- Volume 144, Issue 1, 1998

Summary: RNA polymerase (RNAP) was purified from Methylobacterium extorquens AM1 cells grown on methanol or on succinate. The β, β', α and ω subunits were approximately the same size as those of Escherichia coli, and the identity of the ω subunit was confirmed by N-terminal sequence analysis. N-terminal sequence analysis suggested that two other polypeptides in the purified RNAP preparation might be σ factors, a 40 kDa polypeptide that shared identity with σ32 homologues, and a 97 kDa polypeptide that shared identity with σ70 homologues in other bacteria. The 97 kDa polypeptide did not cross-react with antibody to E. coli σ70. The same complement of putative σ factors was found in RNAP purified from M. extorquens AM1 grown on succinate and those grown on methanol, indicating that no major methanol-inducible σ factor is present in this strain. Run-off assays showed that the purified RNAP was capable of initiating transcription specifically at the transcriptional start site of a methylotrophic gene, mxaF, which encodes the large subunit of methanol dehydrogenase and is found only in methylotrophic bacteria.

Summary: During screening of a gene library of Streptococcus pyogenes type M15 for fibrinogen-binding material, a protein of approximately 100 kDa, encoded outside the vir region, was found. DNA sequencing revealed this component to be identical to protein F, a fibronectin-binding protein. Isolation of the recombinant protein, termed F15, was performed by the use of fibrinogen affinity chromatography. The affinity constant (K a) of protein F15 for fibrinogen, 1.25 x 107 mol−1, was lower than that for fibronectin, 1.8 x 108 mol−1. The fibrinogen-binding domain was located in the N-terminal part of the molecule, while the fibronectin-binding domains, as previously determined, were in the C-terminal portion of protein F. To examine the amino acid sequence heterogeneity of protein F, the 5′ part of the prtF gene, corresponding to the N-terminal variable region of the protein, was amplified by PCR from 12 strains of S. pyogenes belonging to six different M-types. Alignment of these nucleotide sequences indicated that the 5′ portion of the prtF gene had probably undergone a number of intragenic recombination and horizontal gene transfer events, allowing a pattern of structural diversity of protein F observed earlier for some other streptococcal virulence factors. There was no strict correlation between M-type and nucleotide sequence of the variable region of the prtF gene and, compared to streptococcal M protein, the overall variation observed for protein F appeared more limited.

Summary: Cell-surface polypeptide CshA (259 kDa) mediates multiple adherence interactions of Streptococcus gordonii. By generating a chromosomal cshA promoter (p-cshA)-cat gene fusion and measuring both CAT enzyme activity and cat mRNA levels, it was shown that cshA is expressed maximally in cells in the late exponential phase of growth in batch culture. The expression of CAT enzyme activity from the p-cshA-cat promoter fusion was 28% decreased in early stationary phase cell extracts of mutant strain OB528 in which the hppA (oligopeptide-binding lipoprotein) gene was insertionally inactivated. This effect was correlated with proportionally reduced cell-surface expression of CshA protein and with impaired adherence of hppA mutant cells to cells of an oral Actinomyces naeslundii strain. cshA promoter activity was enhanced in streptococcal cells that were incubated in conditioned culture medium as opposed to fresh medium, but this did not occur in an hppA genetic background. It is suggested that HppA is necessary for the response of cells to an extracellular factor that modulates cshA transcription, and hence affects cell-surface CshA expression and streptococcal cell adherence properties.

Summary: PilC is a phase-variable protein associated with pilus-mediated adherence of pathogenic Neisseria to target cells. In this study, 24 strains of Neisseria gonorrhoeae with known epidemiological data were examined for expression of PilC. All strains produced PilC independently of serovar and site of isolation. To investigate whether the PilC protein is conserved or variable among gonococcal strains, the complete nucleotide sequence of pilC in four strains, isolated from either rectum, throat or blood, was determined. The deduced amino acid sequence in these strains differed from each other and from the two PilC proteins of N. gonorrhoeae MS11. These data demonstrate that PilC is commonly expressed, but the PilC sequence may vary among gonococcal strains.

Summary: Opacity (Opa) proteins are a family of antigenically variable outer-membrane proteins of Neisseria meningitidis. ET-37 complex meningococci, defined by multilocus enzyme electrophoresis, have been isolated on different continents. Twenty-six different Opa proteins have been observed within strains of the ET-37 complex isolated between the 1960s and the 1980s, although individual strains have only four opa genes per chromosome. In this work the opa genes of four closely related ET-37 complex N. meningitidis strains recently isolated from Mali, West Africa were characterized and compared with the opa genes of strain FAM18, an ET-37 complex isolate from the USA. DNA sequence analysis and Southern blot experiments indicated that recombinational reassortment, including gene duplication and import by horizontal genetic exchange, has occurred in the opa genes within the ET-37 complex, resulting in two partially different Opa repertoires being present in FAM18 and the Mali isolates. Using synthetic peptides derived from the hypervariable (HV) regions of opa genes, the epitopes for nine mAbs were mapped. These bacteria, isolated on different continents, contain both shared and unique opa HV regions encoding epitopes recognized by mAbs and show evidence of recombinational reassortment of the HV regions.

Summary: Methylobacterium extorquens AM1 is a facultative methylotroph that oxidizes methanol via the pyrroloquinoline quinone (PQQ)-linked enzyme methanol dehydrogenase. In M. extorquens AM1 and other PQQ-synthesizing bacteria, several genes are involved in the synthesis of PQQ and one of these, pqqA, has been proposed to encode a peptide precursor of PQQ. In other PQQ-synthesizing bacteria, pqqA is required for PQQ production. In this study, it is shown that both deletion and insertion mutants of pqqA in M. extorquens AM1 grow normally on methanol and produce PQQ. The level of PQQ production is reduced in the insertion mutant, but it is sufficient to allow normal growth on methanol. These results suggest either that a different peptide in M. extorquens AM1 can substitute for PqqA in pqqA mutants, or that PqqA-like peptides may not be obligatory precursors of PQQ. In addition, it is shown that the methanol oxidation transcriptional regulator gene, mxbM, is required for normal methanol induction of PQQ synthesis.

Summary: Strain ATCC 39006 of Serratia marcescens makes the same carbapenem, (5R)-carbapen-2-em-3-carboxylic acid (Car), as the Erwinia carotovora strain GS101. Unlike E. carotovora, where the onset of production occurs in the late-exponential phase of growth in response to the accumulation of the small diffusible pheromone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), in S. marcescens carbapenem is produced throughout the growth phase and does not appear to involve any diffusible pheromone molecule. Two cosmids capable of restoring antibiotic production in E. carotovora group I carbapenem mutants were isolated from an S. marcescens gene library. These cosmids were shown to contain a homologue of the E. carotovora carR gene, encoding a CarR protein with homology to the LuxR family of transcriptional regulators. The S. marcescens carR was subcloned and shown to be capable of complementing in trans, in the absence of OHHL, an E. carotovora carR carl double mutant, releasing the heterologous E. carotovora host from pheromone dependence for carbapenem production. The apparent OHHL-independence of the S. marcescens CarR explains the constitutive nature of carbapenem production in this strain of S. marcescens.

Summary: The protomer forming the S-layer of Clostridium thermocellum was identified as a 140 kDa protein which was non-covalently bound to the cell wall. Cloning and sequencing of the corresponding gene revealed an open reading frame of 3108 nucleotides encoding a polypeptide of 1036 amino acids, termed SIpA. The amino acid composition of SIpA matches the composition of a previously described exocellular glycoprotein. SIpA shared extensive similarity with the S-layer protein of Bacillus sphaericus and with the outer wall protein of Bacillus brevis. In addition, the amino-terminal region of SIpA contained a segment presenting similarities with segments termed SLH (S-layer homologous), which are found in several bacterial exoproteins. A polypeptide of 209 residues comprising this segment was shown to bind to cell walls extracted from C. thermocellum cells.

Summary: Analysis of the nucleotide sequence of the form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes (cbbL and cbbS) of the non-sulfur purple bacterium Rhodobacter capsulatus indicated that the deduced amino acid sequence of the large subunit was not closely homologous to the large subunit from related organisms. Indeed, phylogenetic analysis suggested that the large subunit protein (CbbL) more closely resembled the enzyme from αlβlγ purple bacteria and cyanobacteria and is within a ‘green-like’ radiation of the RubisCO phylogenetic tree, well separated from CbbL of the related organism Rhodobacter sphaeroides. A cbbQ gene was discovered downstream of cbbS in Rh. capsulatus, a gene arrangement which also appears to be limited to certain organisms containing a ‘green-like’ RubisCO. Upstream, and divergently transcribed from cbbLSQ, is a gene (cbbR I) that encodes a LysR-type transcriptional activator. Phylogenetic analysis of the deduced amino acid sequence of CbbRI also suggests that this protein is quite distinct from the Rh. sphaeroides CbbR protein, and is even distinct from the previously described CbbRII protein, the gene of which is upstream and divergently transcribed from the cbb II operon of Rh. capsulatus. Interestingly, Rh. capsulatus CbbRI is more closely related to CbbR from bacteria whose RubisCO falls within the ‘greenlike’ radiation of the CbbL tree. These studies suggest that the cbbR I-cbbL-cbbS-cbbQ genes were acquired by Rh. capsulatus via horizontal gene transfer from a bacterial species containing a ‘green-like’ RubisCO.

Summary: Twenty-one strains of Verocytotoxin-producing Escherichia coli (VTEC) that hybridized with DNA probe CVD419 were examined for the ability to produce haemolysin. With solid media, all strains produced most haemolysin when grown in blood agar tubes and least when grown on blood agar plates incubated in air. Haemolysin production was increased considerably by incubating blood agar plates in an atmosphere comprising 8% carbon dioxide, 40% hydrogen and 52% nitrogen at 37 °C for 16 h, followed by 6 h at 21 °C in air. Haemolysin production was also increased when strains were grown on L-agar containing the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) prior to subculture on blood agar. Intracellular haemolysin was detected in five out of the 21 strains of E. coli grown on L-agar in the atmosphere described above, but haemolysin was not detected in L-broth culture supernatants. The haemolysins lysed guinea pig, mouse and ferret erythrocytes, but not human, rabbit, rat, turkey or chicken erythrocytes. Also, the addition of calcium ions to culture media was not required for haemolytic activity. It was concluded that haemolysins produced by VTEC appear to be quite distinct from E. coli α-haemolysin and resemble a form of β-haemolysin.

Summary: Forty-six cultures of Listeria monocytogenes (including clinical, food and collection strains) were serotyped, characterized for motility, haemolysis and phospholipase activities and tested for lectin agglutination using a four-lectin set. Lectin reactivity (i.e. agglutination by one or more of the four lectins) was observed in all 12 clinical isolates, 16 of the 23 food isolates and eight of the 11 collection strains. Virulence was evaluated in vitro based on strains' ability to invade the human enterocyte-like cell line Caco-2. In gentamicin survival experiments, recovery of viable intracellular bacteria among lectin-unreactive strains was usually 100-1000-fold lower than among lectin-reactive haemolytic strains, and lower than among nonhaemolytic strains. Considerable cytopathogenic effects were produced by lectin-reactive haemolytic strains in trypan-blue-stained cell monolayers, whereas lectin-unreactive and nonhaemolytic strains produced no detectable cytopathogenic effect. Among lectin-reactive strains, the number of listerial cells associated with Caco-2 monolayers was more than tenfold greater than among lectin-unreactive strains. Cell invasion was inhibited by pretreatment of Caco-2 cells with sugars recognized by the lectins or of listeriae with enzymes which removed the same sugars from the bacterial surface. The results suggest that the study of lectin interactions could be helpful in understanding the pathogenicity potential of isolates of food and environmental origin.