- Volume 129, Issue 12, 1983

Antigen a5 was isolated from strain 830 of Staphylococcus aureus by autolysis in phosphate buffer followed by alcohol precipitation. Purification was principally achieved by affinity chromatography on wheat germ agglutinin ultrogel and on anti-S. aureus teichoic acid immunosorbent. The a5 antigen was weakly immunogenic in rabbits. Chemical analysis showed that a5 is a teichoic acid composed of ribitol phosphate, N-acetylglucosamine and alanine. It has similar physico-chemical properties to the wall β-N-acetylglucosamine ribitol teichoic acid of S. aureus but is serologically distinct.

Chemostat-grown cells of a newly isolated strain of Methylomonas were flocculated by the addition of small amounts of positively charged flocculants. The flocculation ability of the cells was almost completely lost after extracting the cells with 1–5 mm-EDTA, or by reducing pH to below 4·5. Reconstitution of the flocculation ability of EDTA-extracted cells was obtained either by the addition of an excess amount of calcium ions, or by mixing dialysed EDTA extracts with EDTA-extracted cells. The EDTA extract contained mainly proteins. The major protein was purified to homogeneity by gel filtration, and had a molecular size of 65000 Dal and an isoelectric point of 9·0. Immunological techniques indicated that this protein was located on the surface of the bacteria. We, therefore, propose that the surface protein of the Methylomonas sp. is involved in flocculation by positively charged flocculants.

Mutations (cda) leading to non-production of the new calcium-dependent antibiotic (CDA) of Streptomyces coelicolor A3(2) were closely linked on the chromosome. One representative mutation (cda-1) was mapped precisely between nicA and adeC. No cosynthesis of CDA was found in any pairwise combinations of 14 cda mutants. Mutations lacking aerial mycelium (bald mutations), mapping to the four previously described loci (bldA-D), were pleiotropically defective in production of CDA.

The heterokaryon incompatibility system in Aspergillus nidulans has been investigated by parasexual methods. The use of complementary auxotrophs with a repeated serial transfer method or with a protoplast fusion technique has enabled heterokaryons and diploid strains to be recovered from heterokaryon incompatible combinations of strains. The effects of allelic interaction at heterokaryon incompatibility (het) loci on the morphologies of the heterokaryon and diploid colonies isolated are described. Parasexual analyses conducted among strains belonging to the heterokaryon compatibility groups, h-cGl and h-cB, and the two recombinant compatibility classes, have located the hetA and hetB genes to linkage groups V and VI respectively.

Protoplast fusion has made possible the isolation of a diploid strain from haploid parents belonging to heterokaryon compatibility (h-c) groups Q and G1 of Aspergillus nidulans. This diploid was not fully heterozygous as part, or all, of linkage group VI had achieved homozygosity. Heterokaryon compatibility tests conducted between selected pairs of parasexually derived progeny strains facilitated a chromosome assay method for the detection of heterokaryon incompatibility (het) genes. Despite the lack of segregation for the linkage group VI marker, it proved possible to locate het genes on linkage groups III, V, VI and VII. Backcross data detected five het gene differences operating between the h-cQ and h-cGl parental strains. Two het loci were located on linkage group III.

Suppression of virulence in Agrobacterium caused by introduction of the IncW R plasmid pSa into cells containing a Ti plasmid is accompanied by loss of site adherence in the pinto bean infection assay and by loss of site adherence on the part of LPS isolated from these strains. When cured of the pSa plasmid, infectivity and site adherence are restored. This indicates that LPS produced by pSa-containing agrobacteria is sufficiently modified that it will not support site adherence, the initial step of the infection process.

Application of a viscometric assay to the haemagglutination induced by Aeromonas salmonicida strain 438 showed that shear forces can enhance the strength of bacterial adhesion. The D-mannose/L-fucose-sensitive reaction proceeded in two phases, an initial phase in which the degree of aggregation remained constant during shearing and a second stage, induced by shear, in which agglutination was enhanced as shear was maintained. The results strongly paralleled those found in studies of concanavalin A-induced haemagglutination, providing good evidence that adhesion in this species took place via lectin-like molecules. Methyl-α-d-mannoside, which strongly inhibits haemagglutination in this system, would not fully reverse the shear-dependent reaction. EGTA inhibited and reversed both phases, however. The effects of bacterial concentration, temperature, time of growth, pH, and a spectrum of monosaccharide inhibitors were also studied. The results demonstrated that the shear-dependent reaction has a number of features which distinguish it from the initial stage of haemagglutination, implying differences in the underlying biochemical mechanisms involved.

The uridine incorporation technique for assaying phagocytosis is based on the fact that polymorphonuclear leucocytes are impermeable to labelled uridine, and therefore ingested bacteria inside phagocytic vacuoles will be unable to take it up. Extracellular bacteria, including those adherent to the phagocytic cell surface, can do so however. Differences in uptake between bacteria alone and in the presence of phagocytic cells can be used to measure ingestion. The present paper describes the application of this technique to Escherichia coli O-86 as the test organism. It appears that with this test species, the method is unsuccessful, because exposure of the non-ingested bacteria to some soluble product of the triggered polymorphonuclear leucocytes causes a large increase in their uridine uptake rates, over that of the control bacteria. The nature of the product responsible is unknown. It is unconnected with change in the pH of the medium, is heat stable, and is only produced by polymorphonuclear leucocytes which are actively phagocytosing. It may be that a release of phagolysosome contents is responsible.

Rhizobium strain BTAi 1 which was isolated from a stem nodule on Aeschynomene indica was a fast grower with mean generation times of 3·2 and 4·0 h with glucose and mannitol, respectively. Its ability to utilize sucrose and lactose confirmed the similarity of BTAi 1 to other fast growers. However, its inability to utilize rhamnose, dulcitol, raffinose, trehalose, citrate, malate and fumarate linked it to the slow-growing rhizobia. The absence of the pentose phosphate pathway also placed BTAi 1 with the slow-growers as did the presence of 2-keto-3-deoxy-l-arabonate aldolase, its small colony morphology and the production of alkali with most carbon sources. Strain BTAi 1 is thus an intermediate type of Rhizobium, sharing characteristics with both fast and slow growers.

Trimethylamine-N-oxide (TMAO) supported anoxic growth in the non-fermentative marine bacterium Alteromonas sp. NCMB 400 and was reduced quantitatively to trimethylamine. The properties of TMAO reductase were those expected of a terminal reductase in anaerobic respiration. The enzyme was induced in the presence of trimethylamine oxide, repressed at high oxygen tensions and derepressed under anaerobic conditions. The cellular location of TMAO reductase was investigated by determining its distribution with respect to enzymes in cellular fractions prepared after lysozyme/EDTA treatment. A spheroplasting technique was developed, based on the method of Birdsell & Cota-Robles, that gave a high yield of intact spheroplasts from cells of marine alteromonads, and marker enzyme distribution indicated a clean fractionation. TMAO reductase was located in the periplasmic fraction, but it was released less readily than alkaline phosphatase; full release required EDTA treatment. The results are consistent with the role of TMAO as a terminal electron acceptor for anoxic growth, and the location of TMAO reductase does not preclude the possibility that TMAO reduction is linked to enerav conservation.

Foaming promoted the conjugal transfer of IncN plasmid R269N-1 in Escherichia coli; the theoretical basis of this phenomenon is described. The hypothesis that N-mating in conventional (non-foaming) liquid media is precluded by the fragility of the N-pilus is questioned. The environmental significance of foaming is briefly considered.

Analogues of di- and tripeptides in which the peptide backbone is modified have been examined for antibacterial activity in vitro, and for uptake into Escherichia coli. Aminoxy and hydrazino types, in which the peptide linkage is replaced, respectively, by -CO-NHO- or -CO-NH-NH-, were active against E. coli, Staphylococcus aureus, and Salmonella dublin; retro, α-aza, tetrazole, and hydroxamic types were inactive. Highest potency against all three species was found in aminoxy analogues containing d-2-aminoxypropionic acid (d-OAla) residues, Ala-d-OAla being active at < 1 mg 1−1. Uptake into E. coli was seen with all active types, but, with the exception of hydroxamic analogues, not with the inactive types. Following uptake the toxic analogues were rapidly hydrolysed and the constituent amino acid residues underwent exodus. The substrate specificities of the peptide transport systems have been further defined on the basis of our results.

We have examined in Bacillus subtilis how antibiotics that inhibit protein or RNA synthesis affect the stringent response (i.e. accumulation of (p)ppGpp) and the resulting decrease of GTP and sporulation. All antibiotics used inhibited sporulation completely at concentrations at which they inhibited growth only partially and in most cases only slightly. At these concentrations, some antibiotics (chloramphenicol, fusidic acid, lincomycin. tetracycline) abolished the stringent control, others lowered it. Sporulation inhibited by chloramphenicol or fusidic acid could be almost completely restored by addition of 0·2-0·5 mM-decoyinine, an inhibitor of GMP synthetase. The inhibition of sporulation by tetracycline and streptomycin, or by the RNA polymerase inhibitors rifampin and lipiarmycin, was partially counteracted by decoyinine, whereas the inhibition by lincomycin or by compounds that did not interfere with the stringent response could not be overcome by decoyinine addition. In mutants resistant to erythromycin, or lincomycin or thiostrepton, sporulation was no longer inhibited by that particular antibiotic but was still sensitive to the other two.

The results show that some antibiotics inhibited sporulation because they prevented the decrease of GTP needed to initiate sporulation caused by the stringent response. Other antibiotics inhibited sporulation by interfering with specific (ribosomal) functions either needed to maintain a certain metabolic state or specifically required for the synthesis of sporulation-specific proteins.