- Volume 118, Issue 2, 1980

The major polar lipids in cells of Pseudomonas putrefaciens NCIB 10472 grown on nutrient agar were phosphatidylethanolamine, phosphatidylglycerol, a glucosyldiacylglycerol, a glucuronosyldiacylglycerol and an ornithine amide lipid. An additional phospholipid, tentatively identified as acyl phosphatidylglycerol or bis-phosphatidic acid, was a trace component of the wall lipids from broth cultures, which lacked the glycolipids and the ornithine amide lipid. The wall lipids from broth cultures of three further strains of P. putrefaciens (NCIB 10471, NCIB 11156 and NCTC 10737) contained all of the above lipids, and in two cases (strains NCIB 10471, and NCIB 11156) had an unusually high content of free fatty acid. Fatty acid compositions of the extractable lipids were qualitatively similar for all four strains: the major components were iso-pentadecanoic acid, pentadecanoic acid, a cis-heptadecenoic acid and a cis-hexadecenoic acid. Anteiso fatty acids were minor components in strain NCIB 10472. Lipid mixtures in which the ornithine amide lipid was present also contained small amounts of β-hydroxy fatty acids: in strain NCIB 10472 the major ones were the straight-chain and iso-branched C16 acids. Lipopolysaccharides from all four strains had similar, complex fatty acid compositions. The major non-hydroxy acids were the straight-chain and iso-branched C13 acids. β-Hydroxy acids common to all strains included the straight-chain C11, C12, C13, C14 and C15 acids, together with branched-chain C13 and C15 acids probably belonging to the iso series. The lipopolysaccharide from strain NCIB 10472 also contained C12 and C14 hydroxy acids of the same series, and small amounts of C13 and C15 β-hydroxy acids probably belonging to the anteiso series. The close resemblance in both polar lipid and fatty acid compositions between strains of P. putrefaciens and Pseudomonas rubescens is further evidence that these species are synonymous. Significant differences between the lipids and fatty acids of P. putrefaciens and those reported for a strain of Alteromonas haloplanktis do not harmonize with a proposal to transfer the former organism to the genus Alteromonas.

The composition of the cell wall of bacterium NCTC 9742, variously known as Chromobacterium iodinum or Pseudomonas iodinum and considered by some to be a species of Brevibacterium or Arthrobacter, has been studied. Walls isolated from cells grown in nutrient broth consisted predominantly of peptidoglycan and a phosphorylated polymer containing d-mannitol, glycerol, pyruvic acid, d-glucose and d-galactosamine. This phosphorus-rich polymer, which was slowly released on treatment of the walls with cold aqueous trichloroacetic acid, is considered to be a new type of teichoic acid. Quantitative analysis and the results of preliminary degradative studies indicated that the main backbone was a poly(mannitol phosphate) chain to which β-glucopyranosyl substituents and pyruvic acid residues (acetal-linked) were attached. It is suggested that the minor components, glycerophosphate and galactosamine, could form a linkage unit for the covalent attachment of the poly(mannitol phosphate) chain to peptidoglycan. In the walls of cells grown on nutrient agar, it appeared that the phosphorus-rich polymer was partly replaced by another polymer containing galactose and an unidentified component which reacted like a 2-keto-3-deoxyaldonic acid in the periodate/thiobarbituric acid test. The taxonomic implications of the cell-wall composition are discussed.

The highly aggregated proteins precipitated by (NH4)2SO4 from the culture fluid of three strains of Streptococcus mutans gradually released less aggregated glucosyltransferase activities - dextransucrase and mutansucrase - which catalysed the synthesis of watersoluble and insoluble glucans from sucrose. Mutansucrase was eluted from a column of Sepharose 6B before dextransucrase. This activity was lost during subsequent dialysis and gel filtration, but there was a corresponding increase in dextransucrase activity which catalysed the formation of soluble glucan when incubated with sucrose alone, and insoluble glucan when incubated with sucrose and 1·55 m-(NH4)2SO4. Relative rates of synthesis of soluble and insoluble glucan in the presence of 1·55 m-(NH4)2SO4 were dependent upon the enzyme concentration: high concentrations favoured insoluble glucan synthesis. Insoluble glucans synthesized by mutansucrase or by dextransucrase in the presence of 1·55 m-(NH4)2SO4 were more sensitive to hydrolysis by mutanase than by dextranase, but soluble glucans were more extensively hydrolysed by dextranase than by mutanase. Partially purified dextransucrase sedimented through glycerol density gradients as a single symmetrical peak with an apparent molecular weight in the range 100000 to 110000. In the presence of 1·55 m-(NH4)2SO4, part of the activity sedimented rapidly as a high molecular weight aggregate. The results strongly suggest that soluble and insoluble glucans are synthesized by interconvertible forms of the same glucosyltransferase. The aggregated form, mutansucrase, preferentially catalyses (1→3)-α bond formation but dissociates during gel filtration to the dextransucrase form which catalyses (1→6)-α bond formation.

The sequence of appearance of nitrogenase components I and II and leghaemoglobin in root nodules of pea plants inoculated with Rhizobium leguminosarum (strain PRE) was studied using radioimmunoassays. Leghaemoglobin was detected before nitrogenase activity. The MoFe component (I) of nitrogenase was detected at the same time as leghaemoglobin, while the Fe component (II) of nitrogenase and nitrogenase activity appeared 1 and 2d later, respectively. The same order of appearance of nitrogenase activity and leghaemoglobin was found in root nodules of cowpea plants inoculated with Rhizobium sp. 32H1.

The effect of the carbon and nitrogen source on the regulation of glutamine synthetase (GS) has been studied by omitting one or both from mycelium which had been grown in sucrose/ glutamate medium, a condition in which GS is fully induced in its octameric form. The activity and antigen concentration of GS decreased when mycelium was deprived of the carbon source. Even more enzyme was degraded when glutamate was replaced by glutamine and an oligomeric form of GS lower than the octamer was also observed. When the carbon source was restored, some of the enzyme was resynthesized during incubation with glutamate but not with glutamine. In both cases the final predominant oligomeric form of GS was the tetramer. When the carbon and nitrogen sources were both omitted from the medium, degradation of GS again occurred and the restoration of both sources did not result in an increase in GS: no change in the oligomeric state of GS was observed during these shifts. The deprivation of the nitrogen source resulted in the replacement of the octamer by the highly active tetrameric form of GS. These changes were not reversed when either the carbon or the nitrogen source was again supplied.

Protoplasts of Claviceps purpurea (ATCC 20102) were prepared in 0·8 m-sucrose containing 10 mm-CaCl2 and 10 mm-MgCl2. Protoplasts could revert to the filamentous state but not after treatment with water. Most of the protoplasts (about 80%) were highly vacuolated and these were separated from the non-vacuolated protoplasts and cell debris on the basis of their low density. Only the vacuolated protoplasts were able to synthesize ergotamine and ergocryptine de novo. Protoplasts were about 50% less active than the control mycelium. The control mycelium was more active in the uptake of labelled precursors than both protoplasts and freshly harvested mycelium. In the amino acid pool of protoplasts, alanine was present in a concentration which exceeded that of proline by a factor of six and that of phenylalanine by a factor of 100. This finding is consistent with the incorporation ratios of these amino acids into ergotamine when isotope dilution of the added radiolabel is considered. A significant stimulation of incorporation of constituent amino acids into ergotamine and ergocryptine occurred when d-lysergic acid was added to protoplasts and mycelium.

Two presumptive single-step mutants, resistant to penicillin and sensitive to the bactericidal effect of normal human serum, were isolated on penicillin gradient plates from a smooth, penicillin-sensitive, serum-resistant strain of Salmonella enteritidis(O9,12; gal hisEI cys). The chemical composition of their lipopolysaccharides, their phage-sensitivity patterns, and their serological and cultural properties showed one to be ‘part-rough’ and the other smooth. Hfr strains with O antigens O4,5,12 or 01,4,5,12 (two Salmonella typhimurium and one S. abony) and one S. enteritidisF′, O9,12, were crossed with the S. enteritidisO9,12 mutants. The results with the part-rough mutant indicate that its penicillin-resistance, serum-sensitivity and rough phage pattern result from a single mutation between hisEI and the part of the rfbgene cluster determining O specificity, 4 (abequose) or 9 (tyvelose). Transduction experiments confirmed that the mutation is closely linked to the hisoperon. This mutation is inferred to cause an incomplete defect in a transferase for galactose, mannose or rhamnose, the smooth sugars common to O4,5,12 and O9,12. Results from similar crosses to the smooth, serum-sensitive, penicillin-resistant S. enteritidismutant indicate that its serum-sensitivity is not linked to his.The occasional independent segregation of penicillin-resistance and serum-sensitivity suggests that other loci modify penicillin- resistance.

The specialized transducing phage λcysB ( Borck et al., 1976 ) was found to carry about 5 kilobases of Escherichia coli DNA. It was shown to have an intact cysB gene but none of the known neighbouring genetic loci. The phage (which is known to be deficient in its site-specific recombination functions) was shown to integrate into the chromosome of bacterial recipients at the cysB locus. Excision from this site occasionally generated recombinant phages that had exchanged their cysB allele for the one originally present in the host. In this way λcysB derivatives were prepared from lysogens of two strains carrying the amber mutations cysB242 and cysB257; these phages were proved by several tests to contain the expected cysB amber mutations.

A method is described for measuring the syntheses of macromolecules in the filamentous fungus Aspergillus nidulans. It involves growth of mycelium on small filter papers in the presence of radioactively labelled l-leucine to monitor total protein synthesis, and hence growth, and following the incorporation of differently labelled d-uridine (to measure nucleic acid synthesis) or l-leucine (to measure protein synthesis) under various conditions such as starvation for a metabolite or the presence of an inhibitor. Comparison of 3H/14C or 14C/3H labelling ratios (depending on the labelling combination used) shows the effects of the treatments on nucleic acid or protein synthesis. The method allows large numbers of measurements to be made and enables economical use of radioisotopes. Results are presented to show that, under the labelling conditions used, uridine is incorporated mainly into stable RNA. The method is used to demonstrate probable stringent control of stable RNA synthesis in A. nidulans and that inhibitors of protein synthesis such as cycloheximide and anisomycin also inhibit stable RNA synthesis. In contrast, starvation for meso-inositol affects nucleic acid synthesis much more strongly than it affects protein synthesis.

More than 70 conjugative R plasmids have been isolated from wild-type strains originating mainly in South-East Europe and identified as incompatible with the reference plasmid R387 of the incompatibility group K. These plasmids, governing different resistance patterns, have been characterized as PI cotransducible species of between 50 and 60 megadaltons. In contrast to the genetic similarity between all these plasmids and the reference type R387, their DNA revealed different digestion patterns after EcoRI treatment, although a number of common fragments could be identified. The molecular and genetic properties of these plasmid species demonstrate a phylogenetic relatedness.

The incidence of trimethoprim resistance in enterobacteria causing infection in a London hospital increased from 5.6% in 1970 to 16% in 1979. The proportion of gentamicinresistant aerobic Gram-negative bacilli had risen to 6.5% by 1979. During a 5-month period in 1977, during which no epidemic was recognized, all isolates resistant to either trimethoprim, gentamicin, tobramycin or amikacin were studied. The proportion of enterobacteria resistant to both trimethoprim and gentamicin (3.8% of the total) was significantly higher than expected assuming no correlation between acquisition of resistance characters. The resistance was transferable in 23 % of trimethoprim-resistant and 76% of gentamicin-resistant strains. Trimethoprim resistance was carried by plasmids of seven different incompatibility groups and in at least four instances was part of a transposon. Gentamicin resistance was determined by plasmids of three groups - IncC, IncFII and IncW. Transposition of gentamicin resistance was not shown, though this may have been the means of evolution of the gentamicin R plasmids of IncW, which determined aminoglycoside acetyl-transferase, AAC(3). Some bacterial strains with their plasmids were endemic. There was evidence for these plasmids (i) acquiring new resistance genes by transposition, (ii) losing resistance genes by deletion and (iii) being transferred between bacterial species in the hospital.

Clonal Didinium nasutum previously reared on either Paramecium aurelia, P. bursaria (normal and apochlorotic zoochlorellae-containing stocks), P. caudatum, or P. multi-micronucleatum were presented with either 15 cells each of normal or apochlorotic P. bursaria or 30 cells of either stock in 1 ml depression slides. Prey-choice, search period and handling ( = ingestion) times were recorded. Didinia chose apochlorotic prey twice as often as normal paramecia. They located and attacked apochlorotic cells more rapidly than normal prey, and they ingested ‘bleached’ paramecia more quickly than normal cells. There was a consistent pattern of relatively increased search and handling times for primary control didinia reared on paramecia other than P. bursaria, compared with the experimental predators. The frequency distributions of both search and handling times were normal. Predatory efficiency of didinia attacking apochlorotic paramecia was significantly greater (> 10 times) than that exhibited for normal cells as measured by the escape frequency of prey. Similarly, the greatest loss of zoochlorellae by regurgitation or premature defecation occurred after normal prey were ingested. These results support the hypothesis that the mutualistic zoochlorellae within P. bursaria tend to discourage predation by D. nasutum by releasing distasteful metabolites that repel them. The reciprocal nature of this ciliatezoochlorella symbiosis includes a protective function for the latter partner.

Ansamitocins inhibited cell division in Tetrahymena pyriformis W at low concentrations and caused cells to become more rounded. Exposure to ansamitocins for 5 h or more resulted in a burst of synchronous division at 100 min after removal of the antibiotics with a maximum division index of 70 %. A second burst of synchronous division occurred 300 min after removal with a peak division index of 55 %. The rounding of the cell shape was restored by the completion of the first division. When RNA or protein synthesis was blocked by chromomycin A3 or cycloheximide, the first division was suppressed. When DNA synthesis was blocked by methotrexate plus uridine, synchronous division still occurred; furthermore, the DNA content of T. pyriformis cells did not increase before the first division. These findings suggest that the first synchronous division requires RNA and protein synthesis but does not require DNA synthesis. Interference by ansamitocins with the function of microtubule systems in T. pyriformis is discussed.

Formate stimulated growth of the microaerophilic bacterium Campylobacter sputorum subsp. bubulus at reduced dissolved oxygen tensions (d.o.t). At a d.o.t. of 2 kPa the mean doubling time of C. sputorum growing in a complex medium supplemented with formate was 2 h. Growth did not occur in the absence of O2. When C. sputorum was cultured in a complex medium with formate, growth and formate consumption slowed down after a rise of the d.o.t. from 2 to 15 kPa. At the same time the potential respiration rate diminished greatly. The effects of exposure to a d.o.t. of 15 kPa could still be reversed after 4 h by shifting the d.o.t. back to 2 kPa. The decrease in the potential respiration rate could be related to a loss of formate dehydrogenase activity. Its susceptibility to a high d.o.t. was greater when formate was present in the growth medium than in its absence. Formate oxidase had a low affinity for O2 and H2O2 was a product of the oxidation of formate by O2. Formate dehydrogenase appeared to be a membrane-bound enzyme. Of the possible physiological electron acceptors tested, only FAD gave rise to a detectable, although low, activity of formate dehydrogenase. Campylobacter sputorum grown in the complex medium with formate contained cytochromes of the b- and c-type. Cytochrome b was membrane-bound; cytochrome c was largely recovered in the soluble fraction. A CO-binding pigment was identified as a cytochrome of the c-type. Campylobacter sputorum possessed cytochrome c peroxidase activity. Cytochrome oxidases of a known type could not be detected unequivocally in a (reduced-plus-CO minus reduced) difference spectrum. An important role in the microaerophilic nature of C. sputorum is ascribed to H2O2.

Spiroplasmas, the helical wall-free prokaryotes isolated from plants and insects, are capable of translational motility in viscous media, and the speed of swimming increases with viscosity. In consequence, spiroplasmas show ‘viscotactic’ behaviour in viscosity gradients. Motility is energy-dependent and is optimal at pH 7. It has also been found that spiroplasmas are capable of chemotaxis, being attracted towards sugars and some amino acids and repelled by hydrophobic amino acids, some organic acids and heavy metals.