- Volume 121, Issue 2, 1980

Some aspects of protein biosynthesis were investigated in extracts of the obligate intracellular bacterium Coxiella burnetii. Sucrose gradient analysis revealed small quantities of 30S and 50S ribosomal subunits, few 70S ribosomes and no polysomes. Functional endogenous mRNA was not detected. In translation of exogenously added poly(U), extracts required Mg2+ (17 mM) and NH4 + (60 mM) for optimal polyphenylalanine synthesis; the optimum Mg2+ requirement differed from that of Escherichia coli. The translation of coli-phage Qβ RNA by C. burnetii extracts required Mg2+ (13 mM), NH4 + (60 mM) and an energy source for polypeptide synthesis, and was sensitive to chloramphenicol but not to cycloheximide. Under optimal conditions, the translation of Qβ RNA proceeded at a rate and to an extent equal to that obtained in a conventional E. coli system. Electrophoretic analysis of translation products made during incubation of C. burnetii extracts with polycistronic Qβ RNA revealed a major product with a molecular weight of about 14000; this product co-electrophoresed with the coat protein extracted from Qβ phage propagated in E. coli. The results suggested that the extracellular form of the rickettsia-like organism, C. burnetii, possessed the full array of components necessary for the initiation, elongation and termination of polypeptides.

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with the nematodes Neoaplectana and Heterorhabditis, occur in two forms. In general, only one form, designated the primary form, is transmitted into new hosts by the infective stage of the nematode. The significance of the relationship between the two forms has been examined with X. nematophilus, the symbiont of N. feltiae. The forms of X. nematophilus can be differentiated by their colony characteristics but by only two biochemical tests. The two forms of X.nematophilus are equally pathogenic when injected into the haemocoel of Galleria larvae. However, the primary form when injected into Galleria larvae with axenic nematodes provides better conditions for reproduction of the nematodes than the secondary form, for which a role has not been determined. Although the primary form readily converts to the secondary form in vitro and occasionally in vivo, the secondary form is usually stable. Possible causes of the instability have been investigated.

The specific growth rates at various temperatures of 12 bacterial species were measured and plotted as Arrhenius profiles. Temperature characteristics and optimum temperatures for maximum specific growth rates were estimated from these curves. The data reveal that one of two forms of the Arrhenius profile is characteristic of each bacterium: one curve is a simple smooth curve with a single predominant slope at sub-optimum temperatures; the other is a more complex curve with two distinct slopes at sub-optimum temperatures. The simple curve describes bacteria across the entire biokinetic range whereas the more complex curve occurs only with organisms which have optimum temperatures for replication above 37°C. Describing bacteria in terms of these forms of the Arrhenius profile is less arbitrary than is categorization based on optimum temperatures.

The cellular slime mould Dictyostelium discoideum constructs a succession of characteristic structures (grexes) in the multicellular phase of its life cycle. Compared with the wild-type strain, NC-4, grexes of a mutant strain, Ax-3, exhibited a profound prolongation of the early mound stage of development and a premature and exaggerated tendency to construct short, broad and bulbous forms. The aberrant Ax-3 phenotype was partially corrected by increasing the temperature of development, decreasing the plating density, perfusing with fresh air, or by introducing 0.1 mM-formycin B or 3-deazaadenosine into the agar. Mixtures of NC-4 and Ax-3 formed hybrid grexes which often became transformed into toruses by spiral centrifugal movement of the cells. Under other conditions, hybrid mounds developed into binary grex structures, cylindrical columns on top of hemispherical mounds, each component of which completed the morphogenetic sequence independently. The narrow upper structure appeared to be composed primarily of NC-4 cells. These novel patterns of morphogenesis support our hypothesis that the shape of the organism is controlled by the specification of the circumference of loops of cells within the grex.

The nucleotide sequence relationships between 18 species of Mycoplasma and 3 species of Acholeplasma were examined by solution DNA hybridization. Radiolabeled DNAs from strains representing 13 Mycoplasma and 2 Acholeplasma species were used as hybridization probes. The mycoplasmas were heterogeneous but displayed a low degree of conserved information of the order of 3 to 5% of the genome. However, several species within each genus showed 13 to 15% homology. There was no detectable homology between species from the two genera, and M. pneumoniae and M. neurolyticum appeared to be unrelated to any of the other Mycoplasma species or to each other. These results suggest that it may be possible to isolate genes common to most, if not all, Mycoplasma and Acholeplasma species.

Streptococcus faecalis var. zymogenes was grown aerobically on a complex medium containing glycerol as the carbon source. Addition of haematin or bovine liver catalase to the growth medium resulted in a small increment in growth yield. Suspensions of bacteria that had been grown in the presence of haematin or catalase, respectively, translocated 0·83 to 1·98 and 1·33 to 2·53 protons per oxygen atom consumed in glycerol oxidation. Bacteria grown without haematin or catalase had nil or little respiratory-induced proton translocation during glycerol oxidation. Inclusion of haematin in the growth medium caused the bacterium to form a cyanide- and azide-sensitive catalase. Superoxide dismutase activity was similar whether or not haematin was added to the growth medium.

The isolation and pathogenic response of various drug-resistant and auxotrophic mutants of strain PgB3 of Pseudomonas glycinea, the causal agent of bacterial blight of soybean, are reported. A mutagenesis procedure is described which allows efficient recovery of spontaneous, ethyl methanesulphonate-, hycanthone-, 2-aminopurine- and N-methyl-N′-nitro-N-nitrosoguanidine-induced mutants. While many of the auxotrophic mutants were avirulent, fully virulent histidine, adenine and methionine auxotrophic mutants were isolated. The virulence of certain methionine-requiring mutants did not correlate with the amount of methionine required by the mutant. In every instance where revertants to prototrophy were isolated, the revertant regained wild-type virulence.

Two mutants of Salmonella typhimurium were isolated which differ from their respective parental strains in their growth responses to guanine and xanthine. Both mutants had purine phosphoribosyltransferase activities similar to their parental strains. One mutant, CB-3, had a lower guanine uptake rate apparently caused by a genetic lesion in a specific gene (designated guaP) responsible for facilitating the transport of guanine. This gene mapped at 3·5 min in the sequence azi-guaP-nadC. The second mutant, GP103, had a purine carrier molecule with altered specificity, as demonstrated by a competition between hypoxanthine and xanthine for uptake.

Antisera were prepared in rabbits against whole organisms of colony type 1 Neisseria gonorrhoeae strains F62 and B (from gonococcal urethritis) and 7122 (a strain typical of those associated with disseminated gonococcal infection), and against purified outer membrane components from the same strains including pili and principal outer membrane protein. Antibody levels to pili, principal outer membrane protein and lipopolysaccharide were determined using a quantitative enzyme-linked immunosorbent assay. Each antiserum was heat-inactivated and tested for opsonic activity in a quantitative assay of phagocytosis. Each whole-organism antiserum was opsonic for its homologous strain, and this immune-enhanced phagocytosis was decreased by adsorption with homologous purified outer membrane components: pili ≫ lipopolysaccharide ≫ principal outer membrane protein. Opsonic activity was approximately equal for antiserum to purified pili and antiserum to the whole organisms for each of the three strains, and purified antibody to pili was highly opsonic. The F(ab′)2 fragments of antibody to pili were not opsonic, indicating a role for the Fc receptor on the phagocyte membrane in immune-enhanced phagocytosis of gonococci.

The major outer membrane protein patterns of 45 Escherichia coli strains of human origin were compared with that of E. coli K12 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Preparations of the former strains contained between two and five major bands in the molecular weight range between 30000 and 42000. The patterns were very heterogeneous with respect to the numbers and electrophoretic mobilities of the major outer membrane protein bands. In all cases the fastest moving band was characterized as a protein similar to the ompA protein of strain K12 as it was partially degraded by trypsin and reacted specifically with antiserum against the purified ompA protein in a gel immuno-radioassay. All the other major outer membrane proteins are related to the ompC and ompF proteins (the porins) of strain K12 as they were peptidoglycan-associated and reacted with antisera against the purified ompC and/or ompF proteins.

At low pH and with continuous low concentrations of hydrogen peroxide generated in situ, catalase was able to replace peroxidase in the peroxidase/hydrogen peroxide/iodide microbicidal system. The system was effective against Escherichia coli and Mycobacterium tuberculosis. Iodide could not be replaced by chloride. The system was effective in lactate buffer, but not in citrate/phosphate buffer. Strains of M. tuberculosis with high and low virulence were equally susceptible. The observations are discussed in the context of an involvement of host-cell catalase in a possible intracellular killing mechanism against M. tuberculosis.

From an O18ac:K1: H7 CoIV+strain of Escherichia coli (designated MW) that had caused meningitis in a human baby, mutant forms were isolated that lacked different combinations of its O, K and H antigens and its CoIV plasmid. These characters were also transmitted by conjugation to E. coli K12 and the virulence, immunogenicity and other properties of the different forms of both strains were studied.

All the forms of the MW strain that lacked either the O18 or K1 antigens or the CoIV plasmid, but not the H7 antigen, were much less virulent for chickens and mice than the parent form of MW. Another form derived by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment of the parent strain and that possessed all these four characters was also much less virulent. Some of the forms of the K12 strain to which the characters had been transferred were slightly more virulent than the K12 parent, but the virulence of all of them, including one possessing the O18 and K1 antigens and the CoIV plasmid, did not approach that of the MW parent.

Pathogenesis studies in chickens and colostrum-deprived calves revealed that the loss of virulence exhibited by the forms of the MW strain that lacked O18, K1 and CoIV and by the NTG-derived form was associated with decreased ability to invade the body. This was also the reason for the low virulence of the forms of the K12 strain that had acquired these characters.

Possession of both the O18 and K1 antigens was largely responsible for the ability of the different forms of the MW strain to survive in fresh chicken serum; organisms of K12 that possessed the K1 antigen survived as long as those of the parent form of the MW strain.

A substantial degree of immunity against lethal infection with the parent form of the MW strain was produced in chickens and mice by all the forms of the MW and K12 strains that possessed the O18 antigen. The K1 and H7 antigens and the CoIV plasmid produced no detectable immunity.

Saccharomyces cerevisiae S288C/1 was grown in a glucose-limited chemostat at population doubling times (τ) of 80 to 736 min. Estimates of the daughter cycle time (D), the parent cycle time (P) and the budded period (B) were obtained from bud scar analyses and equations derived from the Hartwell & Unger model of asymmetric cell division. D, P and B all showed biphasic linear relationships to τ, quantitatively different from estimates for the same strain in batch culture. Median cell volume and dry weight per cell increased at the faster growth rates, but the average cell density reached a minimum at τ = 150 min. The contiguous array of bud scars on parent cells became increasingly irregular as the doubling time increased from 140 min. At the fastest growth rates a small percentage of filamentous forms were observed due to failure of cells to undergo cell separation.

Acinetobacter calcoaceticus strain EBF65/65 harbours a cryptic plasmid, pAV2, which has been shown by electrophoretic separation on agarose gels to have a molecular mass of approximately 13.5 megadaltons (Md). Transfer of the previously described sex factor pAV1 (Hinchliffe & Vivian, 1980a, b) from the hospital strain JC17 into strains possessing pAV2 occurs only at a low frequency, whereas transfer to similar strains lacking pAV2 occurs at a much higher frequency. In EBF65/65, pAV1 may be present in strains possessing or lacking pAV2; pAV1 strains lacking pAV2 correspond to strains previously described as pAV1a (Hinchliffe & Vivian, 1980b) whereas pAV1 strains which also possess pAV2 correspond to pAV1b strains. The genetic evidence presented here is consistent with the hypothesis that pAV2 specifies a host restriction and modification system that is active against pAV1. Physical evidence from agarose gel electrophoresis indicates that pAV1 corresponds to a band of approximately 85 Md in strain JC17. The corresponding band in strains of EBF65/65 is difficult to distinguish because of the presence of a further cryptic plasmid band of approximately 88 Md, designated pAV3. A small cryptic plasmid of approximately 6 Md, designated pAV4, is reported for EBF65/65.

The naturally occurring plasmid pAV2 restricts the entry of the P class plasmid RP4 and the W class plasmids R388 and S-a into Acinetobacter calcoaceticus strain EBF65/65 from Escherichia coli. The W class plasmids only transfer from E. coli into pAV2− strains. Plasmid RP4 is modified in the presence of pAV2 such that it is no longer restricted on entry into pAV2 recipients of strain EBF65/65.

Transfer of RP4::Mu plasmids from Escherichia coli K12 to Acinetobacter calcoaceticus EBF65/65 was very inefficient compared with RP4 and was only detectable to strains of EBF65/65 lacking pAV2, a cryptic plasmid thought to code for a restriction/modification system. RP4::Mu-derived plasmids transferred from pAV2−strains of EBF65/65 back to E. coli K12 were found to carry defective prophages which had lost the abilityto produce detectable phage particles. Re-transfer of these defective plasmids from hsm k +and hsm k strains of E. coli K12 back to EBF65/65, when compared with the transfer of RP4, provided evidence for a second restriction/modification system in EBF65/65 which affected mainly Mu DNA. Using a mutant Mu prophage, Mu cts62 r23, it was possible to obtain RP4::Mu plasmids in EBF65/65 which were non-defective. These produced viable Mu particles when transferred back to E. coli K12 and could also be thermoinduced in EBF65/65; however, expression of Mu in EBF65/65 was very poor and plaques were only detected on a restrictionless strain of E. coli K12. ‘Plasmid suicide by Mu’ may enable the development of a method for directed chromosome mobilization in A. calcoaceticus.

Haploid strains of Kluyveromyces lactis were used to study the factors that influence mating ability. Analysis of sterol extracts of cells showed that ergosterol acts like a mating-control sterol, depending on the frequency of subculture and the content of intracellular Ca2+. Mating reaction was abolished in yeast cells placed in contact with the ionophore A23187 when Ca2+was absent from the medium. Glucose at concentrations above 1% in the medium repressed the mating process; this repression could be reversed by either dibutyryl cyclic AMP or caffeine. In starved cells glucose stimulated mating ability if provided in the presence of oxygen; in these experiments cyclic AMP and caffeine had no effect.

The kinetic properties of citrate synthase from Candida 107 were examined. Adenine nucleotides were inhibitory to enzyme activity, the order of efficacy being ATP > ADP > AMP. Inhibition by ATP was competitive with respect to acetyl-CoA and mixed with respect to oxaloacetate. Combinations of adenine nucleotides giving simulated energy charge values were also inhibitory, though the total adenine nucleotide concentration was of greater significance than the relative proportions of each in determining the degree of inhibition. When Mg2+was added at a concentration sufficient to saturate the adenine nucleotides, the inhibition was almost entirely relieved. The apparent absence of any rigorous control of citrate synthase by adenine nucleotides in oleaginous micro-organisms is consistent with previous observations that the flow of carbon through the glycolytic and pentose phosphate pathways to pyruvate and thence to citrate should be uninterrupted during the process of lipid accumulation.

Both a penicillinase and a cephalosporinase are present in a strain of Citrobacter freundii (GN7391) resistant to β-lactam antibiotics. The penicillinase was identical to the type Ia penicillinases (Type III by Richmond classification), mediated by Rms212 and R-TEM. A cephalosporinase, typical of enterobacteriaceae chromosomal β-lactamase (Type I by Richmond classification), was purified from the strain. It gave a single protein band on polyacrylamide gel electrophoresis and immunoelectrophoresis; the pI was 8·6 and its molecular weight was approximately 38000. Cysteine was not found among its amino acids. The specific activity was 388 units (mg protein)−1for the hydrolysis of cephaloridine, and the optimal pH was 8·0. Rabbit antiserum obtained against the purified enzyme showed cross-reaction with cephalosporinases produced by strains of Enterobacter cloacae in a neutralization test.