- Volume 140, Issue 8, 1994

The 60 kDa cysteine-rich proteins (CrPs) of Chlamydia are developmentally regulated outer envelope proteins synthesized late in the chlamydial growth cycle. These proteins, found only on the extracellular infectious elementary bodies, elicit major antibody responses in chlamydial infection. We have cloned and expressed in Escherichia coli the complete 60 kDa CrP genes from Chlamydia trachomatis, C. psittaci and C. pneumoniae. The recombinant products were expressed as either ‘native’ proteins or as fusions with the bacteriophage T7 gene 10 protein. Electron microscopy showed that recombinant proteins were produced as insoluble inclusions within the E. coli host cells. The recombinant 60 kDa CrPs were purified and used to raise high titre polyclonal antisera. In immunoblot analysis these antisera reacted with the 60 kDa CrPs from purified elementary bodies of all three chlamydial species in a genus-specific manner. Further molecular analysis allowed the genus-specific cross-reacting epitopes to be localized by using overlapping synthetic peptides covering the C. trachomatis 60 kDa CrP. Immunogold labelling experiments using purified infectious elementary bodies from the three chlamydial species indicated that the 60 kDa CrPs are not surface accessible to antibody binding.

Several strains of extremely halophilic archaeobacteria, both non-alkaliphilic and alkaliphilic, including Halobacterium, Haloferax and Natronobacterium species, were isolated from salt locales in India. The major phospholipids in these strains were the C20-C20-glycerol diether analogues of phosphatidylglycerolmethylphosphate (PGP-Me), phosphatidylglycerol (PG) and phosphatidic acid (PA). In addition, the Halobacterium strains possessed the characteristic glycolipids, sulfated triglycosyl and tetraglycosyl diethers (S-TGD-1 and S-TeGD, respectively) and the unsulfated triglycosyl diether (TGD-1); and the Haloferax strains had the characteristic sulfated and unsulfated diglycosyl glycerol diethers (S-DGD-1 and DGD-1, respectively). The PGP-Me, and PG components of the haloalkaliphiles each occurred as two molecular species with C20-C20- and C20-C25- (isopranoid) glycerol diether lipid cores. In contrast to previous reports of the absence of glycolipids in natronobacteria, the Natronobacterium strains from India were found to contain small amounts of a novel glycolipid identified as glucopyranosyl-1 → 6-glucopyranosyl-1 →1-glycerol diether (DGD-4). The lipid cores of DGD-4 also contained mainly unhydroxylated or hydroxylated C20-C20, C20-C25 and C25-C25 molecular species with unsaturated (isoprenoid) chains. Hydroxylated lipid cores have previously been identified only in some methanogenic archaeobacteria.

The catalytic and regulatory properties of aspartate transcarbamoylase from Pyrococcus abyssi were studied in the GE5 strain isolated from a deep-sea hydrothermal vent located in the North-Fiji Basin in the SW Pacific Ocean. The enzyme from this hyperthermophilic archaeobacterium shows homotropic cooperative interactions between catalytic sites for the utilization of its two substrates, carbamoylphosphate and aspartate. The activity of this enzyme is subject to allosteric regulation. It is feed-back inhibited by the end-product cytidine triphosphate independently of temperature. In contrast, its sensitivity to the feed-back inhibitor uridine triphosphate and to the activator adenosine triphosphate disappears at high temperature. The unusual response of this aspartate transcarbamoylase to carbamoylphosphate analogues suggests a particular mode of binding of this substrate to the catalytic site as compared to the homologous enzymes of other organisms. Aspartate transcarbamoylase of Pyrococcus abyssi exhibits a remarkable stability towards high temperature and pressure.

A trehalose-containing glycolipid was detected in several strains of Mycobacterium fortuitum and characterized as 2,3-di-O-acyltrehalose (DAT) by combined NMR spectroscopy, IR spectroscopy, GLC and GLC-MS. Lipid constituents of the molecule were identified as a mixture of straight-chain (14-18 carbon atoms) and methyl-branched-chain (17-21 carbon atoms) fatty acyl groups. DAT was further fractionated by reverse phase TLC into four fractions that were designated DAT-I-DAT-IV. DAT-I contained 70-75% straight-chain acyl substituents (hexadecanoyl and octadecanoyl predominating) and 25-30% 2-methyl branched substituents (mainly 2-methyl octadecadienoyl). DAT-II was composed of a mixture in which the acyl groups were almost exclusively 2-methyl branched, with 2-methyl octadecadienoyl and 2-methyl octadecen-2-oyl predominating. DAT-III, which was the major isolated fraction, consisted of compounds in which the ratio linear to branched acyl groups varied between 0.8 to 0.9, 2-methyl octadecen-2-oyl, hexadecanoyl and octadecanoyl being the most abundant. Finally, DAT-IV comprised a mixture of DAT molecules containing mostly 2-methyl octadecadienoyl, 2-methyl octadecen-2-oyl, 2-methyl eicosadienoyl and 2-methyl eicosen-2-oyl groups.

The lactate utilizing strain of Selenomonas ruminantium 5934e was found to contain three lactate dehydrogenase (LDH) activities in sonicated cell extracts. One activity, an NAD dependent L-LDH (L-nLDH) was measured at 15-fold greater levels in extracts of cells grown to mid-exponential phase on glucose compared to cells grown to the equivalent growth stage on DL-lactate. A second nLDH activity specific for D-lactate (D-nLDH) was detected at similar levels in both lactate-grown cell extracts and glucose-grown cell extracts. The third activity, an NAD independent DLDH (D-iLDH) was very low in cells grown on glucose but was induced more than 10-fold when DL-lactate was used as the carbon source. The three LDH activities could be separated by gel filtration. Recovery of the activities was low due to the apparent instability of the enzymes at 4 °C, which was most pronounced in the case of the D-iLDH. A Km for lactate of 0.5 mM was estimated for the D-iLDH and this was considerably lower than the values of 45 mM and 70 mM measured for L-nLDH and D-nLDH respectively. It is proposed that the D-iLDH may be largely responsible for the formation of pyruvate in lactate-grown cells of S. ruminantium strain 5934e. Three other lactate utilizing strains of S. ruminantium, HD4, 5521C1 and JW13 exhibited a similar profile of LDH activities to strain 5934e when grown on glucose and DL-lactate.

The metabolic routes of substrate catabolism by intact cells of H. pylori have been investigated by 13C NMR. Real time analyses of metabolic transformations under anaerobic conditions have been obtained with dense cell suspensions incubated with 13C-labelled pyruvate and glucose. In addition, time point studies have been carried out with cells incubated under aerobic conditions. Anaerobically, pyruvate was rapidly metabolized to lactate, ethanol and acetate. In addition, alanine was produced in significant quantities by cells provided with a nitrogen source and the metabolic incorporation of nitrogen from urea was demonstrated. Under aerobic conditions acetate was the major oxidation product from pyruvate; no evidence was obtained for tricarboxylic acid cycle activity. Glucose was metabolized more slowly than pyruvate. Anaerobically, two major products were observed and identified as sorbitol and gluconate by gas chromatography/mass spectrometry. Evidence was obtained for the oxidation of glucose to acetate under aerobic conditions. The fate of the 13C label with glucose substrates labelled in different positions showed that this oxidation takes place via the Entner-Doudoroff pathway.

The csa-15 locus of Bacillus subtilis corresponds to an operon encoding proteins which display features characteristic of the ABC group of transporters. Sequence analysis reveals a very high level of identity to the ribose transport operon of Escherichia coli. This hypothesis is supported by the observation that strains carrying mutagenic insertions in this operon are unable to grow on ribose as sole carbon source. Expression of this operon is directed by a single SigA-type promoter which is negatively regulated by Spo0A during the late-exponential/transition state of the growth cycle. Expression is also subject to catabolite repression and this mode of regulation is dominant to control of expression by Spo0A.

A gene called lplA (lipoprotein-like) has been isolated from a genomic library of Bacillus subtilis expressed in Escherichia coli. Clones carrying the IpIA gene were selected by the ability of the colonies to give visible haloes of starch hydrolysis. The cloned fragment contains an open reading frame (ORF) of 1509 bp encoding a protein of 56 kDa. The protein contains a typical N-terminal signal sequence, a putative transmembrane anchor domain and a leucine zipper at the C-terminus. The expression of this protein in E. coli causes cell lysis, only the N-terminal domain of the LpIA protein being responsible for this phenotype. The mechanism of cell lysis is similar to that previously suggested for the expression in E. coli of the lipoproteins encoded by the Streptococcus pneumoniae genes malX and amiA. The protein is modified with palmitic acid when secreted in E. coli, confirming that it is a typical lipoprotein.

We report the sequence and characterization of the Bacillus subtilis tlpC gene. tlpC encodes a 61.8 kDa polypeptide (TlpC) which exhibits 30% amino acid identity with the Escherichia coli methyl-accepting chemotaxis proteins (MCPs) and 38% identity with B. subtilis MCPs within the C-terminal domain. The putative methylation sites parallel those of the B. subtilis MCPs, rather than those of the E. coli receptors. TlpC is methylated both in vivo and in vitro although the level of methylation is poor. In addition, the E. coli anti-Trg antibody is shown to cross-react with this membrane protein. Inactivation of the tlpC gene confirms that TlpC is not one of the previously characterized MCPs from B. subtilis. Capillary assays were performed using a variety of chemoeffectors, which included all 20 amino acids, several sugars, and several compounds previously classified as repellents. However, no chemotactic defect was observed for any of the chemoeffectors tested. We suggest that TlpC is similar to an evolutionary intermediate from which the major chemotactic transducers from B. subtilis arose.

Prophage induction in Bacillus subtilis strains 168, S31 and W23 is accompanied by synthesis of two endolysins. The synthesis of those of strain 168, with molecular masses of 32 and 34 kDa, was shown to be controlled by the repressor of the defective phage PBSX. The 32 kDa protein corresponds to an N-acetylmuramoyl-L-alanine amidase, and plays the major role in PBSX-mediated lysis. Its structural gene, xlyA, is the last in the PBSX late operon, whose four most distal open reading frames have been cloned and sequenced. Analysis of the nucleotide sequence suggests that the two open reading frames preceding xlyA, designated xhlA and xhlB, encode polypeptides whose combined action could play the role of a holin. The open reading frame upstream of xhlA, designated xepA, encodes an exoprotein. The phage amidase, although endowed with a signal peptide, is apparently, like Xep, exported by a holin-like mechanism which does not involve the cleavage of the signal peptide. The presence on the B. subtilis chromosome of other, similar, genes, and their possible widespread occurrence, is discussed.

Pseudomonas putida strains PaW8 and PRS2000 produce flagellins with apparent molecular masses of 81 kDa and 50 kDa respectively. Two Tn5 insertion mutants of P. putida PaW8 lacking the ability to bind the flagellin-specific monoclonal antibody MLV1 were isolated. Mutant PaW8-flg2 contained a Tn5 insertion within a 2.6 kb EcoRI fragment of the P. putida chromosome carrying putative basal body genes. DNA and deduced protein sequences suggested the presence on this fragment of two complete genes homologous to flgH and flgl from Salmonella typhimurium. The insertion of Tn5 occurred in the flgH locus and appeared severely to reduce expression of the P. putida flagellin gene. A Tn5-containing fragment of DNA from a second mutant, PaW8-flg1, was cloned and found to contain sequences that hybridized strongly with the Pseudomonas aeruginosa flagellin gene. A 2.3 kb HindIII fragment containing all but 62 bp of the P. putida PaW8 flagellin gene was cloned and used as a probe to identify clones carrying the equivalent gene from P. putida PRS2000. Flagellin genes from both P. putida strains were sequenced and their amino acid sequences deduced. Both flagellins were found to contain conserved amino- and carboxy-terminal regions when compared to other flagellins, with the central region being more variable. The epitope for MLV1 is likely to lie within this central region of P. putida PaW8 flagellin. The deduced molecular mass of P. putida PaW8 flagellin (68 kDa) differed significantly from its apparent molecular mass estimated by PAGE, possibly as a consequence of post-translational modification. This was not the case with flagellin from P. putida PRS2000, where the predicted and apparent molecular masses were similar.

The Actinobacillus actinomycetemcomitans population consists of a large number of clones among which the ubiquitous leukotoxin gene operon appears very homogeneous. Population genetic analyses performed by multilocus enzyme electrophoresis together with DNA fingerprinting and analyses of genomic DNA restriction fragment length polymorphisms (RFLP) on 97 strains isolated over a period of 45 years revealed that each of the serotypes a, b, c, d and e comprise genetically isolated subpopulations and that successful horizontal transfer of genomic DNA between strains of different serotypes appears to be extremely rare in vivo. In contrast, recombination between strains of the same serotype in general appears to take place in nature. The results provide evidence that non-serotypeable strains are serotype antigen-deficient variants originating from strains of the known serotypes. Serotype b and c strains may contain transmittable DNA sequences not found in strains of the other serotypes.

Dichelobacter nodosus, a Gram-negative obligate anaerobe and the causative agent of ovine footrot, secretes a number of extracellular proteases, one of which is highly basic in nature. The gene (bprV) encoding this basic protease, from virulent strain 198, has been cloned and sequenced. Clone pBR3KB contained the complete bprV gene which constitutively expressed an active protease using its own promoter, when cloned in Escherichia coli. However, levels of protease expression were low and unstable when the clone was expressed in liquid culture. A range of E. coli strains were examined for stable expression; strains NH274 and SURE™ were found to be better hosts for stable expression than other commonly used E. coli host strains. Stabilization and enhancement of expression was achieved by deletion of the native promoter region and expression from plasmid promoter or promoters, and by modification of culture conditions. The recombinant protease obtained from E. coli was indistinguishable from the native enzyme in size, activity, isoelectric point and immunological properties.

Isolates of enterohaemorrhagic Escherichia coli serotype 0157:H7 do not exhibit β-glucuronidase (GUD) activity; however, they carry nucleotide sequences for the uidA gene that encodes the GUD enzyme. Polymerase chain reaction analysis using uidA-specific primers confirmed that a genetic region homologous in size to the E. coli uidA structural gene, including the regulatory region, was present in E. coli 0157:H7 isolates. DNA sequencing analysis of the regulatory region and the 5′ terminus of the uidA gene of E. coli 0157:H7 showed a base substitution in the putative −10 promotor region and at 93 bases downstream from the initiation codon. Neither base alteration, however, appeared to affect uidA allele gene expression in the 0157:H7 isolates. Immunoblotting of cell extracts with an anti-GUD antibody showed that E. coli 0157:H7 isolates produced an antibody-reactive protein that was homologous in size to E. coli GUD, but no GUD activity was observed in cell-free extracts of these isolates. These results suggest that the antibody-reactive protein produced by E. coli 0157:H7 may be an inactive GUD enzyme. Sequencing of the uidA structural gene in 0157:H7 showed the presence of 18 additional nucleotide base substitutions, but only six altered the amino acid sequence. Also, there were two frame shift mutations, 18 bases apart, that altered the sequence of six consecutive amino acids. These genetic alterations in the uidA structural gene of 0157:H7 may account for the absence of GUD activity in this serotype.