ALBERT

Notes: Comparison of the N-terminus of the heat shock protein Hsp21 of Clostridium acetobutylicum with proteins predicted to be encoded by the genome of this bacterium revealed that this stress protein is encoded by two almost identical open reading frames CAC3597 and CAC3598. These genes encode a rubrerythrin-like protein with the rubredoxin-like FeS4 domain at the N-terminus and the ferritin-like diiron domain (rubrerythrin domain) at the C-terminus. Thus, the order of the two putative functional domains is reversed compared to “normal” rubrerythrins. This protein is proposed to be involved in the oxidative stress response of strict anaerobic bacteria. Northern blot analysis indicated that hsp21 is induced by heat and oxidative stress (air, H2O2). Hsp21 of C. acetobutylicum can be considered as a “reverse” rubrerythrin and a role of this stress protein, which is conserved among clostridia and other strict anaerobic bacteria, in the heat and oxidative stress response is proposed.

Notes: The DnaK chaperone system is involved in various cellular processes such as the control of the folded and oligomeric state of proteins under stress and non-stress conditions. In this study we functionally characterised the homologues of the DnaK system from Clostridium acetobutylicum. DnaK, DnaJ, GrpE and OrfA were heterologously synthesised in Escherichia coli and affinity purified via a His-tag. By optimising the stoichiometry, we were able to refold guanidinium hydrochloride-denatured firefly luciferase in vitro with 22% of the yield obtained with the E. coli DnaK system. In addition, C. acetobutylicum DnaJ could stimulate the E. coli DnaK ATPase by a factor of 55. Furthermore, the DnaK system from C. acetobutylicum was able to prevent the aggregation of OrfA from C. acetobutylicum, which is similar to the repressor HrcA of CIRCE-regulated heat shock genes in Bacillus subtilis.

Notes: Abstract: Characterization of the heat shock response in Clostridium acetobutylicum has indicated that at least 15 proteins are induced by a temperature upshift from 30 to 42°C. These so-called heat shock proteins include DnaK and GroEL, two highly conserved molecular chaperones. Several genes encoding heat shock proteins of C. acetobutylicum have been cloned and analysed. The dnaK operon includes the genes orfA (a heat shock gene with an unknown function), grpE, dnaK, and dnaJ; and the groE operon the genes groES and groEL. The hsp18 gene coding for a cell member of the small heat shock protein family constitutes a monocistronic operon. Interestingly, the heat shock response in this bacterium is regulated by a mechanism, which is obviously different from that found in Escherichia coli. So far, no evidence for a heat shock-specific sigma factor for the RNA polymerase in C. acetobutylicum has been found. In this bacterium, like in many Gram-positive and several Gram-negative bacteria, a conserved inverted repeat is located upstream of chaperone/chaperonin-encoding stress genes such as dnaK and groEL and may be implicated as a cis-acting regulatory site. The inverted repeat is not present in the promoter region of hsp18. Therefore, in C. acetobutylicum there are at least two classes of heat shock genes with respect to the type of regulation. Evidence has been found that a repressor is involved in the regulation of the heat shock response in C. acetobutylicum. However, this regulation seems to be independent of the inverted repeat motif, and the mechanism by which the inverted repeat motif mediates regulation remains to be elucidated. Another protein with a potential regulatory function might be the 21-kDa heat shock protein, which is induced significantly earlier than the majority of heat shock proteins. This protein has similarity to the redox carrier rubredoxin. Interestingly, heat shock genes are expressed in C. acetobutylicum at an increased rate not only after heat stress but also during the initiation of solvent formation. The mRNA level of some heat shock genes, e.g. dnaK, reached a maximum at the same time during the metabolic shift as the mRNA levels of genes necessary for solvent production. Therefore, the heat shock response in C. acetobutylicum might be part of a global regulatory network including different stress responses like heat shock, metabolic switch, and also sporulation.

Notes: In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins form the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7–8 S-layer proteins with a high degree of homology at the 5′ and 3′ ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

Notes: Abstract: The complete nucleotide sequence of two genes from Clostridium thermosulfurogenes EM1 homologous to E. coli genes encoding transport proteins was determined by the dideoxy procedure. The genes were cloned from plasmid pCT4, which contains the α-amylase gene from C. thermosulfurogenes EM1 as a 2.9-kbp XbaI fragment, inserted into the XbaI site of pUC18, to yield plasmid pCT401. The proteins encoded by the two identified complete ORFs are very hydrophobic and thus are probably integral membrane proteins. They show over 50% similarity to the maltose transport proteins MalF and MalG and to the glycerol-3-phosphate uptake proteins UgpA and UgpE of Escherichia coli. Since these genes are located immediately upstream of the α-amylase gene (amyA) of C. thermosulfurogenes EM1, the encoded proteins might be involved in transport of starch degradation products. The genes were tentatively designated amyC and amyD.

Notes: Abstract The complete dnaJ gene of Clostridium acetobutylicum was isolated by chromosome walking using the previously cloned 5′ end of the gene as a probe. Nucleotide sequencing of a positively reacting 2.2-kb HincII fragment, contained in the recombinant plasmid pKG4, revealed that the reading frame of the dnaJ gene of C. acetobutylicum consists of 1125 bp, encoding a protein of 374 amino acids with a calculated Mr of 40376 and an isoelectric points of 9.54. The deduced amino acid sequence showed high similarity to the DnaJ proteins of other bacteria (e.g. Escherichia coli, Bacillus subtilis) as well as of an archaeon (Methanosarcina mazei) and to the corresponding proteins of eukaryotes (Saccharomyces cerevisiae, Homo sapiens). The areas of similarity included a conserved N-terminal domain of about 70 amino acids, a glycine-rich region of about 30 residues, and a central domain containing four repeats of a CXXCXGXG motif, whereas the C-terminal domain was less conserved. Northern (RNA) blot analysis indicated that dnaJ is induced by heat shock and that it is part of the dnaK operon of C. acetobutylicum. The 5′ end (901 bp) of another gene (orfB), downstream of dnaJ and not heat-inducible, showed no significant similarity to other sequences available in EMBL and GenBank databases.

Notes: Summary Clostridium acetobutylicum cells were collected from chemostats which were run at pH 4.3 or 6.0 and which produced either acetone-butanol or acetate-butyrate; they were used to determine the level of enzymes involved either in solvent or in acid formation. The highest activity of phosphotransacetylase, phosphotransbutyrylase, acetate kinase, and butyrate kinase was found in cells which carried out an acetate-butyrate fermentation; these enzymes were present in solvent-producing cells at a level of about 10–50% as compared to acid-producing cells. Hydrogenase activity was detectable in approximately the same amounts in both cell types; however, in solvent-producing cells it was only measurable following a lag-period. Butyraldehyde and butanol dehydrogenases were found in small amounts exclusively in solvent-producing cells. It was demonstrated that the formation of acetone was initiated by the action of a coenzyme A-transferase which transferred coenzyme A from acetoacetyl-CoA to either acetate or butyrate. This coenzyme A-transferase as well as acetoacetate decarboxylase were hardly detectable in acid-producing cells, but reached high levels in solvent producing cells. Similar changes of the activity of the enzymes mentioned were observed when a batch culture was shifted from acid to solvent formation.

Notes: Summary When Clostridium acetobutylicum was grown in continuous culture under glucose limitation at neutral pH and varying dilution rates the only fermentation products formed were acetate, butyrate, carbon dioxide and molecular hydrogen. The Y glucose max and (Y ATP max ) gluc exp values were 48.3 and 23.8 dry weight/mol, respectively. Acetone and butanol were produced when the pH was decreased below 5.0 (optimum at pH 4.3). The addition of butyric acid (20 to 80 mM) to the medium with a pH of 4.3 resulted in a shift of the fermentation from acid, to solvent formation.

Notes: Summary When Clostridium acetobutylicum was grown in continuous culture under phosphate limitation (0.74 mM) at a pH of 4.3, glucose was fermented to butanol, acetone and ethanol as the major products. At a dilution rate of D=0.025 h−1 and a glucose concentration of 300 mM, the maximal butanol and acetone concentrations were 130 mM and 74 mM, respectively. 20% of the glucose remained in the medium. On the basis of these results a two-stage continuous process was developed in which 87.5% of the glucose was converted into butanol, acetone and ethanol. The cells and minor amounts of acetate and butyrate accounted for the remaining 12.5% of the substrate. The first stage was run at D=0.125 h−1 and 37° C and the second stage at D=0.04 h−1 and 33° C. High yields of butanol and acetone were also obtained in batch culture under phosphate limitation.