ALBERT

Notes: The resurgence of tuberculosis and the emergence of multidrug-resistant mycobacteria necessitate the development of new antituberculosis drugs. The biosynthesis of mycolic acids, essential elements of the mycobacterial envelope, is a good target for chemotherapy. Species of the Mycobacterium tuberculosis complex synthesize oxygenated mycolic acids with keto and methoxy functions. In contrast, the fast-growing Mycobacterium smegmatis synthesizes oxygenated mycolic acids with an epoxy function. We describe the isolation and sequencing of a cluster of four genes from Mycobacterium bovis bacillus Calmette–Guérin (BCG), coding for methyl transferases, and which, when transferred into M. smegmatis,allow the synthesis of ketomycolic acid, in addition to an as yet undescribed mycolic acid, hydroxymycolic acid. These oxygenated mycolic acids, unlike the regular mycolic acids of M. smegmatis, and similar to the mycolic acids of M. bovis, are highly cyclopropanated. Furthermore, there is a perfect match between the structures of the keto- and the hydroxy-mycolic acids. We propose a biosynthetic model in which there is a direct relationship between these two types of mycolic acid.

Notes: The gene for CspA, the major cold-shock protein of Escherichia coli is known to be dramatically induced upon temperature downshift. Here, we report that three-base substitutions around the Shine–Dalgarno sequence in the 159-base 5′-untranslated region of the cspA mRNA stabilizes the mRNA 150-fold, resulting in constitutive expression of cspA at 37°C. This stabilization was found to be at least partially due to resistance against RNase E degradation. The cold-shock induction of cspA was also achieved by exchanging its promoter with the non-cold-shock lpp promoter. The results presented indicate that the cspA gene is efficiently transcribed even at 37°C. However, the translation of the cspA mRNA is blocked because of its extreme instability at 37°C. The presented results also demonstrate that the cspA gene is constitutively transcribed at all temperatures; however, its expression at 37°C is prevented by destabilizing its mRNA.

Notes: Ferric citrate induces transcription of the ferric citrate transport genes fecABCDE without entering the cells of Escherichia coli K-12. Point mutants of the outer membrane-receptor protein FecA are affected in induction independent of the FecA transport activity, suggesting that FecA is directly involved in induction. Alignment of FecA with the other ferric siderophore receptors of E. coli reveals an N-terminal extension in FecA that is not found in the receptors whose synthesis is not induced by their cognate ferric siderophores. In this study, we show that excision of the N-terminal region abolished the inducing activity of FecA, but retained its transport activity. Overproduction of the N-terminal FecA fragment inhibited FecA-dependent induction, but not transport. Constitutive expression caused by C-terminally truncated FecR derivatives was not inhibited by the N-terminal FecA fragment. The N-terminal region of FecA was localized in the periplasm, which indicates that FecA probably interacts with FecR, which is involved in signal transduction across the cytoplasmic membrane. Transcription initiation of the fec transport genes required the Ton system, consisting of TonB, ExbB, and ExbD, and was inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), which dissipate the electrochemical potential of the cytoplasmic membrane. fec transcription of mutant fecA4, which displays constitutive fec transcription in the absence of TonB, was not affected by CCCP. The data support a model that proposes initiation of fec transport gene transcription by binding of ferric citrate to FecA. The transcription initiation signal is transferred across the outer membrane through the activity of the Ton system at the expense of the electrochemical potential of the cytoplasmic membrane. The N-terminus of FecA interacts in the periplasm with the C-terminus of FecR, through which the signal is transferred across the cytoplasmic membrane into the cytoplasm, where it increases the activity of the sigma factor FecI, which then directs the RNA polymerase to the fec promoter upstream of fecA.

Notes: The general control transcriptional regulator gene cpcA of Aspergillus niger was cloned by complementation of a Saccharomyces cerevisiaeΔgcn4 mutant strain. The encoded protein conferred resistance to amino acid analogues when expressed in yeast. Disruption of cpcA in A. niger resulted in a strain which is sensitive towards 3-aminotriazole and fails to respond to amino acid starvation. cpcA encodes a transcript of ≈2400 nucleotides in length that includes a 5′ leader region of 900 nucleotides. The 5′ leader region contains two small open reading frames, suggesting translational control of gene expression. Steady-state mRNA levels of cpcA increase by a factor of three upon amino acid starvation. The coding region of cpcA is interrupted by a 57 bp intron and the deduced amino acid sequence displays an ≈30% overall identity to yeast GCN4p and Neurospora crassa cpc-1p. Critical amino acid residues of the transcriptional activation domains of GCN4p are conserved in cpcAp. The basic DNA-binding domain shows up to 70% amino acid sequence identity to other basic zipper (bZIP)-type transcriptional activators. cpcAp binds specifically to a GCN4p recognition element in gel retardation experiments. The C-terminal dimerization domain encodes a leucine zipper with only a single leucine residue.

Notes: Little is known about the energy sources used by rhizobia during colonization, invasion and root nodule formation on leguminous plants. We have recently reported that an impaired proline metabolism in Rhizobium meliloti leads to a reduced nodulation efficiency and competitiveness on alfalfa roots. In the present study we have characterized the R. meliloti proline dehydrogenase gene (putA) and addressed the question of its role in symbiosis. This rhizobial gene encodes a 1224-amino-acid-long polypeptide which is homologous to enteric bacteria, Rhodobacter capsulatus and Bradyrhizobium japonicum PutA proteins.Like the situation in these bacteria, sequence analysis identified the proline dehydrogenase (PDH) and pyrroline-5-carboxylate dehydrogenase (P5CDH) domains in the R. meliloti putA-encoded protein. Beta-galactosidase assays performed with free-living cells carrying a putA–lacZ transcriptional fusion revealed that R. meliloti putA gene expression is induced by proline, autoregulated by its encoded product, and independent of the general nitrogen regulatory system (Ntr). In addition, analysis of putA expression during the different steps of the symbiotic interaction with alfalfa showed that expression of this gene is turned on by the root exudates (RE), during root invasion and nodule formation, but not in differentiated nitrogen-fixing bacteroids. Furthermore, we show that the PutA− phenotype leads to a significant reduction of alfalfa root colonization by R. meliloti.

Notes: The hydrogenosomal malic enzyme (ME) was purified from the anaerobic fungus Neocallimastix frontalis. Using reverse genetics, the corresponding cDNA was isolated and characterized. The deduced amino acid sequence of the ME showed high similarity to ME from metazoa, plants and protists. Putative functional domains for malate and NAD+/NADP+ binding were identified. Phylogenetic analysis of the deduced amino acid sequence of the new ME suggests that it is homologous to reference bacterial and eukaryotic ME. Most interestingly, the cDNA codes for a protein which contains a 27-amino-acid N-terminus which is not present on the purified mature protein. This presequence shares features with known mitochondrial targeting signals, including an enrichment in Ala, Leu, Ser, and Arg, and the presence of an Arg at position –2 relative to amino acid 1 of the mature protein. This is the first report of a mitochondrial-like targeting signal on a hydrogenosomal enzyme from an anaerobic fungus and provides support for the hypothesis that hydrogenosomes in Neocallimastix frontalis might be modified mitochondria.

Notes: The Y. enterocolitica O:8 (YeO8) O-antigen repeat units consist of five sugar residues: N-acetyl-d-galactosamine (GalNAc), d-galactose (Gal), d-mannose (Man), l-fucose (Fuc), and 6-deoxy-d-gulose (6d-Gul). The nucleotide sequence of the O-antigen gene cluster of the YeO8 strain 8081-c was determined. Altogether, 18 open reading frames (ORFs) were identified and shown to be essential for O-antigen biosynthesis. We previously characterized the 3′-end of the O-antigen gene cluster and identified four genes: two for GDP-Man biosynthesis, one for UDP-Gal biosynthesis, and one for O-antigen polymerase. Based on sequence similarity, Tn5-insertion phenotypes and chemical analysis, the 14 new genes were assigned the following functions: four genes are involved in the biosynthesis of CDP-6d-Gul and two in GDP-Fuc biosynthesis. Five gene products were assigned sugar transferase functions and one gene product was similar to Wzx, the O-antigen flippase. Two genes remained unassigned. By genetic complementation we also showed that YeO8 O-antigen biosynthesis was dependent on N-acetyl-glucosaminyl:undecaprenylphosphate transferase (GlcNAc transferase), the WecA (formerly known as Rfe) protein. Data obtained from chemical-composition analysis suggest that in addition to being GlcNAc transferase, WecA may also function as a GalNAc transferase. Using a restriction-deficient derivative of Y. enterocolitica O:8 strain 8081, a rough mutant, designated 8081-R2, was isolated. 8081-R2 was complemented in trans with a cloned O-antigen gene cluster restoring surface O-antigen expression. The virulence of the wild-type strain and that of the complemented strain were significantly higher (approx. 100-fold) than that of the rough mutant in an orally infected mouse model, showing that YeO8 O-antigen is a virulence factor.

Notes: The opa multigene family of Neisseria gonorrhoeae encodes 11 related outer-membrane proteins which phase vary in vitro and in vivo. Illegitimate recombination within direct pentameric DNA repeats, encoding the signal-peptide region of pre-Opas, leads to switches in expression states. Despite the conserved nature of the variation mechanism, specific genes are expressed at high frequencies in the transition from Opa– to Opa+. The genes which are expressed at elevated frequencies differ from the rest of the family with respect to promoter structure, based on sequence comparisons between the opa genes of strain MS11mk. We have analysed transcription of the opa gene family of N. gonorrhoeae MS11mk, focussing on the different promoters found among the 11 genes to determine whether increased levels of expression are associated with increased phase-variation rates. Primer extension and Northern blotting was used to assess the levels of transcription of three representative opa genes (opaA, B and C) in ‘on’ and ‘off’ states. Full-length opa mRNA was detected primarily in strains expressing the homologous gene. Truncated opa mRNA was constitutively expressed from all opa genes regardless of their expression state. Quantitative comparisons in N. gonorrhoeae were complicated by the simultaneous expression of all 11 genes and the cross-reactivity of mRNA probes. Expression levels from the individual promoters were therefore assessed by creating transcriptional and translational lacZ fusions to each of the representative opa promoters which lacked the DNA repeats responsible for variation. The expression levels were compared to the phase-variation rates of translational opa::phoA fusions containing the same promoters in addition to the corresponding coding repeat regions. A strong correlation was found between expression levels from the different promoters and the variation rates at which ‘on’ variants appeared from an ‘off’ population (i.e. opaA 〉 opaB 〉 opaC). These results provide an explanation for the favoured expression of specific Opa proteins and indicate that expression of opa genes may be regulated at the level of transcription.

Notes: Replication of the streptococcal plasmid pLS1 is controlled by two plasmid-encoded gene products: the repressor protein CopG and the antisense RNA, RNA II. Two different mutants in rnaII have been isolated. The 5′-end and the levels of RNA II synthesized by pneumococcal cells harbouring the wild-type pLS1 or mutant plasmids (affected in either genes copG or rnaII ) were analysed. One of the rnaII mutants exhibited a high-copy-number phenotype, whereas an in vitro-constructed mutation, which affects the −10 region of the rnaII promoter, resulted in plasmids lacking copy-number phenotype. The latter mutation had a pleiotropic effect: it abolished RNA II synthesis, but it also affected the initiation of translation signals of the gene encoding the RepB initiator protein. Transcriptional and translational fusions, together with in vitro inhibition of RepB synthesis by specific oligonucleotides, showed translational inhibition of RepB synthesis by RNA II, perhaps by directly blocking the accessibility of the ribosomes to the repB initiation of translation signals.

Notes: The phsA gene encodes phenoxazinone synthase (PHS), which catalyses the penultimate step in the pathway for actinomycin biosynthesis in Streptomyces antibioticus. The phsA promoter strikingly resembles a putative StreptomycesσE cognate promoter, and purified EσE holoenzyme transcribed the phsA promoter in vitro. However, the phsA promoter was still active in an S. antibioticussigE null mutant and the level of PHS activity was unaffected. Despite this, disruption of sigE blocked actinomycin production completely. The loss of actinomycin production correlated with a 10-fold decrease in the activity of actinomycin synthetase I, the enzyme which catalyses the activation of the precursor of the actinomycin chromophore.

Notes: Various mutations were introduced in a conserved helicase domain (motif VI) of the AddA subunit of the Bacillus subtilis ATP-dependent nuclease (AddAB) by site-directed mutagenesis. These mutations affected the helicase activity and the ATP-dependent exonuclease activity on double-stranded DNA (dsDNA) as the substrate to various degrees, but had hardly any effect on the exonuclease activity on single-stranded DNA (ssDNA), suggesting that exonuclease activity on dsDNA of the enzyme requires unwinding of the DNA. This idea was supported by the finding that, initially, the rate and extent of unwinding of the DNA were higher than those of its degradation to acid-soluble products by the exonucleolytic activity. The effects of the mutations on DNA repair and recombination correlated strongly with their effects on helicase activity. Taken together, these results suggest that motif VI is essential for the helicase activity, and that this activity is required for DNA repair and recombination.

Notes: Microcin B17 (MccB17) is a ribosomally encoded DNA-gyrase inhibitor. Ribosomally encoded antibiotics are derived from precursors containing an N-terminal leader, which is removed during maturation, and a C-terminal structural peptide. PreMccB17, the translational product of mcbA, is modified into proMccB17 by the action of three enzymes, McbB, McbC, and McbD. A chromosomally encoded peptidase then converts proMccB17 into MccB17. The role of McbB, McbC, and McbD is to convert glycine, cysteine, and serine residues present in preMccB17 into four thiazole and four oxazole rings. Using a modification-specific antibody rather than antimicrobial activity, we show that the 26-amino-acid N-terminal leader of preMccB17 is essential for the conversion of preMccB17 into proMccB17. Neither a preMccB17 peptide lacking the leader nor a preMccB17–β-galactosidase fusion lacking the leader are post-translationally modified.

Notes: The DNA–membrane complex has been the subject of intensive investigation for over 35 years as the possible site for DNA replication in the prokaryotic cell and the site through which newly synthesized chromosomes are segregated into daughter cells. However, the molecular mechanisms which control these phenomena are, for the most part, poorly understood despite genetic, biochemical, and morphologic evidence in favour of their existence. This is probably due to the transient nature and non-covalent interactions that occur between DNA and the membrane. In addition, there is a paucity of knowledge concerning the nature of the membrane receptors for DNA and whether the membrane plays simply a structural or metabolic role in the two processes. Plasmids can provide important insights into the role of the membrane in replication and partitioning because the plasmid life cycle is relatively simple, with replication occurring during the cell cycle and partitioning during cell division. The replicon model of Jacob et al. (1963, Cold Spring Harbor Symp Quant Biol 28: 329–348) still represents a good conceptual framework (with modifications) to explain how plasmid replication and partitioning are linked by the membrane. In its simplest form, the model focuses on specific membrane binding sites (possibly along the equator of the cell) for plasmid (or bacterial) replication, with the membrane acting as a motive force to separate the newly synthesized replicons and their attached sites into daughter cells. Indeed, proteins involved in both plasmid replication and partitioning have been found in membrane fractions and some plasmids require membrane binding for initiation and an active partitioning. We propose that several factors are critical for both plasmid DNA replication and partitioning. One factor is the extent of negative supercoiling (brought about by an interplay of various topoisomerases, but most importantly by DNA gyrase). Supercoiling is known to be critical for initiation of DNA replication but may also be important for the formation of a partition complex in contact with the cell membrane. Another factor is the presence of specific subdomains of the membrane which can interact specifically with origin DNA and possibly other regions involved in partitioning. Such domains may be induced transiently or be present at all times during the cell cycle.

Notes: KinB is one of the two major histidine kinases that provide phosphate input in the phosphorelay to produce Spo0A∼P, the key transcription factor controlling the initiation of sporulation. A search for insertion mutants affected in activation of KinB-dependent sporulation led to the identification of the lgt locus encoding the lipoprotein glyceryltransferase required for the lipid modification of prolipoproteins before their cleavage and translocation across the cytoplasmic membrane. In parallel, a putative lipoprotein signal peptide cleavage site was detected in KapB, known to be strictly required for KinB-mediated sporulation and located downstream of KinB in a single transcription unit. Using PhoA peptide fusions, we have shown that KapB signal-peptide can direct active alkaline phosphatase to the outer surface of the cytoplasmic membrane in an LGT-dependent manner, strongly suggesting that KapB is a lipoprotein tethered to the outer face of the cytoplasmic membrane via a lipid anchor. As KapB proved to be dispensable for expression of the kinBkapB operon, a chimeric kinase was built consisting of KinA sensor domain fused to KinB kinase domain (KinA′-′B) to assess (i) the involvement of KapB in catalysis of the kinase reaction, and (ii) the ability of KinB to phosphorylate Spo0F in vitro. It was shown that KapB is dispensable for both in vivo and in vitro activation of the phosphorelay by the KinA′-′B chimera and that KinA′-′B phosphorylates Spo0F directly in vitro. Models for the role of KapB in regulating KinB activity are discussed.

Notes: In Erwinia amylovora, the dsp region, required for pathogenicity on the host plant but not for hypersensitive elicitation on tobacco, is separated from the hrp region by 4 kb. The genetic analysis reported in this paper showed that this 4 kb region is not required for pathogenicity on pear seedlings. The environmental conditions allowing expression of a dsp::lacZ fusion were examined: expression was barely detected in rich medium at 30°C, and the highest expression was observed in M9 galactose minimal medium at 25°C. A dsp::uidA fusion appeared to be expressed only in a HrpL-proficient strain, indicating that the dsp region, like the hrp region, is positively controlled via the alternative σ factor HrpL. Sequence analysis revealed that the dsp cluster encodes two genes, dspA (5517 bp) and dspB (420 bp), and that the insertions leading to the dsp::lacZ and the dsp::uidA fusions were within dspA. A HrpL-dependent promoter sequence (GGAACC- N15-CAACA) was identified upstream of dspA, and primer extension analysis detected four transcriptional starts 7, 8, 9 and 10 bp downstream of this sequence. A σ70 promoter sequence (TTGCCC-N16-GATAAT) was observed upstream of dspB. The functionality of this second promoter was confirmed by complementation analysis. This promoter allowed constitutive expression of dspB, as measured by the expression of a dspB::uidA fusion in rich medium. In M9 galactose medium, however, HrpL was shown to activate dspB, as expression of the dspB::uidA fusion was twofold higher in a HrpL+ background than in a HrpL− background. Transposon insertions in either dspA or dspB led to a non-pathogenic phenotype. Thus, both DspA and DspB were required for E. amylovora pathogenicity, as dspB could be expressed independently of dspA. DspA and DspB were visualized as polypeptides with apparent sizes of 190 kDa and 15.5 kDa, respectively, when encoded in the T7 polymerase/promoter system. DspA, which showed homology with the protein predicted from the partial sequence of Pseudomonas syringae pv. tomato avrE transcriptional unit III, was shown to be secreted into the external medium via the Hrp secretion pathway. DspB was predicted to be acidic, like the Syc chaperone of Yersinia. A chaperone role for DspB was suggested further by the fact that DspA secretion required a functional DspB protein.

Notes: Electron microscopic studies have demonstrated that various gliding filamentous cyanobacteria have trichome surfaces with a common structural organization. They contain an S-layer attached to the outer membrane and an array of parallel fibrils on top of the S-layer. In all species studied, the helical arrangement of these fibrils corresponds to the sense of rotation of the organism during the gliding movement. We have investigated the surface fibrils of Phormidium uncinatum using electron microscopic, spectroscopic and biochemical techniques. The fibrils consist of a single rod-shaped protein, which we refer to as oscillin. Oscillin is a 646 amino acid residue protein (Mr 65 807; pI 3.63) and appears to be glycosylated. Sequence analysis reveals a two-domain structure: a 554 residue domain contains 46 repeats of a Ca2+-binding motif; it is followed by a 92 residue C-terminal domain, which might mediate its export. Filaments that do not express oscillin lose their ability to move. Homology studies suggest that similar proteins play comparable roles in other motile cyanobacteria. The structure of oscillin appears to favour a passive role in gliding.

Notes: The chromosomes of Streptomyces species are linear molecules, containing long terminal inverted repeats and covalently bound terminal proteins. These chromosomes undergo spontaneous deletions of the terminal sequences at high frequencies and become circularized in several cases examined. Artificial circularization of the Streptomyces lividans chromosome was also achieved by targeted recombination in vivo, in which the terminal inverted repeats of the chromosome were connected by a kanamycin resistance gene (aphII ). Under kanamycin selection, the circularized chromosomes harboured tandem amplifications of a 20.2 kb sequence that included the aphII gene flanked by direct repeats and deletions nearby. On release from kanamycin selection, the aphII amplifications and the neighbouring sequences were deleted from the chromosomes, rendering all the cultures kanamycin sensitive. The chloramphenicol resistance gene, which was prone to deletion in wild-type S. lividans, became much more stable in the kanamycin-sensitive derivatives. These results indicate that the telomeres and/or certain terminal sequences may be involved in the structural instability of Streptomyces chromosomes.

Notes: Nitrogen metabolism is a highly regulated process in Neurospora crassa. The structural genes that encode nitrogen catabolic enzymes are subject to nitrogen metabolite repression, mediated by the positive-acting NIT2 protein and by the negative-acting NMR protein. NIT2, a globally acting factor, is a member of the GATA family of regulatory proteins and has a single Cys2/Cys2 zinc finger DNA-binding domain. The negative-acting NMR protein interacts via specific protein–protein binding with two distinct regions of the NIT2 protein, a short alpha-helical motif within the NIT2 DNA-binding domain and a second motif at its carboxy terminus. Deletions of segments of NIT2 throughout most of its length result in truncated proteins, which are still functional for activating gene expression; most of these mutant NIT2 proteins still allow proper nitrogen repression of nitrate reductase synthesis. In contrast, deletions or certain amino acid substitutions within the zinc finger and the carboxy-terminal tail result in a loss of nitrogen metabolite repression. Those mutated forms of NIT2 that are insensitive to nitrogen repression have also lost one of the NIT2–NMR protein–protein interactions. These results provide compelling evidence that the specific NIT2–NMR interactions have a regulatory function and play a central role in establishing nitrogen metabolite repression.

Notes: We have examined the effect of a downstream secondary structure (the stem–loop sequence found downstream on the MMTV gag-pro frameshift site) on frameshifting at a bacterial shifty site (U UUC AUA) that responds strongly to a isoleucine-tRNA limitation. Our findings are as follows: (i) the downstream stem–loop has little effect on frameshifting in growing, unstarved cells; (ii) the stem–loop increases the frameshifting effect of isoleucine-tRNA limitation about fourfold, and this synergism is maximal with a distance of 5–9 nucleotides between the ‘hungry’ AUA codon and the stem–loop; and (iii) a stem–loop of different sequence at the same position has the same effect.

Notes: The integration of gene cassettes into integrons is effected by site-specific recombination catalysed by an integrase, IntI, encoded by the integron. The cassette-associated recombination sites, 59-base elements, are not highly conserved and vary in length from 57 to 141 bp. They can be identified by their location and the relationship of over 20 bp at their outer ends to consensus sequences that are imperfect inverted repeats of one another. The recombination cross-over occurs close to one end of the 59-base element, within a conserved core site with the consensus sequence GTTAGGC or GTTRRRY. By introducing single-base changes at each of these positions in the aadB 59-base element, bases that are critical for site activity were identified. The recombination cross-over was also localized to a unique position between the adjacent G and T residues. Changes introduced in the conserved AAC of the inverse core site (GCCTAAC or RYYYAAC) located at the opposite end of the 59-base element also reduced site activity but to a lesser extent. Sequences of rare recombinants revealed an alternative position for strand exchange and led to the conclusion that 59-base elements comprise two simple sites, analogous to those recognized by other integrases, with each simple site made up of a pair of inversely oriented IntI binding domains separated by a spacer of 7 or 8 bp. Re-examination of the sequences of all known 59-base elements revealed that this simple site configuration was present at both the left and right ends in all 59-base elements. The identity of bases in the spacer is not required for efficient recombination and the cross-over is located at one end of the spacer, suggesting that during IntI1-mediated recombination only one strand exchange occurs.

Notes: Mycobacteriophage L5 is a well-characterized temperate phage that forms stable lysogens in Mycobacterium smegmatis. The host range of L5 is, however, unclear because previous reports suggested that it does not infect slow-growing mycobacteria such as Mycobacterium tuberculosis and bacille Calmette-Guérin (BCG). Moreover, luciferase reporter phage derivatives of L5 failed to produce light from BCG, suggesting that infection is blocked at or before the stage of DNA injection. In this study, we demonstrate that L5 infection of slow growing mycobacteria specifically requires a high concentration of Ca2+, conditions that differs from those required for infection of M. smegmatis by L5 and for infection of BCG by the closely related phage D29. In addition, we show that there are specific genetic determinants of L5 that confer the ability to infect slow growing mycobacteria, without altering infection of M. smegmatis. These observations extend the use of phage L5 for the diagnosis and analysis of tuberculosis and other mycobacterial diseases.

Notes: The Escherichia coli chromosome contains about 300 bacterial interspersed mosaic elements (BIMEs). These elements, located at the 3′ end of genes, are composed of three types of alternating repetitive extragenic palindromes (REPs). Based on the type of REP they contain and on their ability to interact with the integration host factor (IHF), BIMEs are subdivided into two families: BIME-1 elements contain an IHF binding site flanked by converging Y and Z1 REPs, whereas BIME-2 elements contain a variable number of alternating Y and Z2 REPs without an IHF site. Although some BIMEs have been implicated in the protection of mRNA against 3′ exonucleolytic degradation, the main role of elements belonging to both families remains to be elucidated. In this paper, we used oxolinic acid, a drug that reveals potential sites of DNA gyrase action, to demonstrate that DNA gyrase interacts in vivo with BIME-2 elements. The frequency of cleavage varied from one element to another, and the cleavage pattern observed in elements containing several REPs indicated that DNA gyrase cut DNA every two REPs. A single cleavage site has been identified in the Y REP in six out of seven instances, and the nucleotide sequence of a 44 bp fragment containing the scission point displayed conserved residues at six positions. The lack of one of the conserved residues accounted for the absence of cleavage in most of the Z2 REPs. Our results also showed that cleaved REPs were always associated with another REP, suggesting that a pair of diverging REPs constitutes the target of DNA gyrase. DNA gyrase cleavage at repetitive BIME-2 elements may have consequences for DNA topology and genomic rearrangements.

Notes: Several bloom-forming cyanobacterial genera produce potent inhibitors of eukaryotic protein phosphatases called microcystins. Microcystins are hepatotoxic cyclic heptapeptides and are presumed to be synthesized non-ribosomally by peptide synthetases. We identified putative peptide synthetase genes in the microcystin-producing strain Microcystis aeruginosa PCC 7806. Non-hepatotoxic strains of M. aeruginosa lack these genes. Strain PCC 7806 was transformed to chloramphenicol resistance. The antibiotic resistance cassette insertionally inactivated a peptide synthetase gene of strain PCC 7806 as revealed by Southern hybridization and DNA amplification. This is the first report of genetic transformation and mutation, by homologous recombination, of a bloom-forming cyanobacterium. Chemical and enzymatic analyses, including high-performance liquid chromatography (HPLC), mass spectrometry, amino acid activation, and protein phosphatase inhibition, revealed the inability of derived mutant cells to produce any variant of microcystin while maintaining their ability to synthesize other small peptides. The disrupted gene therefore encodes a peptide synthetase (microcystin synthetase) that is specifically involved in the biosynthesis of microcystins. Our results confirm that microcystins are synthesized non-ribosomally and that a basic difference between toxic and non-toxic strains of M. aeruginosa is the presence of one or more genes coding for microcystin synthetases.

Notes: The mechanism of transcriptional repression of the aerobactin operon of Escherichia coli by the Fe2+-responsive Fur (ferric uptake regulation) protein has been investigated. In the presence of a divalent metal, such as Mn2+, the Fur protein sequentially occupies two defined sites at the aerobactin promoter region, followed by a looser occupation of upstream DNA sequences. However, binding to the primary target site suffices for the entire repression effect. Comparison of transcription patterns generated with run-off experiments in the presence and absence of heparin showed that access of the RNA polymerase to the principal −35/−10 hexamers of the promoter region was fully prevented by Fur-Mn2+ bound to its primary site. Similarly, promoter-bound RNA polymerase could not be competed out from the DNA even in the presence of a large Fur-Mn2+ excess, although the repressor could immediately bind its target sequence at the region as soon as RNA polymerase moved away from the promoter during transcription. The high affinities of either protein for the promoter produce, in practice, a first-come, first-served effect that helps the system to respond instantly to changes in the iron status of the cells.

Notes: Many fungi undergo a morphological transition to filamentous growth in response to limiting nutrient conditions. Constitutively elongated Saccharomyces cerevisiae mutants (elm) have been isolated; the ELM1 gene encodes a putative serine/threonine protein kinase. A novel allele, elm1-15, has been isolated in an S288C-derived strain, which causes a pleiotropic phenotype, including media-specific growth effects, abnormal morphology and altered stress response, in cells that are auxotrophic for tryptophan. elm1-15 trp1 cells cannot use many nitrogen sources, are sensitive to amino acid analogues, have very low general amino acid permease activity and do not accumulate trehalose. In contrast, haploid elm1-15 TRP1 cells grow well in budding form on all media, are stress resistant and overaccumulate trehalose. Several lines of evidence suggest that Elm1 acts on functions related to the RAS/cAMP pathway. Overexpression of Elm1 partially rescues the ts phenotype of cdc25 and cyr1 mutants. Deletion of ELM1 in low PKA activity mutants increased the severity of their phenotypes, and activation of Ras2 decreases the cell elongation phenotype of elm1 mutants. A ‘signal integration’ model for the complex relationship of Elm1 and the RAS/cAMP pathway in controlling morphogenesis in response to nutrients is proposed.

Notes: Pseudomonas aeruginosa can cause severe life-threatening infections in which the bacterium disseminates rapidly from epithelial colonization sites to the bloodstream. In experimental models, the ability of P. aeruginosa to disseminate is linked to epithelial injury, in vitro cytotoxicity and expression of the exoenzyme S regulon. Using the expression of ExoS as a model, a series of genes that are important for regulation, secretion and, perhaps, intoxication of eukaryotic cells have been identified. Proteins encoded by the exoenzyme S regulon and the Yersinia Yop virulon show a high level of amino acid homology, suggesting that P. aeruginosa may use a contact-mediated translocation mechanism to transfer anti-host factors directly into eukaryotic cells. Potential anti-host factors that may disrupt eukaryotic signal transduction through ADP-ribosylation include ExoS and ExoT. Expression of ExoU, another candidate anti-host factor, has been correlated with acute cytotoxicity and lung epithelial injury. Members of the exoenzyme S regulon represent only a portion of the virulence factor arsenal possessed by P. aeruginosa. It will be important to understand how the exoenzyme S regulon contributes to pathogenesis and whether these factors could serve as potential therapeutic targets.

Notes: We analysed the effects of the overproduction of parts or all of a multisubunit ATP-binding cassette (ABC) transporter, the MalFGK2 complex, involved in the uptake of maltose and maltodextrins in Escherichia coli. We found that production of the MalF protein alone was inducing the phtrA promoter, which is under the control of a recently discovered sigma factor, σ24, involved in the response to extracytoplasmic stresses. The production level, stability and localization of MalF were not altered when produced without its partners, suggesting that the protein was correctly inserted in the membrane. Our results indicate that a large periplasmic loop located between the third and fourth transmembrane segment of MalF, the L3 loop, is responsible for phtrA induction: (i) deleted MalF proteins with no L3 loop or with a L3 loop lacking 120 amino acids do not induce the phtrA promoter; (ii) the export to the periplasm of the L3 loop alone or fused to MalE induces the phtrA promoter. Moreover, the proteolytic sensitivity of MalF is different when it is produced alone and when MalF and MalG are produced together, suggesting a change in the conformation and/or accessibility of MalF. These results suggest that some inner membrane proteins can be sensed outside the cytoplasm by a quality control apparatus or by the export machinery. Moreover, the observation of the phtrA induction by MalF could be a useful new tool for studying the insertion and assembly of the MalFGK2 complex.

Notes: To investigate the temporal and spatial expression patterns of the gene (lat ) encoding lysine ɛ-aminotransferase (LAT) for cephamycin C biosynthesis, a mutant form of green fluorescent protein (mut1GFP) was integrated into the Streptomyces clavuligerus chromosome (strain LH369), resulting in a translational fusion with lat. LAT activity and fluorescence profiles of the recombinant protein paralleled the native LAT enzyme activity profile in wild-type S. clavuligerus, which peaked during exponential growth phase and decreased slowly towards stationary phase. These results indicate that the LAT–Mut1GFP fusion protein retains both LAT and GFP functionality in S. clavuligerus LH369. LH369 produced wild-type levels of cephamycin C in minimal medium culture conditions supplemented with lysine. Time-lapsed confocal microscopy of the S. clavuligerus LH369 strain revealed the temporal and spatial characteristics of lat gene expression and demonstrated that physiological development of S. clavuligerus colonies leading to cephamycin C biosynthesis is limited to the substrate mycelia.

Notes: The 110 kDa haemolysin protoxin (proHlyA) is activated in the Escherichia coli cytosol by acyl carrier protein-dependent fatty acylation of two internal lysine residues, directed by the co-synthesized protein HlyC. Using an in vitro maturation reaction containing purified protoxin peptides and acylACP, we show unambiguously that HlyC possesses an apparently unique acyltransferase activity fully described by Michaelis–Menten analysis. The Vmax of HlyC at saturating levels of both substrates was ≈ 115 nmol acyl group min−1 mg−1 with KmacylACP of 260 nM and KmproHlyA of 27 nM, kinetic parameters sufficient to explain why in vivo HlyC is required at a concentration equimolar to proHlyA. HlyC bound the fatty acyl group from acylACP to generate an acylated HlyC intermediate that was depleted in the presence of proHlyA, but enriched in the presence of proHlyA derivatives lacking acylation target sites. HlyC was also able to bind in vivo 4′-phosphopantetheine. Substitution of conserved amino acids that could act as putative covalent attachment sites did not prevent binding of the fatty acyl or 4′-phosphopantetheine groups. These data and substrate variation analyses suggest that the unique acylation reaction does not involve covalent attachment of fatty acid to the acyltransferase, but rather that it proceeds via a sequential ordered Bi–Bi reaction mechanism, requiring the formation of a non-covalent ternary acylACP–HlyC–proHlyA complex.

Notes: Succinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli is negatively regulated by the arcA and fnr gene products during anaerobic cell growth conditions. The controlled synthesis of this sole membrane-bound enzyme of the tricarboxylic acid cycle allows optimal participation in the aerobic electron transport pathway for the generation of energy via oxidative phosphorylation reactions. To understand how ArcA participates in the anaerobic repression of sdhCDAB expression, a family of sdhC–lacZ fusions was constructed and analysed in vivo. DNase I footprint and gel shift assays using purified ArcA protein revealed the location of four distinct and independent ArcA binding sites in the sdhC promoter region. ArcA sites, designated sites 1 and 2, are centred at −205 bp and −119 bp upstream of the sdhC promoter, respectively, whereas ArcA site 3 overlaps the −35 and −10 regions of the sdhC promoter. A fourth ArcA site is centred at + 257 bp downstream of the sdhC promoter. They are bound with differing affinity by ArcA and ArcA phosphate. The in vivo studies, in combination with the in vitro studies, indicate that ArcA site 3 is necessary and sufficient for the ArcA-dependent repression of sdhC gene expression, while the DNA region containing ArcA site 2 contributes to maximal gene expression. The DNA-containing ArcA sites 1 and 4 provide minor roles in the ArcA regulation of sdhC expression. Lastly, the Fnr-dependent control of sdhCDAB gene expression was shown to occur independently of the ArcA and to require DNA sequences near the start of sdhC transcription.

Notes: Plant calcium can modulate a particular plant–pathogen interaction and have a decisive role in disease development. Enhanced resistance to the phytopathogenic enterobacterium Erwinia carotovora, the causal agent of bacterial soft rot disease, is observed in high-calcium plants. One of the main virulence determinants of E. carotovora, the PehA endopolygalacturonase, is specifically required in the early stages of the infection. Production of PehA was found to be dependent on the calcium concentration in the bacterial environment. An increase in extracellular calcium to mM concentrations repressed pehA gene expression without reducing or even enhancing expression of other extracellular enzyme-encoding genes of this pathogen. An increase in plant calcium levels could be correlated to enhanced resistance to E. carotovora infection and to an inhibition of in planta production of PehA. Ectopic expression of pehA from a calcium-insensitive promoter allowed E. carotovora to overcome this calcium-induced resistance. The results imply that plant calcium can constitute an important signal molecule in plant–pathogen interaction, which acts by modulating the expression of virulence genes of the pathogen.

Notes: In the filamentous bacterium Streptomyces coelicolor, the cell division protein FtsZ is required for the conversion of multinucleoidal aerial hyphae into chains of uninucleoidal spores, although it is not essential for viability. Using immunofluorescence microscopy, we have shown that FtsZ assembles into long, regularly spaced, ladder-like arrays in developing aerial hyphae, with an average spacing of about 1.3 μm. Within individual hyphae, ladder formation was relatively synchronous and extended for distances over 100 μm. These ladders were present only transiently, decreasing in intensity as chromosomes separated into distinct nucleoids and disappearing upon the completion of septum formation. Evidence from the overall intensity of immunofluorescence staining suggested that ladder formation was regulated in part at the level of the accumulation and degradation of FtsZ within individual aerial hyphae. Finally, FtsZ ladder formation was under developmental control in that long arrays of FtsZ rings could not be detected in certain so-called white mutants (whiG, whiH and whiB), which are blocked in spore formation. The assembly of FtsZ into ladders represents the earliest known molecular manifestation of the process of spore formation, and its discovery provides insight into the role of whi genes in the conversion of aerial hyphae into chains of spores. We have also described a novel use of a cell wall-staining technique to visualize apical tip growth in vegetatively growing hyphae.

Notes: A transposon (Tn10dCam) insertion mutant of Escherichia coli K-12 was isolated that exhibited hypersensitivity to zinc(II) and cadmium(II) and, to a lesser extent, cobalt(II) and nickel (II). The mutated gene, located between 75.5 and 76.2 min on the chromosome, is named zntA (for Zn(II) transport or tolerance). The metal-sensitive phenotype was complemented by a genomic DNA clone mapping at 3677.90–3684.60 kb on the physical map. Insertion of a kanamycin resistance (KnR) cassette at a Sal I site in a subcloned fragment generated a plasmid that partially complemented the zinc(II)-sensitive phenotype. DNA sequence analysis revealed that the KnR cassette was located within the putative promoter region of an ORF (o732 or yhhO) predicted to encode a protein of 732 amino acids, similar to cation transport P-type ATPases in the Cpx-type family. Inverse PCR and sequence analysis revealed that the Tn10dCam element was located within o732 in the genome of the zinc(II)-sensitive mutant. The zntA mutant had elevated amounts of intracellular and cell surface-bound Zn(II), consistent with the view that zntA+ encodes a zinc(II) efflux protein. Exposure of the zntA mutant to cobalt(II) and cadmium(II) also resulted in elevated levels of intracellular and cell surface-bound metal ions.

Notes: Enteritis induced by non-typhoid pathogenic Salmonella is characterized by fluid secretion and inflammatory responses in the infected ileum. The inflammatory response provoked by Salmonella initially consists largely of a neutrophil (PMN) migration into the intestinal mucosa and the gut lumen. The interactions between Salmonella and intestinal epithelial cells are known to play an essential role in inducing the inflammatory response. Upon interaction with epithelial cells salmonellae are able to elicit transepithelial signalling to neutrophils. This signalling is recognized as a key virulence feature underlying Salmonella-induced enteritis. However, the nature and mechanism of such signalling has not been clarified to date. Here, we characterize SopB, a novel secreted effector protein of Salmonella dublin, and present data implying that SopB is translocated into eukaryotic cells via a sip-dependent pathway to promote fluid secretion and inflammatory responses in the infected ileum.

Notes: We have developed methods for labelling regions of the Bacillus subtilis chromosome with the nucleotide analogue 5-bromodeoxyuridine (BrdU) and for subcellular visualization of the labelled DNA. Examination of oriC-labelled chromosomes in outgrowing spores has provided direct evidence for active segregation of sister chromosomes. Co-immunodetection of Spo0J and BrdU-labelled DNA has directly confirmed the expected close association between this chromosome partitioning protein and the oriC region of the chromosome. The results provide further support for the notion that bacterial cells use an active mitotic-like mechanism to segregate their chromosomes.

Notes: Previous mutational analysis of the L1 region of the RecA protein suggested that Gly-157 and Glu-158 are ‘hot-spots’ for the occurrence of constitutive LexA co-protease mutants (coprtc). In the present study, we clearly establish that position 157 is a hot-spot for the occurrence of such mutants, as 12 of 14 and 10 of 14 substitutions result in this phenotype for UmuD and LexA cleavage respectively. The frequency of such mutations at position 158 is somewhat lower, 8 of 13 and 5 of 13 for UmuD and LexA respectively. Comparison of the UmuD vs. LexA co-protease activity for all single mutants with substitutions at positions 154, 155, 156, 157 and 158 (47 in total) reveals that, although there is good agreement among most mutants regarding their ability to cleave both LexA and UmuD, there are two in particular (Glu-154→Asp and Glu-154→Gln) that show a clear preference for cleavage of UmuD. We also show that three second-site mutations that completely suppress coprtc activity toward LexA have little or no effect on the coprtc activity of the primary mutant toward UmuD. In addition, we observe a high frequency of second-site suppressor mutations, suggesting a functional interaction among side-chains in this region. Together, these results support the idea that the L1 region of RecA makes up part of the co-protease substrate-binding site.

Notes: Fap1, a fimbriae-associated protein, is involved in fimbriae assembly and adhesion of Streptococcus parasanguis FW213 (Wu et al., 1998). In this study, the sequence of the fap1 gene was resolved using a primer island transposition system. Sequence analysis indicated that fap1 was composed of 7659 nucleotides. The predicted Fap1 protein contains an unusually long signal sequence (50 amino acid residues), a cell wall sorting signal and two repeat regions. Repeat regions I and II have a similar dipeptide composition (E/V/I)S, composed of 28 and 1000 repeats respectively. The two regions combined accounted for 80% of the Fap1 coding region. The experimental amino acid composition and isoelectric point (pI) of Fap1 were similar to that predicted from the deduced Fap1 protein. Results of Northern analyses revealed that the fap1 open reading frame (ORF) was transcribed as a 7.8 kb monocistronic message. Insertional inactivation at the 3′ end, downstream of the fap1 ORF, did not affect Fap1, fimbrial expression or bacterial adhesion. Insertional inactivation of fap1 immediately upstream of the repeat region II abolished expression of Fap1 and fimbriae, and was concurrent with a diminution in adhesion of FW213. Inactivation of the cell wall sorting signal of fap1 also eliminated long fimbrial formation and reduced the ability of FW213 to bind to SHA. Fap1 was no longer anchored on the cell surface. Large quantities of truncated Fap1 were found in the growth medium instead. These results suggest that the fap1 ORF alone is sufficient to support Fap1 expression and adhesion, and demonstrate that anchorage of Fap1 on the cell surface is required for long fimbriae formation. These data further document the role of long fimbriae in adhesion of S. parasanguis FW213 to SHA.

Notes: To help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in the half-life of total pulse-labelled RNA along with decreased half-lives of the rpsO, trxA, lpp and ompA transcripts. In contrast, the transcripts for rne (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes involved in mRNA decay, were stabilized. rnb (RNase II) and rnc (RNase III) transcript levels were unaffected in the presence of increased polyadenylation. Long-term overproduction of PAP I led to slower growth and irreversible cell death. Differential display analysis showed that new RNA species were being polyadenylated after PAP I induction, including the mature 3′-terminus of 23S rRNA, a site that was not tailed in wild-type cells. Quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) demonstrated an almost 20-fold variation in the level of polyadenylation among three different transcripts and that PAP I accounted for between 94% and 98.6% of their poly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polymerized into poly(A) tails in a transcript-dependent manner.

Notes: Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm-grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI-1 (N-(3-oxododecanoyl)-l-homoserine lactone) and PAI-2 (N-butyryl-l-homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (SODs), Mn-SOD and Fe-SOD, and the major catalase, KatA. The expression of sodA (encoding Mn-SOD) was particularly dependent on PAI-1, whereas the influence of autoinducers on Fe-SOD and KatA levels was also apparent but not to the degree observed with Mn-SOD. β-Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI-1/2-deficient double mutant partially restored KatA activity, while the addition of PAI-1 only was sufficient for full restoration of Mn-SOD activity. In biofilm studies, catalase activity in wild-type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild-type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.

Notes: As extracts of poly(A) polymerase I (PAP I) deficient strains of Escherichia coli appeared to contain considerable residual polyadenylating activity, efforts were undertaken to identify a second poly(A) polymerase. Recently, a gene (f310 ) encoding the putative second poly(A) polymerase was cloned and sequenced. Here we have tested the ability of the F310 protein to add poly(A) tails in vivo by measuring total poly(A) levels in both f310 mutants and strains that overproduce F310. In addition, we have visualized poly(A) tails and examined ColE1 plasmid copy number in various genetic backgrounds. We also carried out direct biochemical measurements of AMP incorporation, using cell extracts after amplification of F310. All the data obtained indicate that F310 is not a poly(A) polymerase. Although the presence of two potential ATP binding domains in the F310 protein may account for its apparent ATP binding activity, its true biochemical function remains to be identified. In addition, we show that the f310 gene is transcribed, almost exclusively, during stationary phase from a σs promoter.

Notes: The relapsing fever agent Borrelia turicatae has two antigenically distinct serotypes, A and B, which differ in their variable small proteins (Vsps) and in their degree of virulence and neurotropism in mice. Each Vsp gene (vspA or vspB) had an expression-linked copy that was unique to the serotype expressing it. This was located on one linear plasmid, which was defined by the upstream sequence. The archived copies of vspA and vspB were each located on different linear plasmids that were the same in both serotypes. In this feature, the mechanism of antigenic variation is similar to that of another relapsing fever agent, B. hermsii. However, in other features, the mechanisms of the two organisms differ. The expressed and archived loci for vspA and vspB of B. turicatae were near the centre of linear plasmids instead of near the telomeres. The vspA and vspB expression loci were duplicate copies of their respective silent loci: from the vsp itself to at least 13–14 kb downstream. Despite the extensive interplasmidic duplications and the internal position of the expression locus, the only detectable difference between serotypes A and B was in whether they expressed VspA or VspB.

Notes: SecA, the dimeric ATPase subunit of bacterial protein translocase, catalyses translocation during ATP-driven membrane cycling at SecYEG. We now show that the SecA protomer comprises two structural modules: the ATPase N-domain, containing the nucleotide binding sites NBD1 and NBD2, and the regulatory C-domain. The C-domain binds to the N-domain in each protomer and to the C-domain of another protomer to form SecA dimers. NBD1 is sufficient for single rounds of SecA ATP hydrolysis. Multiple ATP turnovers at NBD1 require both the NBD2 site acting in cis and a conserved C-domain sequence operating in trans. This intramolecular regulator of ATP hydrolysis (IRA) mediates N-/C-domain binding and acts as a molecular switch: it suppresses ATP hydrolysis in cytoplasmic SecA while it releases hydrolysis in SecY-bound SecA during translocation. We propose that the IRA switch couples ATP binding and hydrolysis to SecA membrane insertion/deinsertion and substrate translocation by controlling nucleotide-regulated relative motions between the N-domain and the C-domain. The IRA switch is a novel essential component of the protein translocation catalytic pathway.

Notes: PspF bound to the psp enhancer activates Eσ54 holoenzyme-dependent transcription of the Escherichia coli phage-shock protein (psp ) operon and autogenously represses its own σ70-dependent transcription, thereby keeping its concentration at a low level. It has been demonstrated previously that integration host factor (IHF) bound to a DNA site located between the psp core promoter and the PspF binding sites stimulates psp expression. We show here that wild-type IHF strongly retards DNA containing the psp promoter region. In vitro, PspF binding to the psp enhancer facilitates IHF binding, while IHF binding to the pspF–pspA-E promoter-regulatory region increases the efficacy of PspF binding to the upstream activating sequences (UASs). This is the first demonstration of co-operative binding of an activator and IHF in a σ54-dependent system. In the absence of IHF, in vivo autoregulation of pspF transcription is lifted and, consequently, PspF production is increased, indicating that IHF enhances PspF binding to the psp enhancer in vivo.

Notes: In this report we show that the ENA1/PMR2A gene is under glucose repression. The SNF1 protein kinase, acting independently from the HOG and calcineurin pathways, is essential to release ENA1 from glucose repression. The transcriptional repressor Ssn6p negatively regulates ENA1 expression and, like other glucose repressible genes, this repression is mediated in part by Mig1p. Deletion of a fragment from the ENA1 promoter that includes two Mig1p consensus binding sites gives a high level of expression in glucose without added salt. We suggest that regulation of ENA1 by the SNF1 pathway could be part of a general mechanism through which yeast cells respond to carbon source starvation by activating protective systems against different types of stress.

Notes: Yersinia enterocolitica is a gastrointestinal pathogen of humans and animals. Ail is a 17 kDa cell-surface protein that confers on Y. enterocolitica resistance to serum killing and the ability to attach to and invade cells in vitro. The ail gene of Y. enterocolitica is regulated by temperature and growth phase. In stationary phase, ail transcript is only detected when bacteria are grown at the host temperature of 37°C. Our laboratory previously described a group of mini-Tn10 mutants, which expressed ail in stationary phase at 28°C. In one of these mutants, DP5102::mini-Tn10 3-2, the mini-Tn10 inserted into a gene encoding a protein with 90.3% identity to the ClpP protease subunit from Escherichia coli. Expression of ail in stationary phase at 28°C was also derepressed in a directed Y. enterocolitica clpP mutant. Analysis of ail transcripts in the wild-type and clpP mutant strains indicated that there is a single start site of transcription of ail and that the effect of the clpP mutation was on the initiation of transcription at this site. Similar to E. coli, a clpX homologue was identified downstream of clpP. The Y. enterocolitica clpP gene complemented the clpP mutant phenotype, repressing the expression of both ail transcript levels and cell surface-expressed Ail protein. Thus, ClpP has a role in the modulation of ail transcription in Y. enterocolitica.

Notes: A temperature upshift of 10 or more degrees in the growth temperature of a bacterial culture causes induction of extra rounds of chromosome replication. This heat-induced replication (HIR) initiates at oriC, is transitory, requires RNase H1 and RecA proteins and requires neither RNA polymerase activity nor de novo protein synthesis. The number of origins activated by heat is growth rate and temperature differential dependent. An origin activation higher than 20% increases the DNA:mass ratio around twofold, and this value is kept constant for the subsequent generations of growth at 41°C. We have also shown that HIR is neither related to SDR nor induced by the heat shock response. We suggest that a thermodynamic alteration of oriC structure or of membrane fluidity could explain the observed HIR.

Notes: Escherichia coli group I capsular K antigens are found in two forms on the cell surface. The KLPS form is linked to lipopolysaccharide lipid A core, whereas the high-molecular-weight capsular form is assembled independently of lipid A core. Subgroup IB K antigens are generally co-expressed with either the O8 or O9 antigen and, under the appropriate conditions, with the exopolysaccharide, colanic acid. To examine the relationships between the genetic loci and the synthetic pathways for these various cell-surface polymers, the gene cluster responsible for expression of a prototype group IB K antigen (serotype K40) was cloned and the flanking chromosomal regions characterized. Analysis of the six orfs within the cluster indicates features typical of Wzy (Rfc)-dependent O antigens. Synthesis of group IB K antigens is initiated by WecA (Rfe), a UDP-GlcNAc::undecaprenylphosphate GlcNAc-1-phosphate transferase, and the chain length of K40LPS is determined by the wzz gene product. The his-region of the E. coli O8:K40 prototype is almost exclusively devoted to the expression of three different surface polysaccharides. The rfbK40 cluster is located adjacent to the cps (colanic acid synthesis) and rfbO8 (O8 antigen synthesis) loci in the gene order: his-rfbO8/O9–wzz–ugd–gnd–rfbK40–galF–cps. Thus, rfbK40 is in the location occupied by other Wzy-dependent rfb gene clusters, and rfbO8/O9 represents an additional locus.

Notes: RepA, the initiator protein of plasmid P1, binds to multiple sites (iterons) in the origin. The binding normally requires participation of chaperones, DnaJ, DnaK and GrpE. When purified, RepA appears dimeric and is inactive in iteron binding. On reaction with chaperones, a species active in iteron binding is formed and found to be monomeric. To test whether the chaperones can reduce dimerization, RepA was used to replace the dimerization domain of the λ repressor. The hybrid protein repressed the λ operator efficiently, indicating that RepA can dimerize in vivo. A further increase in repressor activity was seen in dnaJ mutant cells. These results are consistent with a chaperone-mediated reduction of RepA dimerization. We also found that RepA mutants defective in dimerization still depend on DnaJ for iteron binding. Conversely, RepA mutants that no longer require chaperones for iteron binding remain dimerization proficient. These results indicate that the chaperone dependence of RepA activity is not solely owing to RepA dimerization. Our results are most simply explained by a chaperone-mediated conformational change in RepA protomer that activates iteron binding. This conformational change also results in reduced RepA dimerization.

Notes: We have reported previously on seven genes (cps14B–H ) of Streptococcus pneumoniae serotype 14, which are part of the type 14 capsular polysaccharide synthesis (cps14 ) locus. This study describes the cloning and sequencing of the remaining part of the cps14 locus. The entire cps14 gene cluster consists of 12 open reading genes (cps14A to cps14L ), which appear to be arranged as a single transcriptional unit. The flanking regions of the cps14 locus contain vestiges of insertion elements. Moreover, a 115-bp-long repeated DNA element, which is also present in several other intergenic regions on the pneumococcal chromosome, has been identified upstream of cps14AAll 12 open reading frames (ORFs) were inactivated by the insertion of a tetracycline resistance cassette. The cps14A to cps14J and cps14L mutants were unencapsulated, whereas only a limited amount of capsular polysaccharide was expressed by a cps14K insertion mutant. Comparison with DNA and protein sequences available in databases allowed us to predict functions for four out of the five new cps14 gene products. The biosynthetic function of Cps14I was determined experimentally by analysis of intermediates in the synthesis of the type 14 tetrasaccharide subunit, catalysed by membrane preparations of Escherichia coli expressing pneumococcal glycosyltransferases. The cps14I gene encodes the β-1,3-N-acetylglucosaminyltransferase activity necessary for the addition of the third sugar in the synthesis of the type 14 repeating unit. The activity encoded by cps14J was established using a synthetic glycosyltransferase acceptor: cps14J encodes a β-1,4-galactosyltransferase, which requires β-linked GlcNAc as an acceptor. Thus, Cps14J is responsible for the addition of the last (fourth) sugar in the synthesis of the type 14 subunit.

Notes: DNA relaxases play an essential role in the initiation and termination of conjugative DNA transfer. Purification and characterization of relaxases from several plasmids has revealed the reaction mechanism: relaxases nick duplex DNA in a site- and strand-specific manner by catalysing a transesterification. The product of the reaction is a nicked double-stranded DNA molecule with a sequestered 3′-OH and the relaxase covalently bound to the 5′ end of the cleaved strand via a phosphotyrosyl linkage. The relaxase-catalysed transesterification is isoenergetic and reversible; a second transesterification ligates the nicked DNA. However, the covalent nucleoprotein complex is relatively long-lived, a property that is likely to be essential for its role as an intermediate in the process of conjugative DNA transfer. Subsequent unwinding of the nicked DNA intermediate is required to produce the single strand of DNA transferred to the recipient cell. This reaction is catalysed by a DNA helicase, an activity intrinsic to the relaxase protein in some, but not all, plasmid systems. The first relaxase-catalysed transesterification is essential for initiation of conjugative strand transfer, whereas the second is presumably required for termination of the process. The relaxase, in conjunction with several auxiliary proteins, forms the relaxation complex or relaxosome first described nearly 30 years ago as being associated with conjugative and mobilizable plasmids.

Notes: The purpose of this work was to develop a high-resolution analysis of behaviour as an assay of the physiological consequences of mutations in the che genes and also to examine the role of CheZ in chemotaxis. Recent advances in flash photolysis have made it possible to expose cells to an unstable chemical gradient created by a square-wave increase in attractant concentration. The response of individual cells can be tracked in the order of milliseconds using real-time motion analysis. The tumble frequency of wild-type Escherichia coli exposed to photoreleased aspartate falls very quickly to smooth-swimming levels, and the swimming speed of these cells rises. As a consequence of these behavioural changes, there is an increase in the number of bacteria present in the centre of the flashed area, that is the bacteria’s response to the transient gradient generated by flash photolysis was to swim into the centre of the flash area. This allowed the rapid quantitative measurement of chemotaxis. Deletion of various che genes resulted in predictable changes in chemotactic behaviour. cheZ null mutants are non-chemotactic when measured by classical techniques but demonstrate a definite chemotactic response to photoreleased attractant.

Notes: In the absence of oxygen, many bacteria preferentially use nitrate as a terminal electron acceptor for anaerobic respiration. In Escherichia coli, there are two membrane-bound, differentially regulated nitrate reductases. While the physiological basis for this metabolic redundancy is not completely understood, during exponential growth, synthesis of NRA is greatly induced by anaerobiosis plus nitrate, whereas NRZ is expressed at a low level that is not influenced by anaerobiosis or nitrate. In the course of identifying genes controlled by the stationary phase regulatory factor RpoS (σs), we found that the expression of NRZ is induced during entry into stationary phase and highly dependent on this alternative sigma factor. Expression studies, using operon fusions and nitrate reductase assays, revealed that the NRZ operon is controlled mainly at the level of transcription and is induced 10-fold at the onset of stationary phase in rich media. Consistent with previous reports of RpoS expression, the RpoS dependency of NRZ in minimal media was very high (several hundredfold). We also observed a fivefold stationary phase induction of NRZ in an rpoS background, indicating that other regulatory factors, besides RpoS, are probably involved in transcriptional control of NRZ. The RpoS dependence of NRZ expression was confirmed by Northern analyses using RNA extracted from wild-type and rpoS− strains sampled in exponential and stationary phase. In toto, these data indicate that RpoS-mediated regulation of NRZ may be an important physiological adaptation that allows the cell to use nitrate under stress-associated conditions.

Notes: We analysed all major proteins secreted into culture media from Salmonella typhimurium. Proteins in culture supernatants were collected by trichloroacetic acid precipitation, separated in SDS–polyacrylamide gels and analysed by amino acid sequencing. Wild-type strain SJW1103 cells typically gave rise to nine bands in SDS gels: 89, 67, 58, 52, 50, 42, 40, 35 and (sometimes) 28 kDa. A search of the sequences in the available databases revealed that they were either flagellar proteins or virulence factors. Six of them were flagella specific: FlgK or HAP1 (58 kDa), FliC or flagellin (52 kDa), FliD or HAP2 (50 kDa), FlgE or hook protein (42 kDa), FlgL or HAP3 (35 kDa) and FlgD or hook-cap protein (28 kDa). The other four bands were specific for virulence factors: SipA (89 kDa), SipB (67 kDa), SipC (42 kDa) and InvJ (40 kDa). The 42 kDa band was a mixture of FlgE and SipC. We also analysed secreted proteins from more than 30 flagellar mutants, and they were categorized into four groups according to their band patterns: wild type, mot type, polyhook type and master gene type. Virulence factors were constantly secreted at a higher level in all flagellar mutants except a Δmot (motAB deletion) mutant, in which the amounts were greatly reduced. A new morphological pathway of flagellar biogenesis including protein secretion is presented.

Notes: A central technique used to investigate the role of a Candida albicans gene is to study the phenotype of a cell in which both copies of the gene have been deleted. To date, such investigations can only be undertaken if the gene is not essential. We describe the use of the Candida albicans MET3 promoter to express conditionally an essential gene, so that the consequences of depletion of the gene product may be investigated. The effects of environmental conditions on its expression were investigated, using GFP as a reporter gene. The promoter showed an ≈85-fold range of expression, according to the presence or absence of either methionine or cysteine in concentrations in excess of 1 mM. In the presence of either amino acid, expression was reduced to levels that were close to background. We used URA3 as a model to demonstrate that the MET3 promoter could control the expression of an essential gene, provided that a mixture of both methionine and cysteine was used to repress the promoter. We describe an expression vector that may be used to express any gene under the control of the MET3 promoter and a vector that may be used to disrupt a gene and simultaneously place an intact copy under the control of the MET3 promoter. During the course of these experiments, we discovered that directed integration into the RP10 locus gives a high frequency of transformation, providing a means to solve a long-standing problem in this field.

Notes: We describe here a Legionella pneumophila type IV secretion system that is distinct from the previously described icm/dot system. This type IV secretion system contains 11 genes (lvh ) homologous to genes of other type IV secretion systems, arranged in a similar manner. The lvh genes were found to be located on a DNA island with a GC content higher than the L. pneumophila chromosome. In contrast to the icm/dot system that was shown to be required for intracellular growth in HL-60-derived human macrophages and Acanthamoeba castellanii, the lvh system was found to be dispensable for intracellular growth in these two hosts. The lvh system was found to be partially required for RSF1010 conjugation, a process that was previously shown to be completely dependent on several icm/dot genes. However, results obtained from analysis of double mutants in the icm/dot genes and the lvh genes revealed that lvh genes can substitute for some components of the icm/dot system for RSF1010 conjugation, but not for intracellular growth. These results indicate that components of the icm/dot system and components of the lvh type IV secretion system are able to interact with one another.

Notes: The vanB gene cluster mediates glycopeptide resistance by production of peptidoglycan precursors ending in the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac) instead of D-Ala-D-Ala found in susceptible enterococci. Synthesis of D-Ala-D-Lac and hydrolysis of D-Ala-D-Ala is controlled by the VanRBSB two-component regulatory system that activates transcription of the resistance genes in response to vancomycin but not to teicoplanin. Two substitutions (A30→G or D168→Y) in the VanSB sensor kinase resulted in induction by teicoplanin, indicating that the N-terminal domain of the protein was involved in glycopeptide sensing. A substitution (T237→K) located in the vicinity of the putative autophosphorylation site of VanSB (H233) was associated with a constitutive phenotype and affected a conserved residue known to be critical for the phosphatase activity of related kinases. A mutant producing an impaired host D-Ala:D-Ala ligase required vancomycin for growth, since D-Ala-D-Lac was only produced under inducing conditions. The ddl and vanSB mutations, alone or in combination, resulted in various resistance phenotypes that were determined by the amount of D-Ala-D-Ala and D-Ala-D-Lac incorporated into peptidoglycan precursors under different inducing conditions.

Notes: Exogenously provided proline has been shown to serve as an osmoprotectant in Bacillus subtilis. Uptake of proline is under osmotic control and functions independently of the known transport systems for the osmoprotectant glycine betaine. We cloned the structural gene (opuE ) for this proline transport system and constructed a chromosomal opuE mutant by marker replacement. The resulting B. subtilis strain was entirely deficient in osmoregulated proline transport activity and was no longer protected by exogenously provided proline, attesting to the central importance of OpuE for proline uptake in high-osmolarity environments. The transport characteristics and growth properties of the opuE mutant revealed the presence of a second proline transport activity in B. subtilis. DNA sequence analysis of the opuE region showed that the OpuE transporter (492 residues) consists of a single integral membrane protein. Database searches indicated that OpuE is a member of the sodium/solute symporter family, comprising proteins from both prokaryotes and eukaryotes that obligatorily couple substrate uptake to Na+ symport. The highest similarity was detected to the PutP proline permeases, which are used in Escherichia coli, Salmonella typhimurium and Staphylococcus aureus for the acquisition of proline as a carbon and nitrogen source, but not for osmoprotective purposes. An elevation of the osmolarity of the growth medium by either ionic or non-ionic osmolytes resulted in a strong increase in the OpuE-mediated proline uptake. This osmoregulated proline transport activity was entirely dependent on de novo protein synthesis, suggesting a transcriptional control mechanism. Primer extension analysis revealed the presence of two osmoregulated and tightly spaced opuE promoters. The activity of one of these promoters was dependent on sigma A and the second promoter was controlled by the general stress transcription factor sigma B.

Notes: We present evidence for the existence of two large (≈50 kb) excisable segments in the chromosome of Salmonella typhimurium. The two elements — designated Gifsy-1 and Gifsy-2 — cover, respectively, the 57 units and the 24 units of the genetic map where they contribute indicative rare restriction sites. The two elements are closely interrelated and both contain a region of sequence similarity to the recE locus of the Rac prophage of Escherichia coli. Mutations within this region of Gifsy-1 yield the classical ‘Sbc’ phenotype: they suppress the recombination defect of recB mutants, apparently by activating a normally silent recE-like gene. At the same time, these ‘sbcE ’ mutations activate a Xis-type function that promotes excision of one or other of the two elements. Predictably, curing of Gifsy-1 results in the loss of recB mutant suppression. Surprisingly, the suppressor phenotype is also lost in cells cured for Gifsy-2 even though the Gifsy-1-associated sbcE mutation is still present. Moreover, the excision frequency of Gifsy-1 drops dramatically in Gifsy-2-cured cells. Thus, both elements must co-operate in the activation of recombination and excision functions. Overall, the data presented here suggest that Gifsy-1 and Gifsy-2 are cryptic prophages. They are distinct from previously described Fels prophages. Unlike Fels, they are not specific to S. typhimurium strain LT2 since they are both also found in a virulent S. typhimurium isolate (ATCC 14028s).

Notes: In the absence of exogenous promoters, plasmid-mediated complementation of the temperature-sensitive ftsW201 allele requires the presence of the full coding sequence of ftsW plus upstream DNA encompassing the C-terminus of mraY and the full coding sequence of murD. We used molecular and genetic techniques to introduce an insertional inactivation into the chromosomal copy of ftsW, in the presence of the plasmid-borne wild-type ftsW gene under the control of PBAD. In the absence of arabinose, the ftsW-null strain is not viable, and a shift from arabinose- to glucose-containing liquid medium resulted in a block in division, followed by cell lysis. Immunofluorescence microscopy revealed that in ftsW-null filaments, the FtsZ ring is absent in 50–60% of filaments, whilst between one and three Z-rings per filament can be detected in the remainder of the population, with the majority of these containing only one Z-ring per filament. We also demonstrated that the expression of only ftsWS (the smaller of two ftsW open reading frames) from PBAD is sufficient for complementation of the ftsW-null allele. We conclude that FtsW is an essential cell-division protein in Escherichia coli, and that it plays a role in the stabilization of the FtsZ ring during cell division.

Notes: Pseudomonas aeruginosa strains infecting patients with cystic fibrosis (CF) acquire a mucoid phenotype due to overproduction of alginate. The key enzyme in alginate synthesis is AlgD, whose promoter is transcriptionally active in mucoid strains and under the control of several trans-acting factors, including the integration host factor (IHF). The algD promoter (palgD ) contains two IHF-binding sites (ihf1 and ihf2 ). Study of IHF binding to ihf2 of palgD, by electrophoretic mobility-shift assays, led to the discovery of a protein of 36 kDa (p36) able to bind downstream from ihf2, to the 3′ region of palgD. The gene encoding p36 was isolated from the mucoid strain CHA of P. aeruginosa and sequenced. It can encode a 324-amino-acid protein, which shares a high degree of sequence identity (63%) with CysB from Escherichia coli and from Salmonella typhimurium, a transcriptional factor of the LysR superfamily. Furthermore, both p36 and S. typhimurium CysB bind the same site of palgD ; p36 was therefore termed CysB and its structural gene was called cysB. Next to cysB, on the opposite DNA strand, cysH was capable of encoding a protein sharing 26% identity with CysH (PAPS reductase) of E. coli and an even greater identity (54%) with the nucleotide-deduced protein from Arabidopsis. A CysB-deficient mutant of CHA, constructed by insertional inactivation of cysB, was a cysteine auxotroph and was unable to form a specific complex with palgD in vitro. Activity of palgD in the cysB mutant, in CHA and in the non-mucoid strain PAO was assessed by the use of a transcriptional algD–xylE fusion. Cells of PAO and of the cysB mutant grown in minimal media in the presence of 0.3 M NaCl exhibited a palgD activity, which was 10% or less that of the mucoid strain CHA. Thus, P. aeruginosa CysB can act as an activator of algD expression.

Notes: The ability to move in a directed manner may confer distinct advantages upon host-adapted prokaryotes. Potential benefits of motility include increased efficiency of nutrient acquisition, avoidance of toxic substances, the ability to translocate to preferred hosts and access optimal colonization sites within them, and dispersal in the environment during the course of transmission. The costs of motility also may be significant. These include the metabolic burden of synthesizing flagellar components, the energetic expense of fuelling flagellar motors and the presentation of polymeric and highly antigenic targets to the immune system. It is therefore not surprising that synthesis of the motility apparatus is usually subject to strict control. Studies of a variety of bacterial–host interactions demonstrate roles for motility, and its regulation, at points throughout the infectious cycle.

Notes: The mechanism of recombination of tandem repeats in the chromosome of Escherichia coli was investigated by genetic means. Tandem repeats 624 bp long were introduced into the lacZ gene of E. coli and the efficiency of deletion of one repeat was compared in different recombination mutants. No effects of the recA, recBC, recF, ruvA or ruvA recG mutations were detected. Hence, tandem repeat deletion appears to not proceed via the RecBCD or RecF homologous recombination pathways. A new mutant in which RecA-independent recombination is increased 15-fold was isolated. The mutation lies in the dnaE gene coding for the alpha subunit of polymerase III: it is a Gly to Asp change at codon 133. Another dnaE mutation, dnaE486, was tested and also shown to stimulate RecA-independent recombination. It is proposed that tandem-repeat recombination occurs by a replication slippage mechanism. RecA-independent recombination is also enhanced in a rep mutant, in which chromosomal replication is slowed down by the absence of the Rep helicase, suggesting that replication pausing may facilitate slippage.

Notes: The essential cell-division gene ftsZ is transcribed in Escherichia coli from at least six promoters found within the coding regions of the upstream ddlB, ftsQand ftsA genes. The contribution of each one to the final yield of ftsZ transcription has been estimated using transcriptional lacZ fusions. The most proximal promoter, ftsZ2p, contributes less than 5% of the total transcription from the region that reaches ftsZ. The ftsZ4p and ftsZ3p promoters, both located inside ftsA, produce almost 37% of the transcription. An ftsAp promoter within the ftsQ gene yields nearly 12% of total transcription from the region. A large proportion of transcription (≈ 46%) derives from promoters ftsQ2p and ftsQ1p, which are located inside the upstream ddlB gene. Thus, the ftsQAZ genes are to a large extent transcribed as a polycistronic mRNA. However, we find that the ftsZ proximal region is necessary for full expression, which is in agreement with a recent report that mRNA cleavage by RNase E at the end of the ftsA cistron has a significant role in the contol of ftsZ expression.

Notes: In Pseudomonas putida, benzoate and 3-chlorobenzoate are converted to catechol and 3-chlorocatechol, respectively, which are then catabolized to tricarboxylic acid cycle intermediates via the catBCA and clcABD pathways. The catBCA and clcABD operons are regulated by homologous transcriptional activators CatR and ClcR. Previous studies have demonstrated that in addition to sequence similarities, CatR and ClcR share functional similarities which allow catR to complement clcR. In this study, we demonstrate that CatR activates the clcABD promoter in vitro without inducer, but more transcript is produced when inducer is added. DNase I footprinting and DNA-bending analyses demonstrate that CatR binds to and bends the clcABD promoter to the same angle as does ClcR plus its inducer, 2-chloromuconate. This implies that CatR binds to the clc promoter in its active conformation. Transcription of the clcABD promoter by the α-subunit truncation mutant (α-235) of RNA polymerase was sharply reduced, indicating that the α-subunit C-terminal domain is important. However, a small amount of transcript was produced under these conditions, indicating that other contact sites on the RNA polymerase may play a role in activation.

Notes: Cloning and sequencing of the Paracoccus denitrificans ccmG gene indicates that it codes for a periplasmic protein–disulphide oxidoreductase; the presence of the sequence Cys-Pro-Pro-Cys at the CcmG active site suggests that it may act in vivo to reduce disulphide bonds rather than to form them. A CcmG–PhoA fusion confirmed the periplasmic location. Disruption of the ccmG gene resulted in not only the expected phenotype of pleiotropic deficiency in c-type cytochromes, but also loss of spectroscopically detectable cytochrome aa3, cytochrome c oxidase and ascorbate/TMPD oxidase activities; there was also an enhanced sensitivity to growth inhibition by some component of rich media and by oxidized thiol compounds. Dithiothreitol promoted the growth of the ccmG mutant on rich media and substantially restored spectroscopically detectable cytochrome aa3 and cytochrome c oxidase activity, although it did not restore c-type cytochrome biogenesis. Assembly of the disulphide-bridged proteins methanol dehydrogenase and Escherichia coli alkaline phosphatase was unaffected in the ccmG mutant. It is proposed that P. denitrificans CcmG acts in vivo to reduce protein–disulphide bonds in certain protein substrates including c-type cytochrome polypeptides and/or polypeptides involved in c-type cytochrome biogenesis.

Notes: The ‘two-component’ transcriptional activator FixJ controls nitrogen fixation in Sinorhizobium meliloti. Phosphorylation of FixJ induces its dimerization, as evidenced by gel permeation chromatography and equilibrium sedimentation analysis. Phosphorylation-induced dimerization is an intrinsic property of the isolated receiver domain FixJN. Accordingly, chemical phosphorylation of both FixJ and FixJN are second-order reactions with respect to protein concentration. However, the second-order phosphorylation constant is 44-fold higher for FixJN than for FixJ. Therefore, the C-terminal transcriptional activator domain FixJC inhibits the chemical phosphorylation of the receiver domain FixJN. Conversely, FixJN has been shown previously to inhibit FixJC activity ≈ 40-fold, reflecting the interaction between FixJN and FixJC. Therefore, we propose that modulation of FixJ activity involves both its dimerization and the disruption of the interface between FixJN and FixJC, resulting in the opening of the protein structure. Alanine scanning mutagenesis of FixJN indicated that the FixJ~P dimerization interface involves Val-91 and Lys-95 in helix α4. Dimerization was required for high-affinity binding to fixK promoter DNA.

Notes: Some or possibly all Ti plasmids of Agrobacterium tumefaciens encode a bicistronic operon designated virH, which encodes two proteins, VirH1 and VirH2, that resemble a family of cytochrome P450-type monooxygenases. Expression of this operon is induced by a family of phenolic compounds that induce all other operons within the vir regulon. We hypothesized that either or both of these proteins might metabolize some or all of these phenolic compounds. We therefore tested induction of a vir promoter by a variety of phenolic compounds in isogenic strains that express or lack virH1 and virH2. Although some compounds were equally effective inducers regardless of the virH status, other compounds induced vir expression far more effectively in the virH mutant than in the virH-proficient host. For all tested compounds, VirH2 appeared to be solely responsible for this effect. One such compound, ferulic acid, was chosen for biochemical analysis. Ferulic acid was degraded by a VirH-proficient host but not by a VirH mutant. The wild-type strain released large amounts of a more hydrophilic compound into the cell supernatant. This compound was tested by mass spectroscopy, nuclear magnetic resonance and UV spectroscopy and found to consist of caffeic acid. This indicates that wild-type strains convert virtually all added ferulic acid to caffeic acid, and that VirH2 is essential for this O-demethylation reaction. Ferulic acid was far more toxic than caffeic acid to the wild-type strain, although the wild-type strain was more resistant to ferulic acid than was the virH mutant. Caffeic acid was slowly removed from the broth, suggesting further metabolic reactions.

Notes: Most virulence genes of Listeria monocytogenes are activated by the transcriptional regulator PrfA. Previous studies have shown that environmental parameters, such as temperature, pH, stress conditions and medium composition, affect the expression of PrfA and PrfA-dependent proteins. In this report, we demonstrate a threefold increase in PrfA protein synthesis during infection of mammalian cells, which correlates with the increased activity of the plcA promoter, the major prfA promoter. Increased PrfA synthesis begins when L. monocytogenes adheres to host cells. In addition, we show that the observed induction of PrfA during the interaction of L. monocytogenes with mammalian cells can be reproduced in vitro using total cell extracts. Our data suggest a role for host proteinase K-sensitive protein(s) in PrfA upregulation.

Notes: Introduction of anti-host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram-negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia, the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK-mutant strain was found to induce a more rapid YopE-mediated cytotoxic response in HeLa cells as well as in MDCK-1 cells compared to the wild-type strain. We found that this was the result of a cell-contact-dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB-dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK-mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild-type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell-associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.

Notes: Streptococcus pyogenes that produces the bacterial superantigen streptococcal pyrogenic exotoxin A (SpeA) is associated with outbreaks of streptococcal toxic shock syndrome (STSS) in the United States and Europe. SpeA stimulates Vβ2.1, 12.2, 14.1, and 15.1-positive T cells, and the lymphokine production from the activated T cells is believed to result in the symptoms associated with STSS. The T-cell receptor (TCR)–SpeA interaction is crucial for superantigenic activity, and studies were undertaken to determine regions of both SpeA and the TCR involved in the formation of MHC/SpeA/TCR complexes. Previously, recombinant toxins encoded by speA alleles 1, 2, and 3 as well as toxins resulting from 19 distinct point mutations in speA1 were generated. Here, these 22 toxin forms were incubated with human peripheral blood mono- nuclear cells (PBMCs), and the percentages of T-cell blasts bearing Vβ chains 2.1, 12.2, and 14.1 were quantified by flow cytometry. The analysis indicates that the residues of SpeA needed for a productive TCR interaction differ for each Vβ chain examined. An amino acid substitution at only one site significantly affected the toxin’s ability to stimulate Vβ2.1-expressing T cells, three individual amino acid substitutions resulted in significant loss of ability to stimulate Vβ12.2-expressing T cells, and substitution at 13 individual sites significantly affected the ability to stimulate Vβ14.1-expressing T cells. To elucidate the regions of the Vβ chains that interacted with SpeA, synthetic peptides representative of the human Vβ12.2 complementary-determining regions (CDRs) 1, 2, and 4 were used to block the SpeA-mediated proliferation of human PBMCs. The CDR1, CDR2 and CDR4 peptides were each able to block proliferation, with the activity of CDR1 〉 CDR2 〉 CDR4. Combinations of CDR1 peptide with CDR2 or CDR4 peptides allosterically enhanced the ability of each to block proliferation, suggesting SpeA has distinct binding sites for the CDR loops.

Notes: Site-directed mutations in the Escherichia coli ssb gene were tested for the ability to complement a chromosomal ssb deletion for viability, and only the ssb W54→G mutation failed to do so at the pSC101 copy level. Non-aromatic amino acid substitutions for SSB Trp-54 (ssb W54→L and ssb W54→S) produced the greatest effects on in vivo protein function including altered marker linkage subsequent to generalized transduction, extreme UV sensitivity, and a lack of ability to support SOS induction. Additionally, the ssb-113 (ssb P176→S) mutation demonstrated the existence of both uvrA-dependent and uvrA-independent components of SOS induction. Although nucleotide excision repair appeared unaffected by alterations in the SSB protein, the mutational analysis suggests a direct role for SSB in recombinational repair.

Notes: Neisseria gonorrhoeae is able to utilize iron (Fe) from a variety of sources including transferrin (TF) and lactoferrin (LF). To gain insight into the molecular mechanisms used by gonococci to scavenge Fe from TF and LF, we cloned a 3.5 kb segment of wild-type DNA that repaired the defect in tlu mutants, which are unable to take up Fe from either TF or LF despite exhibiting apparently normal ligand binding to the receptor. Nucleotide sequence determination identified three open reading frames (ORFs), designated ORF1, ORF2, and ORF3, which were arranged in tandem. The deduced amino acid sequence of the 852 bp ORF1 encoded a 28 kDa protein that exhibited 26–32% identity with TonB proteins of nine other bacteria. The 663 bp ORF2 predicted a 24 kDa protein and the 435 bp long ORF3 predicted a 15 kDa protein. These predicted protein sequences exhibited 32–38% and 24–36% identity, respectively, with ExbB and ExbD proteins of three other bacteria. Thus, the sequence comparison identified the ORF1, ORF2 and ORF3 as gonococcal homologues of the E. coli tonB, exbB and exbD genes. An insertional mutation in the tonB homologue resulted in the failure of gonococci to grow with TF, LF or human haemoglobin (HB) as sole Fe sources and in the inability to take up 55Fe from TF and LF. The tonB mutation did not prevent the utilization of Fe from citrate (CT) or haemin (HM). Binding of TF, LF and HB to whole cells in a solid-phase binding assay was largely unaffected by the tonB mutation. We conclude that the pathways for utilization of Fe bound to TF, LF and HB but not to HM or CT were dependent on the TonB system.

Notes: Clumping factor of Staphylococcus aureus is a fibrinogen-binding protein that is located on the bacterial cell surface. The protein has an unusual repeat domain (region R) comprising mainly the dipeptide aspartate and serine. To determine if region R has a role in the surface display of the fibrinogen-binding region A domain, deletions lacking the region R encoding region of the clfA gene were generated. To determine the minimum length of region R required for wild-type levels of ClfA expression, variants with truncated region R domains were constructed. S. aureus cells expressing mutated clfA genes were tested for (i) proteins released by lysostaphin treatment that reacted with antisera specific for region A, (ii) clumping in soluble fibrinogen, (iii) adherence to immobilized fibrinogen and (iv) expression of the ClfA antigen on the cell surface by fluorescent activated cell sorting analysis. Each construct expressed three major immunoreactive proteins, two of which were putative N-terminal degradation products. Region R residues greater than 40 were required between region A and W (72 residues between region A and the LPDTG sorting signal) for wild-type levels of clumping in fibrinogen. A stepwise decrease in clumping titre was observed as the distance between region A and LPDTG was decreased from 72 to 4 residues. Similarly, a decrease in binding of anti-ClfA serum and in binding to fibrinogen-coated plastic surfaces was observed with cells expressing ClfA with 40 region R residues or less. Nevertheless, low levels of adherence to fibrinogen and binding to anti-ClfA serum occurred with ClfA derivatives that lacked region R altogether. This indicates that a small proportion of the ClfA molecules are linked to peptidoglycan very close to the cell surface but that residues greater than 72 are needed to allow sufficient ClfA molecules to span the entire cell wall and to display the biologically active A domain in a form that can participate fully in fibrinogen binding.

Notes: The interaction of pathogenic bacteria with host serum and matrix proteins is a common strategy to enhance their virulence. Streptococcus pneumoniae colonizes the human upper respiratory tract in healthy individuals and is also able to cause invasive diseases. Here, we describe a novel pneumococcal surface protein, SpsA, capable of binding specifically to human secretory immunoglobulin A (SIgA). The dissociation constant of SIgA binding to SpsA was 9.3 × 10−9 M. Free secretory component (SC) also binds to S. pneumoniae, whereas serum IgA does not, suggesting that pneumococcal binding to SIgA is mediated by the SC. To our knowledge, this is the first defined interaction of SC with a prokaryotic protein. The spsA gene encodes a polypeptide of 523 amino acids with a predicted molecular mass of 59 151 Da. The SIgA- or SC-binding domain is located in the N-terminal part of SpsA and exhibits no significant homology to any other proteins. The purified SIgA-binding domain of SpsA could completely inhibit the binding of SIgA to pneumococci. SpsA was expressed by 73% of the tested S. pneumoniae isolates and was substantially conserved between different serotypes. The interaction between S. pneumoniae and SC via SpsA represents a novel biological interaction that might increase virulence by the impairment of bacterial clearance.

Notes: The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia-containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule-associated Chlamydia vesicles also collocated with tyrosine-phosphorylated McCoy cell proteins. After migration, the Chlamydia-containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule-dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule-dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR-1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR-1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis.

Notes: The Caulobacter crescentus flagellum serves not only as a motility apparatus, but also as a key landmark in the differentiation of this asymmetrically dividing bacterium. A distinctive aspect of flagellum biosynthesis is the periodic expression of the flagellar genes during the cell cycle in a sequence corresponding to the order of gene product assembly into the growing flagellum. This program of gene expression is achieved in part by the organization of flagellar genes into a four-tiered regulatory hierarchy that controls their expression at both the transcriptional and post-transcriptional levels. Because of the close interconnection of the developmental program to the asymmetric cell-division cycle in C. crescentus, studies of flagellar gene regulation and motility have also begun to reveal basic mechanisms responsible for control of the cell cycle itself. Here, we review recent work on regulation of the flagellar gene hierarchy in C. crescentus and consider regulatory mechanisms that are distinct from those described in Escherichia coli and Salmonella typhimurium.

Notes: Transfer of the conjugative plasmid pCF10 from Enterococcus faecalis donor strains is induced by a peptide pheromone (cCF10) secreted by recipient cells. High-efficiency transfer requires expression of an aggregation protein (Asc10) encoded by the prgB gene and positively regulated by genes in a region 3–5 kb upstream, containing prgQ-R-S. Transcriptional fusion data reported here support the results of recent molecular analysis of the 5′ ends of prgB transcripts which indicated that prgB transcription occurs by readthrough from the prgQ promoter. A 530-nucleotide prgQ-encoded RNA molecule (Ql) with rRNA-like domains is required for Asc10 production. Ql and cCF10 were found to interact with the L6 and S5 ribosomal proteins, respectively. Mutational analysis of Ql indicates that this RNA may also directly interact with 16S RNA. Ql is present in ribosomes translating the prgB message, and pheromone cCF10 may affect the association of this RNA with translation complexes. Results suggest that the positive regulatory molecules act post-transcriptionally on the polycistronic message and modify a ribosome population to enhance pheromone-induced translation of prgB.

Notes: EcoR124I, EcoDXXI and EcoprrI are the known members of the type IC family of DNA restriction and modification systems. The first three are carried on large, conjugative plasmids, while EcoprrI is chromosomally encoded. The enzymes are coded by three genes, hsdR, hsdM and hsdS. Analysis of the DNA sequences upstream and downstream of the type IC hsd loci shows that all are highly homologous to each other and also to sequences present in the bacteriophage P1 genome. The upstream sequences include functional phd and doc genes, which encode an addiction system that stabilizes the P1 prophage state, and extend to and beyond pac, the site at which phage DNA packaging begins. Downstream of the hsd loci, P1 DNA sequences begin at exactly the same place for all of the systems. For EcoDXXI and EcoprrI the P1 homology extends for thousands of base pairs while for EcoR124I an IS1 insertion and an associated deletion have removed most of the P1-homologous sequences. The significance of these results for the evolution of DNA restriction and modification systems is discussed.

Notes: It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O-antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O-antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)-resistance cassette into the rol gene (controlling the modal O-antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP-rhamnose (the precursor of rhamnose in the O-antigen). The mutations had the expected effect on LPS structure. The rol::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol::Km and rfbD::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria-infected HeLa cells stained with fluorescein isothiocyanate (FITC)-phalloidin demonstrated that both the rol::Km and rfbD::Km mutants were defective in F-actin tail formation: the latter mutant showed distorted F-actin tails. Plasma-membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F-actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell-surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O-antigen chains of the rol::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O-antigen chains, and comparing untreated and Sf6c-treated cells by immunofluorescence with anti-IcsA serum.

Notes: The cell wall structure of Salmonella typhimurium has been studied for the first time during transit from free-living to parasitic lifestyles. Peptidoglycan of S. typhimurium proliferating within human epithelial cells contains a high proportion of previously unidentified muropeptides (5–10-fold higher than in extracellular bacteria). Amino acid and mass-spectrometry analyses showed that these new components consist of dimeric cross-linked muropeptides lacking one of the two disaccharide (N-acetyl-glucosamine-β-(1→4)-N-acetyl-muramic acid) molecules. This unique structure suggests an active role for an N-acetyl-muramyl-l-alanine-amidase in remodelling the peptidoglycan of intracellular S. typhimurium. Additional alterations observed included: (i) the absence of glycine-containing muropeptides; (ii) the increase in the relative proportion of muropeptides cross-linked by l(meso)-diaminopimelyl-d(meso)-diaminopimelic acid (l–d) peptide bridges; and, (iii) the decrease in the global cross-linkage of the macromolecule. The structural alterations observed in the peptidoglycan of intracellular bacteria do not produce loss of the cell envelope. These results show that intracellular residence of S. typhimurium within epithelial cells is accompanied by significant changes in the bacterial cell wall. Remodelling of peptidoglycan structure may constitute another sophisticated strategy of this pathogen for adapting to and colonizing the intracellular niche of eukaryotic cells.

Notes: We have previously reported the nucleotide sequence of the first six genes of the Streptococcuspneumoniae type 19F capsular polysaccharide biosynthesis locus (cps19f). In this study we used plasmid insertion/rescue and inverse polymerase chain reaction (PCR) to clone an additional 10 kb downstream region containing the remainder of the cps19f locus, which was then subjected to sequence analysis. The cps19f locus is located in the S. pneumoniae chromosome between dexB and aliA, and consists of 15 open reading frames (ORFs), designated cps19fA to cps19fO, that appear to be arranged as a single transcriptional unit. Insertion-duplication mutants in seven out of the nine new ORFs have been constructed in a smooth type 19F strain, all of which resulted in a rough (non-encapsulated) phenotype, confirming that the operon is essential for capsule production. Comparison with sequence databases has allowed us to propose functions for 12 of the cps19f gene products, and a biosynthetic pathway for type 19F capsular polysaccharide. T7 expression studies confirmed that cps19fH, cps19fK, cps19fL, cps19fM and cps19fN directed the production of polypeptides of the expected size in Escherichia coli. The function of the cps19fK product was confirmed by its ability to complement a mutation in nfrC (rffE ) in E. coli, as judged by restoration of sensitivity to bacteriophage N4. Interestingly, the last four genes of the locus (cps19fL–O ) exhibit very strong homology (up to 70% amino acid identity) to a portion of the Shigella flexneri rfb gene cluster encoding biosynthesis of dTDP-rhamnose. When expressed in E. coli, cps19fL–O were capable of complementing a mutation deleting the respective Shigella flexneri homologues. Southern hybridization analysis indicated that cps19fA and cps19fB were the only cps genes found in all 16 S. pneumoniae serotypes/groups tested. The region from cps19fG to cps19fK was found only in members of serogroup 19, and, within this, cps19fI was unique to type 19F.

Notes: We have identified a third citrate synthase gene in Saccharomyces cerevisiae which we have called CIT3Complementation of a citrate synthase-deficient strain of Escherichia coli by lacZ ::CIT3 gene fusions demonstrated that the CIT3 gene encodes an active citrate synthase. The CIT3 gene seems to be regulated in the same way as CIT1, which encodes the mitochondrial isoform of citrate synthase. Deletion of the CIT3 gene in a Δcit1 background severely reduced growth on the respiratory substrate glycerol, whilst multiple copies of the CIT3 gene in a Δcit1 background significantly improved growth on acetate. In vitro import experiments showed that cit3p is transported into the mitochondria. Taken together, these data show that the CIT3 gene encodes a second mitochondrial isoform of citrate synthase.

Notes: H-NS is a small chromatin-associated protein found in enterobacteria. H-NS has affinity for all types of nucleic acids but binds preferentially to intrinsically curved DNA. The major role of H-NS is to modulate the expression of a large number of genes, mostly by negatively affecting transcription. Many of the H-NS-modulated genes are regulated by environmental signals, and expression of most of these genes is positively regulated by specific transcription factors. Therefore one of the purposes of H-NS could be to repress expression of some genes under conditions characteristic of a non-intestinal environment, but allow expression of specific genes in response to certain stimuli in the intestinal environment. The hns gene is autoregulated. In vivo the H-NS to DNA ratio is fairly constant except during cold shock, when it increases three- to fourfold. In this review we propose that only the preferential binding to intrinsically curved DNA plays a role under normal growth conditions, and we discuss the different mechanisms by which H-NS might affect gene expression and how H-NS could be involved in the response to different stress situations. Finally, we summarize the evolutionary and functional relationship between H-NS and the homologous StpA.

Notes: A crucial step in converting an actively growing Bacillus subtilis cell into a dormant spore is the formation of a cell within a cell. This unusual structure is created by a phagocytosis-like process in which the larger mother cell progressively engulfs the adjacent smaller forespore. Only mutations blocking engulfment at an early stage and affecting genes expressed in the mother cell have been identified. Here we describe a new locus, spoIIQ, which is transcribed in the forespore and which encodes a membrane-bound protein required at a late stage of engulfment. Immunofluorescence microscopy analysis have shown that SpoIIQ is initially targeted to the septum at the boundary between the two cells and then spreads around the entire membrane of the forespore. Septum targeting requires only the first 52 residues of SpoIIQ as well as unidentified forespore-specific components. Electron-microscopy studies of cells engineered to activate the mother-cell program of gene expression independently of the forespore indicate that other as yet uncharacterized genes are involved in engulfment and that this morphological process is driven from both sides of the forespore envelope.

Notes: In Aspergillus nidulans a highly specific l-proline transporter is encoded by the prnB gene which is tightly linked to all other genes involved in proline catabolism. In mycelia, the expression of the prn structural genes is finely co-regulated in response to proline induction and nitrogen/carbon catabolite repression. In this study we establish that prnB expression is also activated during germination of conidiospores. This activation persists until the development of 6 h-old mycelia and it is independent of proline induction mediated by the pathway-specific prnA gene product. We then show that, in mycelia, prnB transcription is activated in response to proline or histidine starvation. This process has two components: a prnA-dependent and a prnA-independent component. A cis-acting element that conforms to the consensus target of the GCN4/CPC1 transcriptional activators mediating amino acid biosynthesis activation in other fungi is involved in the activation of prnB transcription in response to amino acid starvation. We also show that the stimulation of prnB expression in germinating conidiospores is not due exclusively to transient internal amino acid starvation occurring during the transition from conidiospore to mycelium. This is the first report that an amino acid transporter gene is upregulated during development and in response to amino acid starvation and specific amino acid induction.

Notes: We have cloned and sequenced the haemolysin gene locus from Edwardsiella tarda (ETH). This region encoded two open reading frames, designated ethA and ethB. ethA is the haemolysin gene consisting of 4782 bp encoding a product of 165.3 kDa and ethB is an activation/secretion protein gene of 1677 bp that encodes a product of 61.9 kDa. There were two putative ferric uptake regulator (Fur) binding sites on the 5′ upstream region of the ethB gene overlapping the promoter region and ribosome-binding site. The haemolysin produced by the cloned gene was secreted by Escherichia coli. The deduced amino acid sequences of the ethA and ethB genes were found to be homologous to those of the haemolysin and activation/secretion proteins of Haemophilus ducreyi, Proteus mirabilis, and Serratia marcescens. E. coli carrying the ethA gene but not the ethB gene completely lost haemolytic activity, although the ethA gene was transcribed. The protein expressed by E. coli carrying a recombinant plasmid which encoded the ethA gene had haemagglutination activity. The EthB protein was necessary for activation of EthA protein (haemolysin). The ethA and ethB genes were very prevalent in haemolytic E. tarda strains isolated from diseased fish. Transcription of the ethB gene was regulated by iron. The ethA and ethB genes were transcribed independently.

Notes: While catabolite repression by glucose has been studied extensively and is understood in large detail in Enterobacteriaceae, catabolite repression by carbohydrates that are not transported by the phosphotransferase system (PTS) has always remained an enigma. Examples of non-PTS carbohydrates that cause catabolite repression in Escherichia coli are gluconate, lactose and glucose 6-phosphate. In this article it is shown that enzyme IIAGlc of the PTS is not involved in catabolite repression by these carbon sources. Carbon sources that caused strong catabolite repression of β-galactosidase lowered the concentration of both cAMP and the cAMP receptor protein (CRP). A strong correlation was found between the amounts of cAMP and CRP and the strength of the repression. The levels of cAMP and CRP were modulated in various ways. Neither overproduction of CRP nor an increased cAMP concentration could completely relieve the repression by glucose 6-phosphate, lactose and gluconate. Simultaneously increasing the cAMP and the CRP levels was lethal for the cells. In a mutant expressing a constant amount of cAMP-independent CRP* protein, catabolite repression was absent. The same was found in a mutant in which lac transcription is independent of cAMP/CRP. These results, combined with the fact that both the cAMP and the CRP levels are lowered by glucose 6-phosphate, lactose and gluconate, lead to the conclusion that the decreased cAMP and CRP levels are the cause of catabolite repression by these non-PTS carbon sources.

Notes: We demonstrate that horizontal spread of mer operons similar to worldwide spread of antibiotic-resistance genes in medically important bacteria occurred in bacteria found in ores, soils and waters. The spread was mediated by different transposons and plasmids. Some of the spreading transposons were damaged in different ways but this did not prevent their further spread. Certain transposons are mosaics composed of segments belonging to distinct sequence types. These mosaics arose as a result of homologous and site-specific recombination. Our data suggest that the mercury-resistance operons of Gram-negative environmental bacteria can be considered as a worldwide population composed of a relatively small number of distinct recombining clones shared, at least partially, by environmental and clinical bacteria.

Notes: Expression of aggregation protein Asc10 from the prgB gene of conjugative plasmid pCF10 in Enterococcus faecalis is induced by the peptide pheromone cCF10. Genes required for Asc10 production, prgQ and prgS, lie 3–5 kb upstream, but can function at much greater distances. The prgQ transcripts encode a pheromone inhibitor peptide (iCF10) at the extreme 5′ end. Neither production of this peptide nor translation of the 5′ end of prgQ transcripts was found to be necessary for prgB expression. Pheromone cCF10 is required to activate prgB expression, even in the absence of iCF10 production, and does not affect initiation of transcription. The prgS gene encodes a 10.5 kDa protein that appears to be required for translation of prgB, and a non-coding RNA at the 3′ end of prgS may be required for readthrough of transcription to prgB from the prgQ promoter. Although the entire positive control region is transcribed constitutively from the prgQ promoter, translation of PrgS and transcriptional readthrough to prgB occur only after induction with pheromone. The combined data are consistent with a model in which the positive regulatory molecules and pheromone cCF10 activate prgB expression post-transcriptionally.

Notes: The global activator GacA, a highly conserved response regulator in Gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. The gacA gene of Pseudomonas aeruginosa PAO1 was isolated and its role in cell-density-dependent gene expression was characterized. Mutational inactivation of gacA resulted in delayed and reduced formation of the cell-density signal N-butyryl-l-homoserine lactone (BHL), of the cognate transcriptional activator RhlR (VsmR), and of the transcriptional activator LasR, which is known to positively regulate RhlR expression. Amplification of gacA on a multicopy plasmid caused precocious and enhanced production of BHL, RhlR and LasR. In parallel, the gacA gene dosage markedly influenced the BHL/RhlR-dependent formation of the cytotoxic compounds pyocyanin and cyanide and the exoenzyme lipase. However, the concentrations of another known cell-density signal of P. aeruginosa, N-oxododecanoyl-l-homoserine lactone, did not always match BHL concentrations. A model accounting for these observations places GacA function upstream of LasR and RhlR in the complex, cell-density-dependent signal-transduction pathway regulating several exoproducts and virulence factors of P. aeruginosa via BHL.

Notes: Many integral membrane proteins use the same translocation machinery for membrane insertion as secretory proteins use to get across the membrane. This requires that transmembrane segments can be discriminated from other parts of the protein during membrane translocation, and further requires that the transmembrane segments can be moved laterally out of the translocation channel into the surrounding lipid. The molecular basis for this remarkable intramembraneous sorting event is a major focus of current studies of membrane protein biogenesis.

Notes: The energy source for active transport of iron–siderophore complexes and vitamin B12 across the outer membrane in Gram-negative bacteria is the cytoplasmic membrane proton-motive force (pmf). TonB protein is required in this process to transduce cytoplasmic membrane energy to the outer membrane. In this study, Escherichia coli TonB was found to be distributed in sucrose density gradients approximately equally between the cytoplasmic membrane and the outer membrane fractions, while two proteins with which it is known to interact, ExbB and ExbD, as well as the NADH oxidase activity characteristic of the cytoplasmic membrane, were localized in the cytoplasmic membrane fraction. Neither the N-terminus of TonB nor the cytoplasmic membrane pmf, both of which are essential for TonB activity, were required for TonB to associate with the outer membrane. When the TonB C-terminus was absent, TonB was found associated with the cytoplasmic membrane, suggesting that the C-terminus was required for outer membrane association. When ExbB and ExbD, as well as their cross-talk-competent homologues TolQ and TolR, were absent, TonB was found associated with the outer membrane. TetA–TonB protein, which cannot interact with ExbB/D, was likewise found associated with the outer membrane. These results indicated that the role of ExbB/D in energy transduction is to bring TonB that has reached the outer membrane back to associate with the cytoplasmic membrane. Two possible explanations exist for the observations presented in this study. One possibility is that TonB transduces energy by shuttling between membranes, and, at some stages in the energy-transduction cycle, is associated with either the cytoplasmic membrane or the outer membrane, but not with both at the same time. This hypothesis, together with the alternative interpretation that TonB remains localized in the cytoplasmic membrane and changes its affinity for the outer and cytoplasmic membrane during energy transduction, are incorporated with previous observations into two new models, consistent with the novel aspects of this system, that describe a mechanism for TonB-dependent energy transduction.