- Volume 103, Issue 2, 1977

The metabolism of 5-[Me-14C] methyltetrahydrofolate in Lactobacillus casei proceeded oxidatively with incorporation of label into purine and thymidylate derivatives. No labelled methionine was formed. (l)-5-MethyltetrahydrofoIate, the natural isomer, was not a substrate for the L. casei folylpoly-γ-glutamate synthetase although the unnatural (d)-isomer was slowly metabolized to the diglutamate form.

Oxidation of 5-methyltetrahydrofolate to 5,10-methylenetetrahydrofolate was the rate-limiting step in 5-methyltetrahydrofolate metabolism by Lactobacillus casei. The limiting steps in the utilization of suboptimal levels of folate by L. casei were related to the ability of folates to function in purine and/or thymidylate biosynthesis. Folates with glutamate chains of up to at least seven residues were substrates for these biosynthetic enzymes, and comparisons of bacterial growth yields with transport rates for these folates indicated that the polyglutamates were more effective substrates in purine and thymidylate synthesis than the corresponding pteroylmonoglutamate S. Lactobacillus casei contained low levels of a B12-independent, pteroylpolyglutamate-specific methionine synthetase. Its methylene-tetrahydrofolate reductase also functioned more effectively with pteroylpolyglutamate substrates.

Multiple transport systems for l-aspartic acid exist in Streptomyces hydrogenans. The intracellular accumulation of l-aspartate against a concentration gradient was immediately inhibited by proton conductors, such as carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone, 2,4-dinitrophenol or nigericin. Transport activity was gradually lost when inhibitors of protein synthesis were added. l-Aspartate transport had two pH optima at 6·5 and 4·5. At pH 6·5, two saturable transport components with different K m and V max values could be resolved by kinetic studies. A high-affinity system (system I) preferred the l-isomers of the anionic forms of aspartic and glutamic acid. At the same pH, a second, low-affinity system (system II) operated, which was presumably less specific than system I and also able to accept, at high concentrations, neutral amino acids. At pH 4·5, the Lineweaver-Burk plot revealed only a single catalytic component, with K m and V max values similar to those of system II. Again, in contrast to system I, this component showed high affinity for neutral amino acids. The data suggest that l-aspartic acid and l-glutamic acid are transported by this system as neutral zwitterionic molecules.

The isoelectric points of three proteases (I, II and III), separated from culture supernatants of Pseudomonas aeruginosa strain paks-1 by isoelectric focusing, were 8·5, 6·6 and 4·5 respectively. Collagenase activity was not detected. More than 75% of the extracellular protease activity of this strain was due to protease II. This enzyme also possessed elastase activity. When purified by ammonium sulphate precipitation, isoelectric focusing and gel chromatography, protease II showed one band on disc electrophoresis and one band on conventional immunoelectrophoresis. The pH optimum, stability and effect of inhibitors and substrate concentration were examined. The molecular weight was 23000 ± 5000. Protease II was lethal for mice when injected intraperitoneally at a high dose (minimum lethal dose 0·1 mg). Dermonecrosis and subcutaneous haemorrhages were produced in new-born mice upon subcutaneous injection of 10 μg protease II. A sensitive test for cytotoxicity showed no evidence of cytoplasmic membrane damage to HeLa cells or human diploid embryonic lung fibroblasts by protease II. Morphological changes similar to those produced by trypsin were found.

Mutants of Pseudomonas aeruginosa strain paks-1 which are defective in the formation of extracellular protease activity have been characterized. The mutants produced between approximately 1 and 25 % of the protease activity of the wild type and no strains completely lacking extracellular protease were found, even after repeated mutagen treatment. Most mutants also had changed activities of extracellular staphylolytic enzyme, lipase and lecithinase. Four of 13 mutants were unable to release alkaline phosphatase and staphylolytic enzyme into the medium in contrast to the wild type. Serotype, phage type and biochemical reactions were essentially unchanged. The results indicate that some of the mutations affected the cell envelope structure or function leading to decreased ability to release extracellular proteins, and that other mutations possibly affected a common regulatory mechanism for extracellular enzymes.

Antigens specific for Lactobacillus acidophilus were investigated by double immunodiffusion in agar-gel. Antigenic materials were extracted from whole bacteria and some walls with cold trichloroacetic acid. Antisera were prepared by intravenous injection into rabbits of suspensions of whole organisms in solutions of bovine serum albumin, which had been heated and then washed. Four specific antigens were found as precipitinogens and denoted as antigens 11, 12, 13 and 14. Of 43 strains of L. acidophilus studied, 33 strains possessed antigen 11, six strains antigen 12, two strains antigen 13 and two strains antigen 14. Sugar compositions of wall preparations were analysed in an attempt to characterize the determinants of antigens 11 and 12. The walls contained glucose, galactose, hexosamine and sometimes glycerol, but no rhamnose was found. It was considered that α-glucopyranose was the major component of the determinant of antigen 11 since trehalose and maltose significantly inhibited the reaction between antibody 11 and its antigen; the determinant of antigen 12 was not clarified.

Four of nine stock cultures of Agrobacterium tested formed mixtures of two types of colonies, which were distinguished on plates of aniline blue medium because of the difference in their production of two exocellular polysaccharides. The ability of organisms from the white colonies of strain ifo12665 to produce large amounts of water-soluble polysaccharide was unstable; when kept on nutrient agar slants or in inorganic salt solution the bacteria mutated spontaneously to yield blue colonies producing large amounts of curdlan-type polysaccharide instead.

The mode of transport of the complex toxin molecule of Vibrio cholerae (which has a mol. wt of 84000 and consists of several subunits) across the inner and outer membranes of V. cholerae is not known. In this study we found two peptides in the outer and inner membranes of V. cholerae which may be the form in which the toxin subunits are transported across the membrane. We examined two growth conditions: aerobic growth at 37 °C, when most of the synthesized toxin is membrane-bound; and anaerobic growth at 37 °C, when little toxin remains membrane-bound, the toxin being released into the growth medium. When V. cholerae was grown aerobically at 37 °C, the outer and the inner membranes contained two peptides with mol. wts of approximately 22000 and 6000 which were not found in the outer or the inner membrane of anaerobically grown cells. Sodium deoxycholate, which releases membrane-bound toxin, released several peptides including the 22000 and the 6000 mol. wt peptides. Trypsin also released the 22000 and 6000 mol. wt peptides. Purified cholera toxin had three kinds of peptides, of mol. wt 21000 (A1 peptide), 11000 (B subunit) and 5000 (A2 peptide). We postulate that the membrane peptides may be precursors of the A subunit of the toxin molecule.

Cell extracts of Saprolegnia monoica Pringsheim contained enzymes which synthesized glucans from uridine diphosphoglucose. Both MgCl2 and cellobiose stimulated enzyme activity. The pH optimum was about 5·8. High substrate concentrations increased the proportion of alkali-insoluble glucans. Paper chromatography of cellulase-hydrolysed alkali-insoluble glucans revealed cellobiose indicating the presence of a (1→4)-β-glucan (cellulose) synthetase. Glucan synthetases were mainly located in the wall and microsomal fractions. They were more active in branched hyphae than in unbranched mycelium.

Subcellular fractions were characterized by density gradient ultracentrifugation, enzymic tests and electron microscopy. Synthetase activities were associated with dictyosomes which, by histochemical staining, were shown to contain (1→4)-linked polysaccharides. The results were consistent with the current interpretation of the role of the Golgi apparatus in hyphal morphogenesis.

Transduction of resistance from a multiply antibiotic-resistant strain of Staphylococcus epidermidis sub-group II was studied using the typing phage 108. The effect of increasing doses of ultraviolet radiation on the transducing phage was used to indicate the chromosomal or plasmid nature of the genes. Tetracycline and chloramphenicol resistance behaved as plasmid genes and streptomycin resistance as a chromosomal marker. It was also possible to transduce penicillin resistance (Pc) due to penicillinase production (bla +) using a low level of benzylpenicillin (0·03 μg ml−1) for recovery. Approximately 10−5 transductant colonies per phage input were obtained and ultraviolet kinetics indicated that Pc was plasmid carried. Pc transductants fell into two categories. In one group Pc was stable as in the donor strain and transductants had the same phage sensitivity as the recipient. In the other, Pc was unstable at 37 °C and the instability was enhanced by growth at approximately 43·5 °C; these transductants also gained genes for restriction and modification of certain phages. Transductants that subsequently lost bla + also lost the restriction and modification characters.

The ampicillin-resistant isolate Haemophilus influenzae KRE5367 which produced a TEM type β-lactamase contained a 30 megadalton (Mdal) plasmid (pKRE5367) with a copy number of approximately 2 per chromosomal equivalent and a mole fraction guanine plus cytosine content of 0·39. Ampicillin resistance was transferred by cell-to-cell contact, and the transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pKRE5367 DNA proved that the structural gene for the TEM type β-lactamase resided on the plasmid genome. It was shown by molecular hybridization that pKRE5367 probably contained the ampicillin translocation DNA segment (TnA) found on some R factors of enteric origin. The H. influenzae plasmid pKRE5367 had most of its polynucleotide sequences in common with other R factors recently isolated from H. influenzae. DNA-DNA hybridization studies indicated that pKRE5367 shared about 70 % base sequence homology with the two tetracycline-resistance R plasmids pLU 121 (31·5 Mdal) and pFR 16017 (33 Mdal) recently isolated in W. Germany. In addition, pKRE5367 had nearly 100 % of its polynucleotide sequences in common with the 30 Mdal ampicillin-resistance R plasmid RSF007, isolated in the U.S.A.; it shared 70 % base sequence homology with the 31·5 Mdal tetracycline-resistance plasmid pUB701, isolated in the U.K.; and it had 65 % of its polynucleotide sequences in common with the 38 Mdal tetracycline- and chloramphenicol-resistance R plasmid pR1234 isolated in the Netherlands. Despite the broad geographical range, the R plasmids of H. influenzae appeared to be closely related.

Thiamin-deficient mutants of Bacillus subtilis were characterized by their growth responses to the pyrimidine and thiazole moieties of the vitamin molecule and by cross-feeding tests. All mutants growing on the thiazole moiety and all mutants with an absolute requirement for thiamin fed all those growing on the pyrimidine moiety. No other cross-feeding effects were observed. From the culture fluid of a mutant growing on the thiazole moiety, two compounds were isolated which supported growth of mutants requiring the pyrimidine moiety. These compounds were identified by chromatographic, bioautographic and spectrophotometric procedures as 4-amino-5-hydroxymethyl-2-methylpyrimidine and its monophosphate derivative.

The shapes of tips of rapidly growing hyphae approximate more closely to half ellipsoids of revolution than to hemispheres. It is concluded that provided growth is isotropic the specific rate of wall area expansion will be more nearly proportional to the cotangent of the position angle than to its cosine. Certainly better qualitative fits to the observed data are given by cotangent rather than by cosine curves.

Adding Chloramphenicol to cultures of Schizosaccharomyces pombe, growing exponentially on glycerol as carbon source, resulted in a heterogeneous population of abnormally large cells. After removal of the inhibitor and resuspension in fresh medium, the cells increased in size and the cell volumes became more normally distributed. Cells then underwent two synchronous divisions separated by 5 h; this is significantly shorter than the doubling times for culture extinction and total cell volume which are both about 7 h. There was an overall decrease in mean cell volume. The chloramphenicol-induced cell cycle was shorter than the first complete cell cycle in synchronous cultures prepared by size-selection.

Dialysis culture was used to investigate the extent to which growth inhibition in bacterial cultures may be caused by accumulation of metabolites. Escherichia coli B was grown in a glucose/salts medium. A concentrated nutrient solution was pumped at a constant rate into the growing culture to ensure that growth was not limited by exhaustion of nutrients. In this way the only difference between growth conditions in dialysis and non-dialysis cultures was the transfer of dialysable metabolites from the culture vessel to the reservoir in the dialysis culture system. By adjusting the glucose concentration in the feed and maintaining a constant rate of feeding, glucose-limited growth could be achieved. Under these conditions, with oxygen in excess, bacterial yields of 140 to 150 g dry wt 1−1 were obtained in dialysis culture compared with 30 to 40 g 1−1 in non-dialysis culture. The high yields in dialysis culture depended on the removal of end-products of glucose metabolism. Growth inhibition was demonstrated to be the result of the combined influence of acetate, lactate, pyruvate, succinate, propionate and isobutyrate in concentrations found at the end of growth in non-dialysis cultures of Escherichia coli b.

Yields of Escherichia coli B grown on glucose were determined in dialysis and non-dialysis culture. The molar growth yields were compared under conditions of excess glucose and oxygen as well as glucose- and oxygen-limiting conditions. The molar growth yields on glucose (Y G) were determined for different periods during growth in non-dialysis cultures. A rapid decrease of Y G was observed and growth ceased even in the presence of high concentrations of glucose and dissolved oxygen in the culture liquid. The decrease in Y G was delayed in dialysis cultures where a high Y G could be maintained at very high cell concentrations. The inhibition of growth depended on the accumulation of end-products of fermentative degradation of glucose. These products interfered with the oxidative phosphorylation. A large proportion of the glucose was fermented even in the presence of high concentrations of dissolved oxygen in the culture liquid. A decrease in the growth yield per g glucose was also observed.