- Volume 129, Issue 9, 1983

Properties of stalks isolated from Caulobacter crescentus were studied. Isolated stalks possessed a density close to that of the cellular outer membrane, and contained a very low activity of succinate dehydrogenase. The protein patterns were similar to those of the outer membrane. These results suggest that the membrane component of the C. crescentus stalk comprises mainly outer membrane. The patterns of penicillin-binding proteins (PBPs) in isolated stalks were different from those of total cell envelopes. The stalks possessed neither PBP 1A nor PBP 3 which is localized in the inner membrane of growing cells. PBP X (mol. wt 93000) and PBP Y (mol. wt 85000), which are minor PBPs in the total cell envelope fraction were greatly enriched in the isolated stalks. A mecillinam-resistant, stalk-defective mutant, C. crescentus CB15 mcr-816, lacked both PBP X and PBP Y. It is concluded that PBP X and PBP Y are localized mainly in the stalk. Whether they are involved in the stalk development remains to be investigated.

ATP:citrate lyase has been purified some 20-fold from the oleaginous yeast, Lipomyces starkeyi CBS 1809. The enzyme, which is labile, was stabilized by the addition of ATP and citrate to extraction buffers. Michaelis-Menten kinetics were exhibited towards ATP and citrate. The molecular weight of the enzyme was determined to be approximately 510000. There was a specific requirement for ATP for activity. No activity was observed in the absence of Mg2+, although Co2+ and Mn2+ could partially substitute for Mg2+. ADP inhibited activity (50% inhibition at 1 mM), as did long-chain fatty acyl-CoA esters (50% inhibition with 4m oleoyl-CoA). The possibility of control of ATP:citrate lyase activity by fluctuations in the prevailing energy charge or by changes in the intracellular concentrations of long-chain fatty acyl-CoA esters is discussed.

Cell-free extracts of an itaconic acid producing strain of Aspergillus terreus contained NADP-dependent isocitrate dehydrogenase activity but no NAD-dependent activity was detected. During the acid-producing phase the activity of the NADP-linked enzyme fell to one-eighth of its value during the growth phase, whereas citrate synthase activity increased. Intact mitochondria were isolated from growth and production phase mycelium. The preparation from the growth phase oxidized exogenous NADH and all tricarboxylic acid cycle intermediates tested. That from the production phase oxidized tricarboxylic acid cycle intermediates at a reduced rate but exogenous NADH oxidation remained high. [2-14C]Acetate was incorporated into itaconic acid and over 90 % of the incorporated radioactivity was located in the methylene carbon. [1-14C]-Acetate was a poorer precursor and did not preferentially label the methylene carbon. These results support the view that itaconic acid biosynthesis in A. terreus occurs by decarboxylation of aconitic acid, the latter produced by the normal reactions of the tricarboxylic acid cycle.

Electrophoretically demonstrable variation in 12 enzymes was studied in more than 1600 isolates of Escherichia coli from human and animal sources and in 123 strains of the four species of Shigella. All 12 enzymes were polymorphic; and the number of allozymes (mobility variants), which were equated with alleles, averaged 9·3 per locus in E. coli. For Shigella species, the mean number of alleles was 2·9 per locus. Some 77% of the allozymes recorded in Shigella were shared with E. coli. A total of 302 unique genotypic combinations of alleles over the 12 loci (electrophoretic types, ETs) was distinguished, of which 279 represented E. coli and 23 were Shigella. Among electrophoretic types, mean allelic diversity per locus was 0·52 for E. coli and 0·29 for Shigella. It was estimated that there are, on the average, about 0·3 detectable codon differences per locus between pairs of strains of E. coli and Shigella, which is roughly equivalent to 1·2 amino acid differences per enzyme. Evidence that the enzyme loci studied are a random sample of the genome is provided by a significant positive correlation between estimates of genetic divergence between pairs of strains obtained by DNA reassociation tests and estimates of genetic distance between the same strains based on electrophoresis.

A principal components analysis of allozyme profiles revealed that the 302 ETs fall into three overlapping clusters, reflecting strong non-random associations of alleles, largely at four loci. Each of the four ETs of E. coli that have been most frequently recovered from natural populations has an allozyme profile that is very similar to, or identical with, the hypothetical modal ET of one of the groups. ETs of Shigella fall into two of the groups. No biological significance can at present be attributed to the genetic structure revealed by multilocus electrophoretic techniques. The electrophoretic data are fully compatible with other molecular and more conventional evidence of a close affinity between E. coli and Shigella, and they raise questions regarding the present assignments of certain strains to species. In support of evidence from DNA reassociation tests and serotyping, the present study suggests that S. sonnei is homogeneous in chromosomal genotype.

Spores formed by Bacillus subtilis carrying the mutation ger-36 are lysozyme-sensitive and germination-defective. The coats of these spores lack a number of polypeptides normally found in the spore coat, and four additional polypeptides are present that are not normally found in the coats of wild-type spores. The ger-36 mutation also prevents the synthesis (at about stage V) and the incorporation into the spore outer layers, of an intracellular protease (protease B, approx. mol wt. of monomer 25000). A partial revertant of a strain carrying ger-36 was isolated and this had an additional mutation in a locus distinct from gerE. The suppressing mutation restored the protease activity, but not the germination defect. It partly restored resistance to lysozyme and the spores formed had apparently normal coat protein composition except for the absence of one polypeptide (mol wt. 36000).

A-factor is a potent pleiotropic effector produced by Streptomyces griseus and is essential for streptomycin production and spore formation in this organism. Its production is widely distributed among various actinomycetes including Streptomyces coelicolor A3(2). Genetic analysis of A-factor production was carried out with S. coelicolor A3(2), and two closely linked loci for A-factor mutations (afsA and B) were identified between cysD and leuB on the chromosomal linkage map. In contrast, genetic crosses of A-factor-negative mutants of S. griseus, using a protoplast fusion technique, failed to give a fixed locus for A-factor gene(s) and suggested involvement of an extrachromosomal or transposable genetic element in A-factor synthesis in this organism.

A 34 MDal plasmid harboured by Pseudomonas fluorescens B69 carried a gene coding for the mercuric reductase enzyme, thus promoting mercury resistance in the host strain. Mercury-sensitive variants were isolated when a hypersensitive, cured derivative of strain B69 was exposed to 10 g mercury (II) ml−1. The alteration in mercury transport appeared to be the result of a mutation, since a transconjugant of the mutant, which carried the mercury-resistance plasmid, showed a reduced rate of mercury volatilization compared to the parental resistant strain. In addition, more mercury was tightly bound to the mutant cells. This phenomenon represents a new aspect of bacterial resistance to mercurials.

A bla gene encoding OXA-2 β-lactamase has been transposed from the Salmonella typhimurium R plasmid R1767. This transposable element which was designated Tn2410 encoded sulphonamide and mercury resistance in addition to the bla gene. It had a size of 18·5 kb and appeared to be flanked by small inverted repeats. Restriction enzyme analysis resulted in a detailed map of Tn2410, which showed a high degree of similarity with the published map of Tn2603, a transposon encoding OXA-1 β-lactamase. The ampicillin and sulphonamide resistance genes on Tn2410 were mapped in a cluster on the region between the centre and the right end of the map.

Partial smooth-rough transition was observed in 30 strains of Yersinia enterocolitica O:3 cultivated at 37 °C. Immunodiffusion and haemagglutination inhibition tests with eight patients sera demonstrated that the immunogenicity of the lipopolysaccharide of Yersinia enterocolitica in vivo was similar to that of the bacteria grown in vitro at 25 °C.

The enterotoxigenic Escherichia coli strains 1676, 1706, 1751 and KEC96a, which do not produce fimbrial adhesive antigens of the K88, K99 or 987P antigen type reacted both in vitro and in vivo with antiserum to F41 fimbriae in an indirect immunofluorescent antibody technique. Antiserum used to demonstrate material B, an adhesive antigen thought to mediate the adhesive and mannose-resistant (MR) haemagglutinating properties of E. coli strains 1676, 1706 and 1751, reacted in vitro with an F41+ strain. The antiserum also inhibited the MR haemagglutinating activity of F41 antigen and gave an anionic precipitation line in immunoelectrophoresis experiments with an extract containing F41 antigen.

The MR haemagglutinating properties of an antigen extract containing material B from E. coli strain 1706 was neutralized by antiserum to F41 fimbriae and by OK antisera to E. coli strains that produce both F41 and K99 fimbriae. These sera also gave an anionic precipitation line with the MR haemagglutinin from E. coli strain 1706 and the MR haemagglutinin gave a line of identity with F41 in gel diffusion experiments with antiserum to F41 fimbriae. OK antisera to K99+ F41− bacteria and OK antisera to K88+ bacteria and 987P+ bacteria did not react with this haemagglutinin.

Transmission electron microscopy on the ileum of newborn gnotobiotic piglets infected with E. coli strain 1706 showed irregular, poorly defined filamentous material surrounding some, though not all, bacteria but regular fimbrial structures were not visible. Fine thread-like material was visible surrounding most of the bacteria in ultra thin sections from a piglet infected with an E. coli strain that produces both F41 and K99. Staining with ruthenium red suggested. that this material was associated with polysaccharide.

Monoclonal antibodies have been produced with both type-specific and broadly cross-reacting properties against the variant pili of Neisseria gonorrhoeae P9. Competitive radioimmunoassays demonstrated that at least three separate epitopes contribute to type-specificity within the strain. ELISA using purified pili from clinical isolates showed that some of the type-specific epitopes are randomly distributed among variants of single strains isolated from different sites in sexual partners. Cross-reacting antibody SMI recognized an epitope present on all gonococcal isolates. This epitope was also present on a large proportion of meningococci and could be directly demonstrated in CSF from a patient with meningococcal meningitis. Commensal Neisseria and other Gram-negative bacteria tested failed to react. The meningococci which lack the epitope recognized by the antibody SMI were nevertheless shown by electron microscopy to be pilated, suggesting that pili from meningococci may be antigenically more diverse than those from gonococci.

The filamentous haemagglutinin of Bordetella pertussis has been purified from static, liquid culture supernatants and from extracts of cells grown on a solid medium. SDS-PAGE of the purified protein has shown multiple polypeptides with molecular weights ranging from 220000 to about 58000. By transferring the SDS-dissociated polypeptides to nitrocellulose paper and reacting with several monoclonal antibodies, it has been shown that many of the polypeptides are probably fragments of the polypeptide of highest molecular weight.

The adhesion of two Staphylococcus epidermidis strains and one Staphylococcus saprophyticus strain on to poly(tetrafluorethylene-co-hexafluorpropylene) (FEP)-fluorocarbon and cellulose acetate was studied in vitro. Both S. epidermidis strains showed a more hydrophobic character than the encapsulated S. saprophyticus as determined by the bacterial affinity towards xylene. Staphylococcus epidermidis showed a significantly higher adhesion on to the hydrophobic FEP than S. saprophyticus. The adhesion of staphylococci on to the more hydrophilic cellulose acetate was always low. Treatment of S. epidermidis with pepsin or extraction with aqueous phenol yielded cells with a decreased hydrophobicity, which resulted in a decreased adhesion on to FEP. Cells with a decreased hydrophobicity showed a lower rate of reaggregation in suspension. The hydrophobicity and the adhesion on to FEP of S. epidermidis were not affected by exposure to a subminimal inhibitory concentration of penicillin. The strong interaction between S. epidermidis and FEP, which appeared not to be influenced by the age or the metabolic stage of the bacteria, is mainly caused by hydrophobic bonding.

The physiological changes that occurred in chemostat cultures of an oleaginous yeast, Lipomyces starkeyi CBS 1809 undergoing transitions from steady-state carbon-limiting to steady-state nitrogen-limiting conditions have been investigated. A sequence of events has been identified initiated by the presentation of excess glucose to the culture and culminating in the accumulation of substantial quantities of intracellular lipid. The intracellular concentrations of adenine nucleotides and citrate have been determined and their possible regulatory significance in the control of catabolic and anabolic pathways is discussed.