can be rescued phenotypically when co-developed with wild-type cells, whereas the sporulation efficiency of cells carrying the ORF2 insertion was not improved when mixed with wild-type cells. Thus, the asgE insertion mutant appears to belong to a class of developmental mutants that are unable to produce cell–cell signals required for M. xanthus development, but they retain the ability to respond to them when they are provided by wild-type cells. Several lines of evidence indicate that asgE cells fail to produce normal levels of A-factor, a cell density signal. A-factor consists of a mixture of heat-stable amino acids and peptides, and at least two heat-labile extracellular proteases. The asgE mutant yielded about 10-fold less heat-labile A-factor and about twofold less heat-stable A-factor than wild-type cells, suggesting that the primary defect of asgE cells is in the production or release of heat-labile A-factor.

Notes: We describe here 20 families of secondary (pmf-driven) carriers which, in addition to nine families within the ATP-dependent ABC superfamily, and seven families of Gram-negative bacterial outer membrane porins, largely account for the stereospecific transport of sugars and their derivatives into and out of all living cells on earth. Family characteristics as well as struc-tural and functional properties of the family constituents are described. By reference to our website (), phylogenetic relationships, detailed substrate specificity information and both primary and secondary references are also available. This review provides a comprehensive guide to the diversity of carriers that mediate the transport of sugar-containing molecules across cell and organellar membranes.

Notes: This study shows that characterization of the molecular mobility in the cytoplasm of pollen provides a new understanding of the effects of moisture and temperature on ageing rates. Using EPR spectroscopy, we determined the rotational motion of the polar spin probe, 3-carboxy-proxyl, in the cytoplasm of Typha latifolia pollen, under different temperature and moisture content conditions. Increasing the temperature resulted in faster rotational motion, analogous to faster ageing rates. With decreasing moisture content, rotational motion first decreased until a minimum was reached, after which rotational motion slightly increased again. The moisture content at which this minimal rotational motion was observed increased with decreasing temperature, comparable to the pattern of ageing rate. A significant linear relationship was found between ageing rates and rotational motion in the cytoplasm, suggesting that these parameters are causally linked. Upon melting of the intracellular glass, a twofold increase in activation energy of rotational motion and ageing rate was observed. In contrast, melting of the sucrose glass resulted in an increase in rotational motion of five orders of magnitude. The difference in rotational motion upon melting glasses of pollen or sucrose suggests that other molecules beside sugars play a role in intracellular glass formation in pollen.

Notes: Stomata have a fundamental role in controlling plant photosynthesis and transpiration, but very little is known about factors controlling stomatal differentiation and development. Lines of soybean that contain a specific flavonol glycoside, kaempferol-3-O-2-glycosyl-gentiobioside (K9), as well as greatly reduced stomatal density, especially on the adaxial epidermis, have been identified. The specific effects of blue light photoreceptors on stomatal development in K9 lines and their isoline pairs containing no K9 were studied. Low irradiances of blue light (7% of total photosynthetically active radiation) added to high irradiances from low-pressure sodium lamps strongly inhibited stomatal development on the adaxial epidermis of K9 lines, but not in isoline pairs differing putatively in only one gene and lacking K9. Overall, blue light slightly increased stomatal density on the abaxial epidermis in all isolines, demonstrating differential regulation of stomatal development in the upper and lower epidermis. Blue light also caused an increase in leaf area in all isolines, indicating that changes in stomatal density were not the non-specific result of alterations in leaf area. Morphological studies revealed that the blue light-induced reduction in stomatal density in K9 lines was due to reduced stomatal initiation as well as aborted or abnormal stomatal development. As the phytochrome photostationary state was kept constant, the results indicate that one or more blue light receptors are involved in the control of stomatal development. This system should be useful for the study of mechanisms controlling stomatal development, even if the photo-inhibitory response is unique to K9 lines.

Notes: Isoprene emission from plants is highly temperature sensitive and is common in forest canopy species that experience rapid leaf temperature fluctuations. Isoprene emission declines with temperature above 35 °C but the temperature at which the decline begins varies between 35 and 44 °C. This variability is caused by the rate at which leaf temperature is increased during measurement with lower temperatures associated with longer measurement cycles. To investigate this we exposed leaves of red oak (Quercus rubra L.) to temperature regimes of 35–45 °C for periods of 20–60 min. Isoprene emission increased during the first 10 min of high temperature exposure and then decreased over the next 10 min until it reached steady state. This phenomenon was common at temperatures above 35 °C but was not noticeable at temperatures below that. The response was reversible within 30 min by lowering leaf temperature to 30 °C. Because there is no storage of isoprene inside the leaf, this behaviour indicates regulation of isoprene synthesis in the leaf. We demonstrated that the variability in isoprene decline results from regulation and explains the variability in the temperature response. This is consistent with our theory that isoprene protects leaves from damage caused by rapid temperature fluctuations.

Notes: The mechanisms controlling the photosynthetic performance of C4 plants at low temperature were investigated using ecotypes of Bouteloua gracilis Lag. from high (3000 m) and low (1500 m) elevation sites in the Rocky Mountains of Colorado. Plants were grown in controlled-environment cabinets at a photon flux density of 700 μmol m−2 s−1 and day/night temperatures of 26/16 °C or 14/7 °C. The thermal response of the net CO2 assimilation rate (A) was evaluated using leaf gas-exchange analysis and activity assays of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPCase) and pyruvate,orthophosphate dikinase (PPDK). In both ecotypes, a reduction in measurement temperature caused the CO2-saturated rate of photosynthesis to decline to a greater degree than the initial slope of A versus the intercellular CO2 response, thereby reducing the photosynthetic CO2 saturation point. As a consequence, A in normal air was CO2-saturated at sub-optimal temperatures. Ecotypic variation was low when grown at 26/16 °C, with the major difference between the ecotypes being that the low-elevation plants had higher A; however, the ecotypes responded differently when grown at cool temperature. At temperatures below the thermal optimum, A in high-elevation plants grown at 14/7 °C was enhanced relative to plants grown at 26/16 °C, while A in low-elevation plants grown at 14/7 °C was reduced compared to 26/16 °C-grown plants. Photoinhibition at low growth temperature was minor in both ecotypes as indicated by small reductions in dark-adapted Fv/Fm. In both ecotypes, the activity of Rubisco was equivalent to A below 17 °C but well in excess of A above 25 °C. Activities of PEPCase and PPDK responded to temperature in a similar proportion relative to Rubisco, and showed no evidence for dissociation that would cause them to become principal limitations at low temperature. Because of the similar temperature response of Rubisco and A, we propose that Rubisco is a major limitation on C4 photosynthesis in B. gracilis below 17 °C. Based on these results and for theoretical reasons associated with how C4 plants use Rubisco, we further suggest that Rubisco capacity may be a widespread limitation upon C4 photosynthesis at low temperature.

Notes: Monitoring the light–shadow windows of a tree via a grid system on the ground was performed on sunny summer days at high spatial resolution using a custom-built, inexpensive scanner. The measurements were taken with two goals: (1) to quickly and remotely quantify the overall, short-wave solar radiation (300–1100 nm) intercepted by the tree canopy, and (2) to yield such crown geometric traits as shape, size and the number of theoretical canopy leaf layers (leaf layer index, LLI) in relation to the section orthogonal to sunbeam direction (sun window). The ground readings at each measurement over the day were used to project a digitized shadow image. Image processing was applied and the intercepted radiation was calculated as the difference from the corresponding incoming radiation above the canopy. Tree-crown size and shape were profiled via computer imaging by analysing the different shadow images acquired at the various solar positions during the day. It is notable that these combined images yielded the crown features without having to parameterize such canopy characteristics as foliage extension and spatial distribution.

Notes: The physiological reason that higher plants are green is unknown. Other photosynthetic organisms utilize pigments that strongly absorb green light; therefore, there must have been natural forces that ‘selected’ the photosynthetic pigments found in higher plants. Based on previously published data and our recent findings about green light and photosynthesis within leaves (Sun et al.), a specific functional role is described for the primary photosynthetic pigments of higher plants, that were derived from green algal progenitors. The particular absorptive characteristics of chlorophylls a and b appear to perform two contradictory, but necessary functions in higher plants. Firstly, chlorophylls a and b absorb light for maximum utilization under non-saturating conditions, a function that is well understood. Secondly, they can act as protective pigments under over-saturating light conditions, when absorbed light is dissipated as heat. Under such conditions, a significant portion of light can also be efficiently utilized, especially in the bottom portion of the leaf, that is mainly illuminated by green light and not down-regulated. The second function may have been the selective force that gave rise to the extremely successful terrestrial plants, that evolved from green algae.

Notes: DNA endoreduplication in Zea mays L. (cv. A619 × W64A) endosperm peaks between 16 and 18 d after pollination (DAP). The physiological function of DNA endoreduplication is not known but it is believed to be important in maize kernel development. In the present study, we investigated how 2, 4 or 6 d of high temperature (35 °C) affected DNA endoreduplication and maize kernel development in comparison with control kernels grown at 25 °C. Data were collected on fresh weight (FW), nuclei number, mitotic index, and DNA endoreduplication. Maize endosperm FW and nuclei number were reduced by exposure to 4 or 6 d of high temperature. At 18 DAP, the 2 d high temperature treatment (HTT) caused a reduction in FW and nuclei number, but had no effect on DNA endoreduplication and average DNA content per endosperm. However, when the exposure to high temperature was increased to 4 or 6 d, FW, nuclei number and the magnitude of DNA endoreduplication were progressively reduced, and the peak mitotic index was delayed compared with the control endosperm. At 18 DAP, the 4 d treatment showed 54·7% of the cells were 3 or 6 C, whereas only 41·2% were 12 C or higher. Six days of high temperature also resulted in a reduction in endosperm FW, nuclei number and a delay in the peak of mitotic index. DNA endoreduplication occurred in the kernels exposed to this treatment, although the magnitude was severely reduced compared with the control kernels. Nuclear DNA content was highly correlated (r= 0·93) with kernel FW, suggesting an important role of DNA endoreduplication in determining endosperm FW. The data suggest that high temperature during endosperm cell division exerted negative effects on DNA endoreduplication by dramatically reducing the nuclei number, leaving fewer nuclei available for DNA endoreduplication. However, the data also suggest that prolonged exposure to high temperature restricts entry of mitotic cells into the endoreduplication phase of the cell cycle.

Notes: A mutant of Synechocystis sp. strain PCC6803 was obtained by random cartridge mutagenesis, which could not grow at low sodium concentrations. Genetic analyses revealed that partial deletion of the sll0273 gene, encoding a putative Na+/H+ exchanger, was responsible for this defect. Physiological characterization indicated that the sll0273 mutant exhibited an increased sensitivity towards K+, even at low concentrations, which was compensated for by enhanced concentrations of Na+. This enhanced Na+ demand could also be met by Li+. Furthermore, addition of monensin, an ionophore mediating electroneutral Na+/H+ exchange, supported growth of the mutant at unfavourable Na+/K+ ratios. Measurement of internal Na+ and K+ contents of wild-type and mutant cells revealed a decreased Na+/K+ ratio in mutant cells pre-incubated at a low external Na+/K+ ratio, while it remained at the level of the wild type after pre-incubation at a high external Na+/K+ ratio. We conclude that the Sll0273 protein is required for Na+ influx, especially at low external Na+ concentrations or low Na+/K+ ratios. This system may be part of a sodium cycle and may permit re-entry of Na+ into the cells, if nutrient/Na+ symporters are not functional or operating.

Notes: NAD kinase is thought to play an important role in the plant cellular responses to biotic and abiotic stress as one of the isoforms of the enzyme is activated by the Ca2+–calmodulin (CaM) complex. NAD kinase activity was measured after short-term NaCl stress applied to isolated cells from Lycopersicon esculentum, var. Volgogradskij (NaCl-sensitive tomato) and L. pimpinellifolium, acc. PE2 (NaCl-tolerant species). NAD kinase activity remained constant in the sensitive species, whereas a sharp decrease was observed in the tolerant one. After salt treatment, an induction of the calmodulin gene(s) was observed in the two species, together with a 30–50% decrease in ‘active’ CaM content, i.e. CaM able to activate purified NAD kinase, in L. pimpinellifolium. The decrease in NAD kinase activity could not, however, be fully explained by this decrease in active CaM content. A similar decrease in NAD kinase activity was also recorded with other ionic stresses and exposure to high temperatures, but not in the case of drought, exposure to low temperatures, hormonal (indole-3-acetic acid and abscisic acid) or H2O2 treatments. External Ca2+ certainly plays a role in the biochemical mechanism(s) leading to NAD kinase inhibition, while no role could be shown for intracellular Ca2+. In addition, after salt stress, a modification of the redox state of NAD kinase seems to be responsible for the inhibition of the enzyme.

Notes: Diurnal changes in photosynthetic gas exchange and chlorophyll fluorescence were measured under full sunlight to reveal diffusional and non-diffusional limitations to diurnal assimilation in leaves of Arisaema heterophyllum Blume plants grown either in a riparian forest understorey (shade leaves) or in an adjacent deforested open site (sun leaves). Midday depressions of assimilation rate (A) and leaf conductance of water vapour were remarkably deeper in shade leaves than in sun leaves. To evaluate the diffusional (i.e. stomatal and leaf internal) limitation to assimilation, we used an index [1–A/A350], in which A350 is A at a chloroplast CO2 concentration of 350 μmol mol−1. A350 was estimated from the electron transport rate (JT), determined fluorometrically, and the specificity factor of Rubisco (S), determined by gas exchange techniques. In sun leaves under saturating light, the index obtained after the ‘peak’ of diurnal assimilation was 70% greater than that obtained before the ‘peak’, but in shade leaves, it was only 20% greater. The photochemical efficiency of photosystem II (ΔF/Fm′) and thus JT was considerably lower in shade leaves than in sun leaves, especially after the ‘peak’. In shade leaves but not in sun leaves, A at a photosynthetically active photon flux density (PPFD) 〉 500 μmol m−2 s−1 depended positively on JT throughout the day. Electron flows used by the carboxylation and oxygenation (JO) of RuBP were estimated from A and JT. In sun leaves, the JO/JT ratio was significantly higher after the ‘peak’, but little difference was found in shade leaves. Photorespiratory CO2 efflux in the absence of atmospheric CO2 was about three times higher in sun leaves than in shade leaves. We attribute the midday depression of assimilation in sun leaves to the increased rate of photorespiration caused by stomatal closure, and that in shade leaves to severe photoinhibition. Thus, for sun leaves, increased capacities for photorespiration and non-photochemical quenching are essential to avoid photoinhibitory damage and to tolerate high leaf temperatures and water stress under excess light. The increased Rubisco content in sun leaves, which has been recognized as raising photosynthetic assimilation capacity, also contributes to increase in the capacity for photorespiration.

Notes: Phytochromes are photoreceptors in plants which can exist in two different conformations: the red light-absorbing form (Pr) and the far-red light-absorbing form (Pfr), depending on the light quality. The Pfr form is the physiologically active conformation. To attenuate the Pfr signal for phytochrome A (phyA), at least two different mechanisms exist: destruction of the molecule and dark reversion. Destruction is an active process leading to the degradation of Pfr. Dark reversion is the light-independent conversion of physiologically active Pfr into inactive Pr. Here, we show that dark reversion is not only an intrinsic property of the phytochrome molecule but is modulated by cellular components. Furthermore, we demonstrate that dark reversion of phyA may be observed in Arabidopsis ecotype RLD but not in other Arabidopsis ecotypes. For the first time, we have identified mutants with altered dark reversion and destruction in a set of previously isolated loss of function PHYA alleles (Xu et al. Plant Cell 1995, 7, 1433–1443). Therefore, the dynamics of the phytochrome molecule itself need to be considered during the characterization of signal transduction mutants.

Notes: Many abiotic environmental factors elicit the production of stress-ethylene in higher plants. To elucidate the molecular mechanisms underlying the regulation of stress-ethylene production and the physiological roles played by stress-ethylene in stress responses of plants, we studied the gene expression of ACC synthase in tobacco plants that had been subjected to environmental stresses. Four new tobacco ACC synthase cDNA fragments, NT-ACS2, NT-ACS3, NT-ACS4 and NT-ACS5, were identified and sequenced. It was found that NT-ACS2 could be induced by wounding, cold temperature and, especially, sunlight. NT-ACS4 was induced at a faster kinetics by wounding. The multiple environmental stress-responsive (MESR) NT-ACS2 gene was found to contain three introns and four exons and encode a polypeptide of 484 amino acids, 54·6 kDa and pI 6·87. Computer analysis of the 3·4 kb 5′ flanking region upstream of the ACS coding region revealed the existence of a group of putative cis-acting regulatory elements potentially conferring wounding, chilling, and UV light inducibility. Phylogenetic analysis of ACC synthase genes of different plant origins indicated that the chill-inducible NT-ACS2 gene is closely related to a chilling-inducible citrus ACS gene.

Notes: Growth in stagnant, oxygen-deficient nutrient solution increased porosity in adventitious roots of two monocotyledonous (Carex acuta and Juncus effusus) and three dicotyledonous species (Caltha palustris, Ranunculus sceleratus and Rumex palustris) wetland species from 10 to 30% under aerated conditions to 20–45%. The spatial patterns of radial oxygen loss (ROL), determined with root-sleeving oxygen electrodes, indicated a strong constitutive ‘barrier’ to ROL in the basal root zones of the two monocotyledonous species. In contrast, roots of the dicotyledonous species showed no significant ‘barrier’ to ROL when grown in aerated solution, and only a partial ‘barrier’ when grown in stagnant conditions. This partial ‘barrier’ was strongest in C. palustris, so that ROL from basal zones of roots of R. sceleratus and R. palustris was substantial when compared to the monocotyledonous species. ROL from the basal zones would decrease longitudinal diffusion of oxygen to the root apex, and therefore limit the maximum penetration depth of these roots into anaerobic soil. Further studies of a larger number of dicotyledonous wetland species from a range of substrates are required to elucidate the ecophysiological consequences of developing a partial, rather than a strong, ‘barrier’ to ROL.

Notes: A total of 244 plants from two species, Lythrum salicaria and Epilobium glandulosum, were grown individually in hydroponic sand culture from seed for 36 d. Until day 27 all plants experienced an irradiance of 550 μmol m−2 s−1 PFD and on day 27 half of the plants were subjected to a neutral shade treatment in which irradiance was reduced to 100 μmol m−2 s−1 photon fluy density (PFD). Measures of relative growth rate, net assimilation rate, specific leaf area, biomass partitioning to leaves, roots, structural tissues (i.e. stems, petioles and inflorescences) and tissue density were obtained from intensive harvests three or four times per day. The shade treatment caused an immediate decrease in relative growth rate and net assimilation rate. Within hours the specific leaf area of the shaded plants increased and leaf tissue density decreased, thus partially offsetting the decrease in relative growth rate. Biomass partitioning was not affected.

Notes: Compartmentation of heavy metals on or within mycorrhizal fungi may serve as a protective function for the roots of forest trees growing in soils containing elevated concentrations of metals such as Cd and Zn. In this paper we present the first quantitative measurements by X-ray microanalysis of heavy metals in high-pressure frozen and cryosectioned ectomycorrhizal fungal hyphae. We used this technique to analyse the main sites of Cd and Zn in fungal cells of mantle and Hartig net hyphae and in cortical root cells of symbiotic Picea abies – Hebeloma crustuliniforme associations to gain new insights into the mechanisms of detoxification of these two metals in Norway spruce seedlings. The mycorrhizal seedlings were exposed in growth pouches to either 1 mM Cd or 2 mM Zn for 5 weeks. The microanalytical data revealed that two distinct Cd- and Zn-binding mechanisms are involved in cellular compartmentation of Cd and Zn in the mycobiont. Whereas extracellular complexation of Cd occurred predominantly in the Hartig net hyphae, both extracellular complexation and cytosolic sequestration of Zn occurred in the fungal tissue. The vacuoles were presumed not to be a significant pool for Cd and Zn storage. Cadmium was almost exclusively localized in the cell walls of the Hartig net (up to 161 mmol kg−1 DW) compared with significantly lower concentrations in the cell walls of mantle hyphae (22 mmol kg−1 DW) and in the cell walls of cortical cells (15 mmol kg−1 DW). This suggests that the apoplast of the Hartig net is a primary accumulation site for Cd. Zinc accumulated mainly in the cell walls of the mantle hyphae (111 mmol kg−1 DW), the Hartig net hyphae (130 mmol kg−1 DW) and the cortical cells (152 mmol kg−1 DW). In addition, Zn occurred in high concentrations in the cytoplasm of the fungal mantle hyphae (up to 164 mmol kg−1 DW) suggesting that both the cell walls and the cytoplasm of fungal tissue are the main accumulation sites for Zn in P. abies resulting in decreased Zn transfer from the fungus to the root.

Notes: This study tested the hypothesis that antagonistic interactions between abscisic acid (ABA) and ethylene mediate the effects of soil compaction on shoot growth. Isogenic wild-type (Ailsa Craig), ABA-deficient (notabilis) and a transgenic (ACO1AS) tomato genotype with a reduced capacity to synthesize ethylene were examined. Exogenous ABA was also applied. Leaf area was comparable when Ailsa Craig and ACO1AS were grown in uncompacted (1·1 g cm−3) or compacted (1·5 g cm−3) soil, but was lower in notabilis. However, a 1·1/1·5 g cm−3 split-pot treatment invoked marked genotypic differences, whereby leaf area was comparable to 1·1 g cm−3 control plants in ACO1AS but was intermediate between the 1·1 and 1·5 g cm−3 treatments in Ailsa Craig and notabilis. ABA may be discounted as the root-sourced signal responsible for reducing leaf area when the roots encountered compacted soil as Ailsa Craig and ACO1AS showed differing responses despite similar increases in xylem sap ABA concentration; leaf area was invariably lower in notabilis. These genotypic differences were correlated with ethylene evolution; thus the greater leaf area in ACO1AS was associated with its reduced ability to synthesize ethylene, whereas the reductions in leaf expansion observed when Ailsa Craig and notabilis encountered compacted soil were accompanied by increased ethylene production. Application of ABA had little effect on ACO1AS, but promoted a recovery of leaf expansion in notabilis, and more surprisingly in Ailsa Craig. These results suggest that antagonistic interactions between ABA and ethylene may regulate leaf expansion when the root system simultaneously encounters uncompacted and compacted soil.

Notes: Studies of pollination ecology have been hindered by an absence of biochemical information about the basis of polymorphism. Using model plants and mutant lines described by molecular genetics may circumvent this difficulty. Mutation of genes controlling petal colour and petal epidermal cell shape in Antirrhinum majus was previously shown to influence fruit set. White flowers set less fruit than magenta flowers and mutants with flat petal epidermal cells set less fruit than flowers with conical cells. Here we analyse the causal pathway underlying this phenomenon through a study of floral characteristics and bee behaviour. Results indicate that bees recognized plants with magenta conical-celled flowers at a distance and did not approach white flowers or magenta flat-celled flowers so frequently. Petal cell shape interacted with colour in determining whether an approaching bee landed on a flower within a plot and whether a bee landing on a flower would probe it. The intrafloral temperature of flowers with conical petal cells was shown to increase with solar irradiance, unlike the intrafloral temperature of flowers with flat petal cells. The difference in fruit set may reflect pollinator discrimination between genotypes as a consequence of the effect of intrafloral temperature on nectar quality and quantity.

Notes: Although Arabidopsis halleri (=Cardaminopsis halleri) is known as a Zn hyperaccumulator, there have been no detailed studies on Zn accumulation, tolerance and cellular distribution in this species. In a hydroponic experiment, A. halleri grew healthily with Zn concentrations varying from 1 to 1000 μM, without showing phytotoxicity or reduction in root or shoot dry weights. The concentration of Zn in the shoots increased from 300 μg g−1 dry weight in the 1 μM Zn treatment to 32 000 μg g−1 in the 1000 μM Zn treatment. Approximately 60% of the total Zn in the shoots were water-soluble, and there was no evidence of Zn and P co-precipitation. Both citric and malic acid concentrations in the shoots were not significantly affected by the Zn treatments, whereas in the roots there was a positive response in both organic acids to increasing Zn in solution. Cellular distribution of Zn, Ca and K in frozen hydrated leaf tissues was examined using energy-dispersive X-ray microanalysis. Zinc was sequestered in the base of trichomes, whereas the middle and upper parts of trichomes were highly enriched with Ca. Mesophyll cells appeared to have more Zn than the epidermis, probably because the latter were very small in size. Similarities and differences between A. halleri and the other well-known Zn hyperaccumulator, Thlaspi caerulescens, are discussed.

Notes: This work explores the influence of cadmium on a suspension cell culture of Nicotiana tabacum (TBY-2) by examining cell morphology, viability and DNA integrity. Changes in these parameters were strikingly dependent on concentration of cadmium in the culture medium: a concentration of 50–100 mmol m−3 CdSO4 induced apoptotic changes including DNA fragmentation into oligonucleosomal units, while 1 mol m−3 Cd2+ showed strong cytotoxicity, but no fragmentation of DNA. Low cadmium concentrations (below 10 mmol m−3) affected neither cell viability nor DNA integrity. A detailed kinetic study showed a significant delay in the onset of apoptosis after the application of high concentrations of cadmium. From days 0–3 after the application of 50 mmol m−3 CdSO4, the morphology of the cells, their viability and growth were indistinguishable between control and treated cells, and ‘domain’ DNA fragmentation into 50–200 kb fragments was observed at the DNA level. After this (days 4–7), there was a characteristic and rapid decrease in cell viability, distinct changes in cell morphology and oligonucleosomal fragmentation. The results suggest that chronic exposure of plant cells to cadmium can trigger programmed cell death.

Notes: Cotton (Gossypium hirsutum L. cv. CS50) plants were grown at two levels of relative humidity (RH) and sprayed daily with abscisic acid (ABA) at four concentrations. Plants grown at lower humidity had higher transpiration rates, lower leaf temperatures and lower stomatal conductance. Plant biomass was also reduced at low humidity. Within each humidity environment, increasing ABA concentration generally reduced stomatal conductance, evaporation rates, superficial leaf density and plant biomass, and increased leaf temperature and specific leaf area. As expected, decreased stomatal conductance resulted in decreased carbon isotope discrimination in leaf material (Δ13Cl). Plants grown at low humidity were more enriched in 18O than those grown at high RH, as theory predicts. Within each humidity environment, increasing ABA concentration increased oxygen isotope enrichment of leaf cellulose (Δ18Oc) and whole-leaf tissue (Δ18Ol). Values of Δ13Cl and Δ18Ol predicted by theoretical models were close to those observed, accounting for 79% of the measured variation in Δ13Cl and 95% of the measured variation in Δ18Ol. Supporting theory, Δ13Cl and Δ18Ol in whole-leaf tissue were negatively related.

Notes: Solute transport systems are one of the major ways in which organisms interact with their environment. Typically, transport is catalysed by integral membrane proteins, of which one of the largest groups is the ATP-binding cassette (ABC) proteins. On the basis of sequence similarities, a large family of ABC proteins has been identified in Arabidopsis. A total of 60 open reading frames (ORFs) encoding ABC proteins were identified by BLAST homology searching of the nuclear genome. These 60 putative proteins include 89 ABC domains. Based on the assignment of transmembrane domains (TMDs), at least 49 of the 60 proteins identified are ABC transporters. Of these 49 proteins, 28 are full-length ABC transporters (eight of which have been described previously), and 21 are uncharacterized half-transporters. Three of the remaining proteins identified appear to be soluble, lacking identifiable TMDs, and most likely have non-transport functions. The eight other ORFs have homology to the nucleotide-binding and transmembrane components of multi-subunit permeases. The majority of ABC proteins found in Arabidopsis can, on the basis of sequence homology, be assigned to subfamilies equivalent to those found in the yeast genome. This assignment of the Arabidopsis ABC proteins into easily recognizable subfamilies (with distinguishable subclusters) is an important first step in the elucidation of their functional role in higher plants.

Notes: The decline in above-ground net primary productivity (Pa) that is usually observed in forest stands has been variously attributed to respiration, nutrient or hydraulic limitations. A novel model is proposed to explain the phenomenon and the co-occurring changes in the balance between foliage, conducting sapwood and fine roots. The model is based on the hypothesis that a functional homeostasis in water transport is maintained irrespective of age: hydraulic resistances through the plant must be finely tuned to transpiration rates so as to avoid extremely negative water potentials that could result in diffuse xylem embolism and foliage dieback, in agreement with experimental evidence. As the plant grows taller, allocation is predicted to shift from foliage to transport tissues, most notably to fine roots. Higher respiration and fine root turnover would result in the observed decline in Pa. The predictions of the model have been compared with experimental data from a chronosequence of Pinus sylvestris stands. The observed reduction in Pa is conveniently explained by concurrent modifications in leaf area index and plant structure. Changes in allometry and shoot hydraulic conductance with age are successfully predicted by the principle of functional homeostasis.

Notes: A structural analysis was conducted to determine whether glycoprotein-containing intercellular space occlusions are involved in medium-term regulation of O2 diffusion in soybean (Glycine max) nodules. Alterations in O2 diffusion were induced by a 3 h detopping treatment, and glycoprotein was immunolocalized with the monoclonal antibodies MAC236 and MAC265. Western blots of unstressed nodules revealed that these antibodies recognize antigens with two different molecular weights in soybean nodules. Tissue printing of halved nodules showed that both antigens were present in fresh nodules from control and 3 h detopped plants. The main localization appeared to be the inner cortex, but some immunolabelling also occurred in the infected region. ELISAs demonstrated a significant increase in total nodule concentration of intercellular glycoprotein following detopping, and cryosections of fresh nodules from this treatment also showed localization of antigens within the intercellular spaces of the infected region. The production of intercellular space occlusions in both the mid-cortex and infected regions after 3 h detopping was confirmed by light microscopy and silver-enhanced immunolabelling; cortical changes were quantified by image analysis techniques. Electron microscopy revealed that the occlusions within the infected region were less dense and less heavily labelled than those in the cortex. These results are discussed in relation to O2 diffusion regulation in soybean nodules

Notes: Variation in stomatal conductance is typically explained in relation to environmental conditions. However, tree height may also contribute to the variability in mean stomatal conductance. Mean canopy stomatal conductance of individual tree crowns (GSi) was estimated using sap flux measurements in Fagus sylvatica L., and the hypothesis that GSi decreases with tree height was tested. Over 13 d of the growing season during which soil moisture was not limiting, GSi decreased linearly with the natural logarithm of vapour pressure deficit (D), and increased exponentially to saturation with photosynthetic photon flux density (Qo). Under conditions of D= 1 kPa and saturating Qo, GSi decreased by approximately 60% with 30 m increase in tree height. Over the same range in height, sapwood-to-leaf area ratio (AS:AL) doubled. A simple hydraulic model explained the variation in GSi based on an inverse relationship with height, and a linear relationship with AS:AL. Thus, in F. sylvatica, adjustments in AS:AL partially compensate for the negative effect of increased flow-path length on leaf conductance. Furthermore, because stomata with low conductance are less sensitive to D, gas exchange of tall trees is reduced less by high D. Despite these compensations, decreasing hydraulic conductance with tree height in F. sylvatica reduces carbon uptake through a corresponding decrease in stomatal conductance.

Notes: Diurnal variation in petiole specific hydraulic conductivity and simultaneous measurements of leaf water potential were recorded in red maple, tulip tree and fox grape. Petiole specific conductivity was determined from in situ measurements of water flow into the distal (leaf-bearing) end of an attached petiole as a function of applied hydrostatic pressure and petiole dimensions. The hydraulic properties of the petiole dominated the measurements, indicating that this technique can be used for rapid estimates of petiole hydraulic conductivity. There was a significant decrease in petiole specific conductivity associated with increasingly more negative leaf water potentials in maple and tulip tree, but not in grape. Petiole specific conductivity increased during the afternoon while the plant was actively transpiring and the xylem sap was under tension. The recovery of petiole conductivity during the afternoon suggests that hydraulic conductivity reflects a dynamic balance between a loss of hydraulic conductivity with increasing water stress, and its restoration as tension within the xylem decreases. Three experimental manipulations were applied to red maple and tulip tree to examine the sensitivity of diurnal changes in petiole conductivity to various physiological perturbations. Both phloem girdling and application of HgCl2 to the transpiration stream resulted in a marked decrease in the degree to which petiole specific conductivity recovered as xylem tension relaxed during the afternoon. Delivery of a surfactant to the xylem, however, did not significantly alter the relation between leaf water potential and petiole hydraulic conductivity.

Notes: The cessation of tomato fruit growth is thought to be induced by an increase in the activity of enzymes which rigidify cell walls in the fruit skin. Peroxidase could catalyse such wall-stiffening reactions, and marked rises in peroxidase activity were recently reported in skin cell walls towards fruit maturity. Peroxidase isoforms in the fruit are here analysed using native gel electrophoresis. New isoforms of apparent Mr 44, 48 and 53 kDa are shown to appear in cell walls of the fruit skin at around the time of cessation of growth. It is inferred that these isozymes are present in the cell wall in vivo. Fruit from a range of non-ripening mutants were also examined. Some of these do not soften or ripen for many weeks after achieving their final size. The new isozymes were found in skin cell walls of mature fruit in each of these mutants, as in the wild-type and commercial varieties. It is concluded that the late-appearing isozymes are not associated with fruit ripening or softening, and are probably not ethylene-induced. They may act to control fruit growth by cross-linking wall polymers within the fruit skin, thus mechanically stiffening the walls and terminating growth.

Notes: Chilling injury to crop plants was first described 70 years ago and has been systematically investigated with electron microscopy since the late 1960s. Chloroplasts are the first and most severely impacted organelle. Thylakoids swell and distort, starch granules disappear, and a peripheral reticulum (vesicles arising from inner membrane of chloroplast envelope) appears. Chloroplast disintegration follows prolonged chilling. Mitochondria, nuclei and other organelles are less susceptible to chilling injury. Organellar development and ontogeny may also be disrupted. The inherent chilling sensitivity of a plant, as well as the ability of some species to acclimate to chilling, influence the timing and appearance of ultrastructural injury with the resulting outcome being mild, moderate, or severe. Other environmental factors that exacerbate injury are irradiance, chilling duration, and water status. The physiological basis for chloroplast swelling may be linked to chilling-stable starch-degrading enzymes that produce soluble sugars thus lowering stromal water potential at a time when chloroplast photosynthate export is reduced. Thylakoid dilation appears to be related to photo-oxidative conditions produced during chilling in the light. The peripheral reticulum is proposed to increase surface area of the transport-limiting membrane (chloroplast inner membrane) in response to the chilling-induced reduction in metabolite transport. Many of the ultrastructural symptoms appearing during moderate stress resemble those seen in programmed cell death. Future research directions are discussed.

Notes: Wheat seedlings exhibited a differential ability to utilize P from a range of organic P substrates when grown in agar culture under sterile conditions. Plants showed limited ability to obtain P from inositol hexaphosphate (IHP), whereas other monoester substrates such as glucose 1-phosphate (G1P), were equivalent sources of P for plant growth as compared with inorganic phosphate (Pi). Poor utilization of IHP was exemplified by significantly lower rates of dry matter accumulation and reduced P content of tissues, which were generally not significantly different to control plants that were grown in the absence of added P. The inability of wheat seedlings to obtain P from IHP was not associated with poor substrate availability but was due to either insufficient root phytase activity or inappropriate localization of phytase within root tissues. Phytase activities of 4 and 24 mU g−1 root fresh weight (FW) were determined for crude root extracts prepared from plants that were grown with either adequate P or under deficient conditions, respectively. Similar levels of phytase activity (approximately 12 mU g−1 FW) were observed in assays using intact roots, although no secreted activity was detected. By comparison, a secreted acid phosphomonoesterase activity was observed, and activities of between 466 and 1029 mU phosphomonoesterase g−1 root FW were measured for intact roots. On the basis of the differences in enzyme activity, and the observed differences in the ability of wheat seedlings to utilize G1P and IHP, it is evident that low intrinsic levels of phytase activity in wheat roots is a critical factor that limits the ability of wheat to obtain P from phytate when supplied in agar under non-limiting conditions. This hypothesis was further supported by the observation that the ability of wheat to obtain P from IHP was significantly improved when the seedlings were inoculated with a soil bacterium (Pseudomonas sp. strain CCAR59) that possesses phytase activity.

Notes: A spectrum of models that estimate assimilation rate A from intercellular carbon dioxide concentration (Ci) and measured stomatal conductance to CO2 (gc) were investigated using leaf-level gas exchange measurements. The gas exchange measurements were performed in a uniform loblolly pine stand (Pinus taeda L.) using the Free Air CO2 Enrichment (FACE) facility under ambient and elevated atmospheric CO2 for 3 years. These measurements were also used to test a newly proposed framework that combines basic properties of the A–Ci curve with a Fickian diffusion transport model to predict the relationship between Ci/Ca and gc, where Ca is atmospheric carbon dioxide concentration. The widely used Ball–Berry model and five other models as well as the biochemical model proposed by Farquhar et al. (1980) were also reformulated to express variations in Ci/Ca as a function of their corresponding driving mechanisms. To assess the predictive capabilities of these approaches, their respective parameters were estimated from independent measurements of long-term stable carbon isotope determinations (δ13C), meteorological variables, and ensemble A–Ci curves. All eight approaches reproduced the measured A reasonably well, in an ensemble sense, from measured water vapour conductance and modeled Ci/Ca. However, the scatter in the instantaneous A estimates was sufficiently large for both ambient and elevated Ca to suggest that other transient processes were not explicitly resolved by all eight parameterizations. An important finding from our analysis is that added physiological complexity in modeling Ci/Ca (when gc is known) need not always translate to increased accuracy in predicting A. Finally, the broader utility of these approaches to estimate assimilation and net ecosystem exchange is discussed in relation to elevated atmospheric CO2.

Notes: Seasonal changes in tissue water relations of Erica arborea L., Myrtus communis L. and Juniperus communis L., grown in a Mediterranean environment, were analysed under field conditions over a 12 month period by comparing plants grown in the proximity of a natural CO2 spring (about 700 μmol mol−1 atmospheric CO2 concentration, [CO2]) with plants in ambient conditions. Tissue water relations varied in response to changes in water availability, but the seasonal course of tissue water relations parameters was also related to ontogeny. Tissue water relations of these co-occurring shrubs were not alike. Osmotic potentials and saturated mass/dry mass ratio were lowest during peak drought stress periods. Diurnal changes in osmotic potential at the point of turgor loss were least early in the season, maximal in mid-season, and decreased again in autumn. Turgor potentials decreased as drought progressed and were highest in late fall and mid-winter. Symplastic water fraction was highest in mid-spring for E. arborea and M. communis and decreased during the summer, while the opposite was observed for J. communis. Common to all species, under elevated [CO2], was an increase of turgor pressure, particularly during the summer months. Other parameters showed species-specific responses to long-term elevated [CO2]. In particular, exposure to elevated [CO2] increased osmotic potentials in E. arborea under drought, while the opposite was the case for J. communis. Site differences in predawn to midday shifts were not strong in any of the species. Differences in tissue water relations suggest that the coexistence of these shrubs in the same environment with similar water availability are partially based on differential water relations strategies and water use patterns. Regardless of the mechanisms, growth of these shrubs in elevated [CO2] may be either less, similarly or more affected by drought stress than plants in ambient [CO2] depending on the species and season.

Notes: Signalling process of water stress-induced abscisic acid (ABA) accumulation was investigated in maize (Zea mays L.) leaf and root tissues. Potent free-radical scavengers and reducing agents, N-acetyl cysteine (NAC) and cystein (Cys), significantly inhibited or nearly completely blocked dehydration-induced ABA accumulation. Dithiothreitol (DTT), a reducing agent but not a free-radical scavenger, also significantly inhibited such accumulation whereas solely free-radical scavengers, dimethyl sulphoxide (DMSO) and melatonin (Mela), had no effects. Moreover, water stress-induced ABA accumulation was not affected either by free radicals, such as superoxide anion and hydrogen peroxide, or by oxidants such as KIO4. These observations suggest that the blocking of water stress-induced ABA accumulation resulted from the reducing effect, rather than from anything associated with free radicals. The disulphide bond might be crucial to the reactivity of some signal element(s) in the signalling process of water stress-induced ABA accumulation. To test the hypothesis, we used a sulfhydryl modifier, iodoacetamide (IOA), and found that it nearly totally blocked the water stress-induced ABA accumulation. Furthermore, an impermeable sulfhydryl modifier, p-chloromercuriphenylsulphonic acid (PCMBS), could also inhibit the water stress-induced ABA accumulation in the leaf tissues. These results indicate that water stress-perception protein(s) or receptor(s) may be located on the plasmalemma and a sulfhydryl group in the extracellular domain is critical to the reactivity of the speculated water stress receptors. Cys, DTT and IOA did not lead to a decrease of the baseline ABA level, i.e. in non-stressed roots. Result indicates that their blocking of water stress-induced ABA accumulation occurred upstream of the ABA biosynthesis pathway, i.e. in the signalling process that initiates such accumulation.

Notes: Miniaturized pulse-amplitude modulated photosynthesis yield analysers are primarily designed for measuring effective quantum yield (ΔF/Fm′) of photosystem II under momentary ambient light conditions in the field. Although this provides important ecophysiological information, it is often necessary to learn more about the potential intrinsic capacities of leaves by measuring light-response curves. Thus, instruments provide light-curve programmes, where light intensities are increased in short intervals and instant light-response curves are recorded within a few minutes. This method can be criticized because photosynthesis will most likely not be in steady state. This technical report shows that with the appropriate precautions instant light curves can nevertheless provide reliable information about cardinal points of photosynthesis. First, the geometry of the light source of the instrument in relation to the quantum sensor must be considered and quantum sensor readings must be corrected. Second, the measurements of the light-response curves must be compared with readings of effective quantum yield of photosystem II under ambient light conditions where photosynthesis is in steady state. This may show that in the critical range of the light curves either both measurements perfectly coincide or are offset against each other by a constant value (examples are given here). In the first case results of light curves can be taken at face values, and in the second case a simple correction can be applied. With these precautions and careful interpretations instant light-response curves can be an enormous advantage in ecophysiological field work.

Notes: Although exogenous electric fields have been reported to influence the orientation of plant root growth, reports of the ultimate direction of differential growth have been contradictory. Using a high-resolution image analysis approach, the kinetics of electrotropic curvature in Vigna mungo L. roots were investigated. It was found that curvature occurred in the same root toward both the anode and cathode. However, these two responses occurred in two different regions of the root, the central elongation zone (CEZ) and distal elongation zone (DEZ), respectively. These oppositely directed responses could be reproduced individually by a localized electric field application to the region of response. This indicates that both are true responses to the electric field, rather than one being a secondary response to an induced gravitropic stimulation. The individual responses differed in the type of differential growth giving rise to curvature. In the CEZ, curvature was driven by inhibition of elongation, whereas curvature in the DEZ was primarily due to stimulation of elongation. This stimulation of elongation is consistent with the growth response of the DEZ to other environmental stimuli.

Notes: Strain 21gr from Chlamydomonas reinhardtii is a cryptic mutant defective in the Nit5 gene related to the biosynthesis of molybdenum cofactor (MoCo). In spite of this mutation, this strain has active MoCo and can grow on nitrate media. In genetic crosses, the Nit5 mutation cosegregated with a phenotype of resistance to high concentrations of molybdate and tungstate. Molybdate/tungstate toxicity was much higher in nitrate than in ammonium media. Strain 21gr showed lower amounts of MoCo activity than the wild type both when grown in nitrate and after growth in ammonium and nitrate induction. However, nitrate reductase (NR) specific activity was similar in wild type and 21gr cells. Tungstate, either at nanomolar concentrations in nitrate media or at micromolar concentrations during growth in ammonium and nitrate induction, strongly decreased MoCo and NR amounts in wild-type cells but had a slight effect in 21gr cells. Molybdate uptake activity of ammonium-grown cells from both the wild-type and 21gr strains was small and blocked by sulphate 0·3 mM. However, cells from nitrate medium showed a molybdate uptake activity insensitive to sulphate. This uptake activity was much higher and more sensitive to inhibition by tungstate in the wild type than in strain 21gr. These results suggest that strain 21gr has a high affinity and low capacity molybdate transport system able to discriminate efficiently tungstate, and lacks a high capacity molybdate/tungstate transport system, which operates in wild-type cells upon nitrate induction. This high capacity molybdate transport system would account for both the stimulating effect of molybdate on MoCo amounts and the toxic effects of tungstate and molybdate when present at high concentrations.