- Volume 128, Issue 12, 1982

Intact cells of the methane-oxidizing organism Methylococcus capsulatus (Bath) assimilated CO2 for several hours in the absence of methane, provided that an alternative energy source such as hydrogen and/or formate was available. Despite the presence of ribulose bisphosphate carboxylase and a ribulose monophosphate pathway in this organism, autotrophic growth in the presence of a suitable energy source could not be demonstrated. Radiolabelling studies suggested that the CO2 was incorporated via C3 carboxylation and ribulose bisphosphate carboxylase in the presence of both methane and hydrogen plus formate. However, in the presence of methane the CO2 was further metabolized into sugar phosphates, whereas in the absence of methane, but with hydrogen plus formate as an energy source, the sugar phosphates were not labelled to any significant extent. Of the methane-oxidizing bacteria tested, ribulose bisphosphate carboxylase was found only in M. capsulatus strains (Bath and Foster & Davis). The ribulose monophosphate pathway in M. capsulatus (Bath) probably uses the keto-deoxy-6-phosphogluconate route for the cleavage of fructose 6-phosphate into two C3 molecules rather than via phosphofructokinase and fructose bisphosphate aldolase which have low activities in this organism. The function of the ribulose bisphosphate carboxylase may be to provide an alternative cleavage pathway for the synthesis of 3-phosphoglycerate during growth on methane.

Azospirillum brasilense strain Cd was grown in a medium containing NH4 + in a chemostat at a range of constant dissolved oxygen tensions (d.o.t.) (0·070–0·18 atm). Poly-β-hydroxybutyrate (up to 12% of the cell dry weight) increased under oxygen limitation and moderate dilution rate (D = 0·14 h−1). The highest carotenoid content was observed at high d.o.t. and dilution rates up to 0·12 h−1. The amount of protein varied with d.o.t. from 0·29 mg protein (mg dry wt)−1 at 0·007 atm to 0·54 mg at 0·18 atm. The yield efficiency and respiration rate were highest at low d.o.t. and decreased significantly at a d.o.t. of 0·18 atm. Succinate dehydrogenase and malate dehydrogenase activities increased 2·5-fold at 0·10-0·18 atm O2, whereas succinate oxidase and NADH oxidase activities increased consistently with increasing d.o.t. Azospirillum brasilense showed a low specific activity for catalase; the specific activity of superoxide dismutase increased sharply above 0·16 atm O2.

Cerulenin at a concentration of 10 g ml−1 only slightly inhibited growth of Bacillus subtilis YY88 while at 50μg ml−1 it inhibited the growth rate by 20–35% in complex media, and by 50–60% in mineral medium containing a single carbon source. Cerulenin (50μg ml−1) only partially prevented the secretion of α-amylase, proteases and levansucrase when added to bacteria growing in complex medium, but completely prevented the secretion of these enzymes in the mineral medium. Lower concentrations of cerulenin (10μg ml−1) inhibited the secretion of the enzymes in the mineral medium by up to 40%. When the cells were incubated with l-[35S]-methionine (plus a complete amino acid mixture) in the presence of cerulenin (50μg ml−1) protein secretion was lowered by 68%. Cerulenin inhibited translocation of membrane-bound forms of the secreted enzymes to 18% of the control value and the incorporation of radioactive methionine into membranes to 46%. These results suggest that the membrane-bound forms of secreted proteins play an important role in the secretion process in B. subtilis.

The fate of radiolabeled cyanide added to washed non-proliferating suspensions of the cyanogenic organism Chromobacterium violaceum was studied. Within 6 h of addition, over 90% of the added cyanide had been metabolized to other compounds, except when l-glutamate alone was included in the suspension medium; in this case only 50% of the added cyanide had been metabolized after 6h incubation. Less than 2% of radioactivity was incorporated into cell material; the rest remained in the suspension medium. The only identified compound into which radioactivity accumulated was β-cyanoalanine. Formation of β-cyanoalanine was stimulated by inclusion of l-glutamate and either O-acetyl-l-serine or l-serine in the incubation medium. Under optimal conditions, approximately 50% of added radiolabel accumulated in β-cyanoalanine. Compounds capable of supplying carbon for growth of C. violaceum were able to substitute, to some extent, for l-glutamate in stimulating β-cyanoalanine synthesis. Both O-acetyl-l-serine and l-serine inhibited β-cyanoalanine formation when included at concentrations in excess of 3 mm. l-Cysteine, l-threonine and l-alanine were unable to substitute for O-acetyl-l-serine or l-serine, and l-cysteine inhibited β-cyanoalanine synthesis. l-Methionine inhibited β-cyanoalanine formation by cells resuspended in cyanide, l-glutamate and O-acetyl-l-serine or l-serine. The effect of growth conditions on the ability of resuspended cells to synthesize β-cyanoalanine was studied.

By using plasmid pTB90, penicillinase genes, penP and penl, from both the wild-type and constitutive strains of Bacillus licheniformis 9945A were cloned and expressed in a thermophile Bacillus stearothermophilus. Bacillus stearothermophilus carrying the recombinant plasmid produced a large amount of penicillinase [3300 U (mg cells)−1] at 48 C, whereas the amount of enzyme produced at 55 C and 60 C was reduced to 660 U (mg cells) −1 and 39 U (mg cells) −1, respectively. Active repressor for penicillinase functioned normally. About 10 to 20% of the total penicillinase was secreted into the culture medium by the transformant of B. stearothermophilus.

The chemical and functional stability of Klebsiella pneumoniae mRNAs was dependent on the nitrogen status of the organism. During exponential growth on N2 or NH4 + as N-source the half-lives of mRNAs were 10 and 4 min, respectively. When NH4 +-grown cells were starved of NH4 + for 3 h, under which conditions the nitrogen fixation (nif) genes were derepressed, the half-life of mRNAs increased to 20 min. Addition of NH4 + to such N-starved suspensions rapidly destabilized mRNAs with a resultant half-life of 9 min. This destabilization occurred even in the presence of transcription inhibitors, which indicated metabolic control of mRNA degradation. Under most conditions studied the functional decay of nif-specific mRNAs paralleled the decay of bulk mRNAs.

Growth of Clostridium perfringens with low concentrations of benzylpenicillin inhibited septum formation and division of the organisms. This resulted in continued growth of the organisms as aseptate filaments. The effect was reversed on removal of the antibiotic. The composition of walls isolated from organisms grown with the antibiotic was similar to that of walls from untreated bacteria. In addition, both contained non-N-acetylated glucosamine residues in their peptidoglycan. No differences were detected in the degree of cross-linkage of peptidoglycan. Clostridium perfringens contains six membrane-associated penicillin-binding proteins (PBPs) which have different affinities for [3H]benzylpenicillin. Concentrations of the antibiotic which were sufficient to cause filamentation of apparently all organisms in a culture caused almost complete saturation of PBPs 3, 4, 5 and 6. At these concentrations there was no measurable interaction with PBPs 1 and 2. Thus interaction of the antibiotic with the lower molecular weight PBPs is correlated with the inhibition of septum formation in C. perfringens.

Bacillus cereus consistently formed multiple growth bands in gel-stabilized gradient systems. The development of inoculated and uninoculated systems was followed by gel scanning and by measuring physico-chemical parameters. Factors which influenced band formation included the availability of oxygen and the presence of a glucose gradient. The relative position of bands was dependent on the glucose concentration in the source layer and the strength of the basal medium. Other factors such as agar type and concentration were relatively unimportant. The generation of steep pH gradients in the gel profoundly affected band formation. Thus, in the presence of a high buffering capacity changes in band pattern and pH profile occurred. No bands and no pH gradient were observed in gels incubated anaerobically. Addition of an alkaline overlayer, however, resulted in a pH gradient and band formation. Although pH gradients were responsible for sub-surface bands, it was not clear what caused the ‘chopping’ of growth into discrete bands. Computer simulations suggested that an asymmetric activation threshold may be responsible, and the mechanism may involve sporulation and germination.

The malate dehydrogenase (MDH) electrophoretic mobilities of 128 strains of bacteroides belonging to 17 species, including three subspecies of Bacteroides melaninogenicus and two subspecies of Bacteroides ruminicola, were examined. Amongst the pigmented bacteroides, the migration of this enzyme correlated well with recognized taxa, and only one strain, VPI 9085 was clearly different. Other species such as B. oralis, B. buccalis, B. denticola, B. pentosaceus, B. bivius, B. disiens and B. ruminicola were delineated by the combined use of MDH and glutamate dehydrogenase. Forty-three strains belonging to the ‘B. fragilis group’ differed from the above species in possessing glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and reference strains as well as fresh isolates were assigned to the correct species by the mobility pattern of these two enzymes.

Other properties of MDH such as the pH optima for the oxidation of malate or the reduction of oxaloacetate were of limited taxonomic value. However, the alkaline stability of this enzyme at pH 9, 10 and 11 clearly differentiates the saccharolytic from the non-saccharolytic species of pigmented bacteroides with the latter showing highly stable enzymes with a half life greater than 50 min.

The indirect haemagglutination reaction was evaluated in the classification of conjugative plasmids. A simple and sensitive method was worked out using pili and in some cases whole bacteria as antigens. Antibodies were prepared against pili coded for by plasmids from incompatibility groups IncFI, IncFII, IncIα and IncN. The antisera were tested against pili from 35 strains harbouring plasmids. The test differentiated clearly between plasmids from unrelated incompatibility groups, whereas cross-reaction occurred with closely related groups such as FI and FII. Minor antigenic variation could be seen within the IncFII group.