- Volume 139, Issue 8, 1993

SUMMARY: The class 1 outer-membrane protein of Neisseria menigitidis is the target for subtype-specific, bactericidal monoclonal antibodies (mAbs). The epitopes recognized by these antibodies have been mapped previously to linear peptides corresponding to the sequences thought to be exposed at the apices of surface-exposed loops of the protein. In this work several synthetic peptides containing the subtype PI. 16b epitope have been synthesized with the aim of inducing a polyclonal immune response resembling the reactivity of the mAbs. Initially, peptides of 9 and 15 amino acid residues were synthesized and used for immunization after coupling to a carrier protein. The reactivity of the resulting antisera, with synthetic linear decapeptides, resembled that seen in previous epitope mapping experiments with the protective mAbs. However, despite the induction of antibodies having the desired specificity, the antisera reacted poorly with the native protein in outer membranes, and were non-bactericidal. A 36mer peptide, consisting of the entire surface-exposed loop 4 of the class 1 protein was then synthesized and used for immunization as (i) free peptide, (ii) peptide coupled to carrier and (iii) peptide subjected to cyclization, in an attempt to restrict it to conformations that might more closely resemble the native loop structure. In contrast to antisera raised against linear peptides, antibodies raised by immunization with the 36mer cyclic peptide, did not react with linear peptides recognized by the mAbs, but instead appeared to recognize conformational determinants. This antiserum promoted complement-mediated bactericidal killing of the homologous meningococcal strain, demonstrating the potential of synthetic peptide immunogens for inducing a protective immune response against group B meningococci.

SUMMARY: A fibrin clot model for intra-abdominal abscess formation was used to study the migratory properties of peritoneal cells from rats during the early stages of infection. Peritoneal cells and fibrin clot remnant were harvested 6 h after implantation of a sterile, singly infected (Escherichia coli or Bacteroides fragilis) or mixed infected (E. coli and B. fragilis) fibrin clot. Histological study of fibrin clots, removed 6 h after implantation, showed a deeper infiltration by host cells of B. fragilis infected clots compared to the others. This difference in infiltration by peritoneal cells was not due to differences in fibrinolytic activity of the bacterial strains. Differential cell counts of the peritoneal cells from rats implanted with sterile, singly and mixed infected fibrin clots showed distribution over subpopulations to be independent of the bacterial content of the infected clots used. In vitro migration assays showed no significant differences in migration by peritoneal cells from rats implanted with clots containing a different bacterial composition. Since B. fragilis infected fibrin clots were more deeply infiltrated by host defence cells than the other clots, and only mixed infected clots led to persistent abscesses in this model, we conclude that local conditions within the fibrin matrix rather than intrinsic cellular capacities of the host cells are important for the process of abscess formation.

SUMMARY: Propionibacterium acnes is the predominant microbial resident within the pilosebaceous follicles of sebum-rich areas of human skin. This study investigated the effects of known hydrophobic components of sebum on the physiology and nutrition of this microorganism, grown anaerobically at 33 °C, under defined conditions using continuous culture techniques. The medium used was chemically defined, comprising eight amino acids, with glucose as the main carbon energy source, and the culture pH was maintained at 5.6. The range of sebum lipids assayed was based on the C18 monounsaturated fatty acid 9-cis-octadecenoic acid (oleic acid). Stock micronized solutions were aseptically pulsed into continuous cultures in the presence and absence of glucose, and nutritional effects monitored. None of the lipid substrates significantly affected P. acnes growth either in terms of maximum specific growth rate (μmax) or final culture biomass yield. Glycerol (3 mg ml-1) was found to be a poor carbon/energy source in comparison to glucose. Bacterial cells did, however, adhere with varying degrees, to the different lipid species, with maximum adherence occurring with the free fatty acid. This observation was confirmed by preliminary uptake experiments using [14C]oleic acid. The interactive site for cell adherence may be the lipid-fibrillar layer associated with the cell surface of P. acnes, as discerned in electron microscopical studies. The findings of this investigation suggest that one function of the P. acnes lipase may be to aid colonization within the pilosebaceous follicle, by promoting cell adherence to components such as oleic acid.

SUMMARY: The formate dehydrogenases of Escherichia coli involved in electron transfer from formate to nitrite (Nrf activity: nitrite reduction by formate) have been identified. No previously undescribed selenoprotein was detected in bacteria grown under conditions optimal for the expression of Nrf activity. The Nrf activities of single mutants defective in either FdhN or FdhH were between 50 and 60% that of the parental strain. A double mutant defective in both FdhN and FdhH retained less than 10 % of the activity of the FdhN+FdhH+ strain. No Nrf activity was detected in a triple mutant defective in FdhN, FdhH and FdhO or in the selC strain. It is concluded that all three of the known formate dehydrogenases of E. coli can contribute to the transfer of electrons from formate to the Nrf pathway. Mutants defective in Nrf activity and cytochrome c 552 synthesis were isolated by insertion mutagenesis or identified amongst strains received from the E. coli Genetic Stock Center. The mutations were located in at least three regions of the chromosome, including the 92 to 94 minute region which includes fdhF, the gene encoding FdhH required for formate hydrogenlyase activity. Fine structure mapping by P1 transduction established that the nrf mutations in the fdhF region were due to defects in three separable loci, all of which were independent of but close to fdhF. Clones were isolated from a cosmid library that complemented a deletion extending from fdhF into a region essential for Nrf activity. From these clones, plasmids were isolated that complemented only some of the Nrf− mutations in the 92 to 94 minute region, confirming the presence of different operons essential for Nrf activity and cytochrome c 552 synthesis in this region. Suggested reasons for this genetic complexity include the need for proteins involved in electron transfer from the various formate dehydrogenases to cytochrome c 552′ for the attachment of the haem group to the apocytochrome and for cytochrome c 552 export into the periplasm.

SUMMARY: Three different co-cultures composed of a methanogenic and a strictly aerobic bacterium were grown under O2-limitation in continuous cultures. The combinations used were (1) Methanobacterium formicicum with the aerobic heterotroph Comamonas testosteroni; (2) M. formicicum with a methanotrophic Methylocystis species; and (3) Methanosavcina barkeri with C. testosteroni. Although true steady-states were not obtained, growth and metabolic activity of the methanogenic and aerobic organisms occurred during O2-limited growth of these mixed cultures over extended periods of time. Co-cultures with C. testosteroni were considerably more stable than those with Methylocystis. Co-cultures with M. barkeri were less O2-sensitive than those with M. formicicum. C. testosteroni exhibited a higher O2-affinity than Methylocystis, resulting in a lower dissolved oxygen tension and a superior protection of the methanogenic bacteria against O2-poisoning than in mixed cultures with Methylocystis. The dissolved O2-concentrations in the mixed cultures were below the detection limit of the O2-probes used (0.2 μM). Calculations based on growth properties of pure cultures of C. testosteroni, M. barkeri and M. formicicum suggested that the dissolved O2-concentrations in the mixed cultures, as well as the O2-inhibition constants (apparent K 1 o2) of the methanogens were in the nanomolar range.

SUMMARY: The growth characteristics of Eubactevium limosum, an anaerobic acetogen, have been described kinetically for fermentation of glucose. A short-lived exponential growth phase was associated with a homoacetogenic fermentation in which all catabolic CO2 liberated was reassimilated via the acetyl-CoA pathway. During this phase no additional products were observed. A major shift in the metabolism leading to a mixed-acid fermentation pattern (acetate and butyrate) occurred coincident with the onset of the linear growth phase. Significantly diminished yields of acetate in this decelerating growth period were attributed to the limited capacity of the enzymes responsible for reductive generation of methyl donor within the acetyl-CoA pathway: CO2 and H2 accumulated in the gas phase. Pyruvate overflow (and to a lesser extent lactate, glycerate and carbon monoxide accumulation) were also observed, though pyruvate-ferredoxin oxidoreductase specific activity remained constant. In all experiments specific biomass formation rates were directly related to the rate of acetyl-CoA formation. The exponential growth phase was prolonged and the production of all compounds other than acetate was diminished when the medium was supplemented with tungsten, suggesting that formate dehydrogenase may be the enzyme limiting the correct functioning of the acetyl-CoA pathway.

SUMMARY: The moderately halophilic eubacterium Pseudomonas halosacchavolytica has been grown at salinities over the range 5-25 % (w/v), equivalent to 0.7-3.5 M-NaCl, and the fatty acid composition determined in the late-exponential and stationary phases of batch culture. There was an increase in the proportion of cyclopropane fatty acids (CFA) as the cultures went into stationary phase at all salinities; the overall proportion of CFA was higher in the media containing more salt. The biosynthesis of CFA in P. halosacchavolytica was determined using radiolabelled S-adenosylmethionine as the precursor incubated in cell-free extracts prepared by breaking bacteria with a French press. Compared with the activity obtained in 100 mM-phosphate buffer, the activity of CFA synthetase was inhibited by the addition of NaC1 or KC1, but stimulated up to 12-fold by added glycinebetaine, with maximum activity at 3 M. Although the specific activity of CFA synthetase in lysates from cultures grown in 0.7 or 2.1 M-NaC1 were similar in the presence of 3 M-glycinebetaine, the enzyme activity in low-salinity cultures was better adapted to function in 1 M-glycinebetaine. Shift-up experiments, in which CFA synthetase activity was assayed in cell-free extracts prepared at different times after increasing culture salinity from 0.7 to 2.1 M-NaC1, showed that the activity of the enzyme was immediately responsive to compatible solute concentration changes and indicated that enzyme induction would not be required to achieve the salt-dependent alterations in membrane lipid CFA composition in vivo. A range of other compatible organic solutes stimulated CFA synthetase activity to a much lesser extent (1.8-fold) compared with glycinebetaine. It is suggested that a compatible solute, which is normally accumulated during osmo(halo)adaptation by an organism in order to contribute towards osmotic balance, does not behave passively towards intracellular proteins but can also stimulate enzyme activity.

SUMMARY: The distribution of muropeptides formed by muramidase digestion of peptidoglycan from Staphylococcus aureus H was determined by gel-filtration HPLC. The observed crosslinking pattern supports the conclusion that incorporation of peptidoglycan in S. aureus proceeds by a similar mechanism to that proposed earlier for Bacillus megaterium. In this mechanism single glycan-peptide strands are incorporated into the sacculus by crosslinking reactions that take place only between the monomer muropeptide units of the incoming glycopeptide and muropeptides present in the innermost region of wall at the wall-membrane interface: such crosslinking reactions take place only during incorporation and no other crosslinking reactions occur. This assembly process has now been termed restricted monomer addition. The present analysis shows that the distribution of muropeptides in S. aureus peptidoglycan is in excellent agreement with that predicted by this mechanism. We propose that cell wall assembly in S. aureus proceeds via restricted monomer addition without any requirement for the secondary crosslinking reactions that have been suggested to occur in this organism. The high degree of crosslinking in S. aureus, 80% in this study, may result mainly from the freedom for crosslinking provided by the pentaglycine bridge peptide.

SUMMARY: N-AcetyI-D-[1-14C]glucosamine ([14C]GlcNAc) was used to label specifically the cell wall in Streptomyces antibioticus. Lysozyme solubilized 86.5% of the radioactivity present in the trichloroacetic acid-precipitated and RNAase plus trypsin-digested fraction of hyphae pulse-labelled with [14C]GlcNAc for 3 min in a medium containing glucose, yeast extract and a mixture of nine amino acids. Experiments with [14C]GlcNAc-labelled hyphae revealed that growth of Streptomyces occurs without turnover of peptidoglycan. Autoradiographic analysis of S. antibioticus hyphae indicated that cell wall elongation occurs by intercalation of newly synthesized wall polymers at the tip, but also over a relatively broad zone that, depending on the hyphal length, extends up to 6-10 μm from the tip.

SUMMARY: Spores of Bacillus subtilis 1604 can be induced to germinate by incubation in L-Ala (the ALA pathway) or in a combination of β-D-gIucose (Glc), β-fructose (Fru), L-Asn and K+ (the GFAK pathway). Biochemical analysis of the germination response of a gerA mutant deficient in the ALA pathway revealed that L-Ala can replace L-Asn in the GFAK pathway (the GFAlaK pathway). In contrast to the ALA pathway of both the wild-type and of a gerB mutant, the GFAlaK pathway w as insensitive to D-Ala and showed the same overall inhibitor profile as the GFAK pathway of wild-type and gerA spores. It is deduced that a second L-Ala receptor with different characteristics to that functioning in the ALA pathway is present in wild-type spores. Analysis of the germination response of a gerB mutant showed that whilst the rate of ALA germination could be stimulated by Glc as well as by Fru in the presence of Glc, the spores could not germinate in GFAK. In addition, Glc and Fru were unable to reverse D-Ala inhibition of L-Ala germination which they do in the wild-type. Thus, in the gerB mutant, the L-Ala/L-Asn receptor in the GFAK pathway is defective. It is concluded that the germination receptors in the ALA and GFAK pathways can functionally interact with each other to initiate B. subtilis spore germination. This conclusion is discussed in relation to proposed models of triggering of spore germination.