- Volume 142, Issue 1, 1996

Identification of antigenic variants of the PorA protein of Neisseria meningitidis with specific mAbs (serosubtyping) is used in meningococcal strain characterization and the resultant data has been exploited in the design of novel multivalent vaccines against this important pathogen. The reactivity of the P1.10 serosubtyping mAb MN20F4.17 with eight members of the meningococcal P1.10 variable region (VR) family (prototype P1.10 and variants P1.10a-P1.10g), identified by nucleotide sequence analysis of porA genes, was investigated. Analysis of overlapping synthetic octapeptides by ELISA demonstrated that the peptide sequence, QNQRPTL, present only in the prototype P1.10, was sufficient for binding of the mAb. A linear peptide of 14 amino acids, containing the minimum epitope, inhibited binding of mAb MN20F4.17 to whole cells in a competitive ELISA. This binding was weak compared with a tethered peptide or the native protein. In whole-cell ELISA or dot-blot assays using low concentrations of mAb MN20F4.17 only the prototype P1.10 was detected. However, when higher concentrations of antibody were used the prototype P1.10 was detected, together with variants P1.10a, P1.10c and P1.10e by whole-cell ELISA and P1.10a and P1.10c by the immunoblot technique. The variants P1.10b, P1.10d, P1.10f and P1.10g showed no reactivity with mAb under any of the conditions tested. A survey of the porA genes in serogroup B and C strains revealed that the P1.10a variant, rather than the prototype P1.10, was the most common member of the P1.10 VR family in England and Wales. These data illustrate: (i) the problems associated with epidemiological analyses that rely solely on monoclonal antibodies; (ii) the importance of using defined assay conditions for serosubtyping; and (iii) that genetical analyses provide more reliable information than serological data based on murine reagents for the design of candidate vaccines that include PorA.

The major outer-membrane protein (MOMP) of Pasteurella multocida serotype D strain P210, with an apparent molecular mass of 32 kDa, was purified and characterized. The purification method involved selective extraction of MOMP with N-lauroylsarcosine and SDS, followed by immunoaffinity chromatography using a murine monoclonal antibody (mAb). The N-terminal sequence and amino acid composition of the MOMP showed considerable similarity to other Gram-negative bacterial porins, notably to the 37 kDa MOMP (porin H) of P. multocida. Immunoelectron microscopy and colony blotting assays were used to demonstrate the surface localization of the 32 kDa MOMP on bacterial cells. The colony blotting assay provided a simple, sensitive and rapid screening method for visualizing accessibility of the antibody on the cells. In a Western blot assay, murine polyclonal hyperimmune serum against the purified 32 kDa MOMP recognized both serotype B and D strains bearing either a 32 kDa or a 37 kDa MOMP, whereas the mAb recognized only serotype D strains bearing a 32 kDa but not a 37 kDa MOMP. The present data indicate that the 32 kDa MOMPs of P. multocida are antigenically heterogeneous and possess both specific and cross-reacting epitopes. Detection of type-specific epitopes on the 32 kDa MOMP using an mAb may have potential implications regarding the feasibility of developing a serotyping system for P. multocida.

Lactic acid is the major end-product of glycolysis by Streptococcus mutans under conditions of sugar excess or low environmental pH. However, the mechanism of lactic acid excretion by S. mutans is unknown. To characterize lactic acid efflux in S. mutans the transmembrane movement of radiolabelled lactate was monitored in de-energized cells. Lactate was found to equilibrate across the membrane in accordance with artificially imposed transmembrane pH gradient (Δψ). The imposition of a transmembrane electrical potential (Δψ) upon de-energized cells did not cause an accumulation of lactate within the cell. The efflux of lactate from lactate-loaded, deenergized cells created a ΔpH, but did not create a Δψ, indicating that lactate crosses the cell membrane in an electroneutral process, as lactic acid. ΔpH and Δψ were determined by the transmembrane equilibration of [14C]benzoic acid and [14C]tetraphenylphosphonium ion (TPP), respectively. The presence of a membrane carrier for lactic acid in S. mutans was suggested by counterflow. Enzymic determination of the intra- and extracellular lactate concentrations of S. mutans cells glycolysing at pHo 6.8 and 5.5 showed that lactate distributed across the cell membrane in accordance with the equation ΔpH = log[lact]i/[lact]o. The addition of high extracellular concentrations of lactate to glycolysing S. mutans at acidic pH resulted in a fall in ΔpH and a subsequent decrease in glycolysis. The fall in ΔpH was attributed to the F1F0 ATPase being unable to raise the pHi back to its initial level due to the build up of lactate anion within the cell creating a large Δψ. The increase in Δψ resulted in the overall proton motive force remaining constant at about −110 mV. The results demonstrate that lactate is transported across the cell membrane of S. mutans as lactic acid in an electroneutral process that is independent of metabolic energy and as such has important bioenergetic implications for the cell.

Covalently bound haem proteins and cytochromes were analysed in Rhizobium leguminosarum biovar viciae free-living cells and nitrogen-fixing bacteroids isolated from pea nodules. Increased levels of spectroscopically detectable cytochrome c in bacteroids were correlated with the appearance of two proteins of Mr 30000 and 28000 that contained covalently bound haem. Conversely, bacteroids had undetectable levels of a periplasmic cytochrome c of M r 14000 that is normally present in free-living bacteria. Difference spectra confirmed that the terminal oxidases, cytochromes aa 3 and d, were absent, and photodissociation spectra revealed novel components that may be due to a bacteroid terminal oxidase.

QUTA is a positively acting regulatory protein that regulates the expression of the eight genes comprising the quinic acid utilization gene (qut) gene cluster in Aspergillus nidulans. It has been proposed that the QUTA protein is composed of two domains that are related to the N-terminal two domains-dehydroquinate (DHQ) synthase and 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase - of the pentadomain AROM protein. The AROM protein is an enzyme catalysing five consecutive steps in the shikimate pathway, two of which are common to the qut pathway. A genetic and molecular analysis of non-inducible qutA mutants showed that all 23 mutations analysed map within the N-terminal half of the encoded QUTA protein. One dominant mutation (qutA382) introduces a stop codon at the boundary between the two domains that were identified on the basis of amino acid sequence alignments between the QUTA protein and the N-terminal two domains of the pentafunctional AROM protein. The truncated protein encoded by mutant qutA382 has DNA-binding ability but no transcription activation function. A second dominant mutation (in strain qutA214) is missense, changing 457E K in a region of localized high negative charge and potentially identifies a transcription activation domain in the N-terminus of the EPSP-synthase-like domain of the QUTA protein. A series of qualitative and quantitative Northern blot experiments with mRNA derived from wild-type and mutant qutA strains supported the view that the QUTA protein regulates the expression of the qut gene cluster, including the qutA gene which encodes it. A series of Western blot and zinc-binding experiments demonstrated that a putative zinc binuclear cluster motif located within the N-terminus of the QUTA protein is able to bind zinc in vitro.

A cluster of arginine biosynthetic genes of Corynebacterium glutamicum ATCC 13032, comprising argJ, argB and argD as well as part of argC and argF, has been cloned by heterologous complementation of an Escherichia coli argE mutant. The gene order has been established as argCJBDF by sequencing the entire 4.4 kb cloned DNA fragment. The C. glutamicum argB gene can be transcribed in E. coli cells from an internal promoter located in the coding part of the preceding argJ gene, whereas transcription of the argJ gene appears vector-dependent. Expression of the corynebacterial argB gene is repressed by arginine in the native host but not in recombinant E. coli cells. Feedback inhibition of the corresponding N-acetylglutamate kinase activity was observed both in cell extracts of C. glutamicum and in recombinant E. coli argB auxotrophic strains. Extracts of E. coli cells carrying cloned corynebacterial DNA display an ornithine acetyltransferase activity (encoded by argJ) which alleviates the acetylornithinase (encoded by argE) deficiency of the enterobacterial host. In contrast to Bacillus stearothermophilus ornithine acetyltransferase which also exhibits acetylglutamate synthase activity, C. glutamicum ornithine acetyltransferase appears monofunctional. ArgA and ArgB proteins from different sources share highly significant similarities. The evolutionary implications of these data are discussed.

A protocol was developed for the introduction of foreign plasmid DNA into various Bifidobacterium strains. The method, which is applicable to all Bifidobacterium species tested so far, is based on electroporation of bacteria made competent by preincubation in electroporation buffer for several hours at 4 °C. Transformation of Bifidobacterium could be achieved with a plasmid vector originating from Bifidobacterium and with plasmid vectors from Corynebacterium, but not with vectors carrying replicons from Lactococcus or Lactobacillus.

Pasteurella multocida strain P1059 is a highly virulent bacterium which causes fowl cholera in turkeys and chickens. A genomic library of P. multocida P1059 DNA was constructed using pUC19, expressed in Escherichia coli DH5α, and screened with chicken antisera generated against P. multocida P1059. Twelve out of the 4100 clones screened were immunoreactive. Plasmids isolated from these twelve clones were transformed into E. coli CSR603 for maxicell analysis. Five proteins, with molecular masses of 34, 37, 43, 46 and 55 kDa, were expressed. Further work focused on the 43 kDa protein because it was expressed at levels detectable by SDS-PAGE and immunoblot analysis. The nucleotide sequence of the 1.8 kbp insert containing the gene encoding this protein was determined. The sequence contained three open reading frames (ORFs). The first ORF (ORF1) did not appear to code for any known protein. The second ORF (ORF2) encoded a protein of 403 amino acids (43662 Da). The deduced amino acid sequence showed 77% identity (84% similarity) with the tryptophan synthase β subunit (TrpB) of Salmonella typhimurium and Vibrio parahaemolyticus. The eight conserved regions of TrpB are observed in the P. multocida enzyme, including the conserved lysine (Lys-88) and consensus sequence (GGGSNA) implicated in pyridoxal phosphate binding. The expression and identity of the P. multocida TrpB were confirmed by complementation studies using E. coli W3110 tnaA2 trpB9578. The third ORF (ORF3) consisted of the first 77 nucleotides of the gene encoding the β-subunit of tryptophan synthase (trpA), and overlapped the 3'-end of trpB by 14 nucleotides. The deduced amino acid sequence of the 77 nucleotides of the P. multocida TrpA had 68% identity (92% similarity) with the analogous region of TrpA from Klebsiella aerogenes (K. pneumoniae).

DNA from a region downstream of and overlapping the polyketide synthase (PKS) gene cluster for jadomycin B biosynthesis in Streptomyces venezuelae was cloned and sequenced. Analysis of the nucleotide sequence located one complete ORF (ORF6), an incomplete one representing the 3' region of ORF4 in the PKS cluster, and a second incomplete one (ORF7). The deduced amino acid sequences for ORFs 6 and 7 resemble those of oxygenases. Since a plausible biosynthetic pathway for jadomycin B includes an angular polyketide intermediate that undergoes oxidative ring fission before condensation with an amino acid, we subcloned one of the presumptive oxygenase genes (ORF6) in a segregationally unstable shuttle vector (pHJL400) and disrupted it by inserting the gene for apramycin resistance. Transformation of S. venezuelae with the disruption vector and selection for apramycin resistance gave mutants blocked in jadomycin biosynthesis. Southern hybridization confirmed that gene replacement had occurred. Cultures of the mutants accumulated a metabolite identified by comparison with an authentic sample as rabelomycin, a non-nitrogenous polyketide-derived antibiotic originally isolated from Streptomyces olivaceus.

Streptococcus gordonii gave rise to β-haemolytic variants (Bhp+ for beta-haemolysin production) at frequencies of 10-4-10-3 on agar medium containing washed horse erythrocytes. Bhp+ variants reverted to the wild-type α-haemolytic phenotype (Bhp-) at the same frequencies. There was a significant probability (> 0.1) that phase variation in Bhp and phase variation in the previously described Spp (sucrose promoted phenotype) would occur concomitantly, but there was no correlation between these phenotypes. There was evidence also of independent phase variation in adhesion to saliva-coated hydroxyapatite (Asp for adhesion to salivary pellicles), in lactose-sensitive coaggregation (Cls for coaggregation, lactose-sensitive) and in the concentrations of particular cell surface antigens (Cap for cell antigen profile) in strains that had undergone phase changes in Spp and/or Bhp. Phase variation in all these phenotypes were transitions between high and low levels of activity and each appeared to occur as an independent event. Significant associations (P ⩽ 0.0001 by contingency table analysis) between particular phenotypes such as Bhp and Asp and between Asp, Cls and Cap phenotypes, however, were apparent. The results suggest that S. gordonil cells become predisposed to phase variation and that the resulting independent phenotypic changes may give rise to phenotypically diverse streptococcal populations able to accommodate rapid and transient environmental changes in the mouth.

The α-toxin produced by the type strain of Clostridium perfringens (NCTC 8237) was shown to differ from the α-toxins produced by most strains of C. perfringens isolated from man and from calves with respect to reactivity with a neutralizing monoclonal antibody (DY2F5D11). The difference in antibody binding correlated with three differences in the deduced amino acid sequence (Ala174 to Asp174; Thr177 to Ala177; Ser335 to Pro335) of the α-toxins. Using octapeptides synthesized on the basis of the amino acid sequences from these regions of variability, it was shown that the Ala174 to Asp174 change had the greatest effect on reducing the binding of monoclonal antibody DY2F5D11 to the α-toxin. These differences did not affect the enzymic or toxic properties of the protein. However, the phospholipase C activity of the α-toxin produced by strain NCTC 8237 was more susceptible to inactivation by chymotrypsin. The changes in amino acid sequence did not affect the ability of a C-terminal domain vaccine, derived from the α-toxin of strain NCTC 8237, to induce protection against the α-toxin from a bovine enteric strain of C. perfringens.