- Volume 88, Issue 1, 1975

SUMMARY: Polyacrylamide gel electrophoresis and isoelectric focusing techniques have been used to compare NAD-dependent l(+)lactate dehydrogenases (LDH) from ten different strains of Mycoplasma mycoides var. mycoides. The enzymes were not distinguished from one another, or from normal bovine LDH 1 by these methods.

The kinetic behaviour of LDH from M. mycoides (t 1 vaccine strain) suggested that the enzyme could readily reduce pyruvate or oxidize lactate in a manner which, in vertebrates, requires two different isoenzymes.

SUMMARY: Polyphosphate kinase, an enzyme which incorporated the γ-phosphate of ATP into long-chain polyphosphate molecules, was purified more than 700-fold from Arthrobacter atrocyaneus by ammonium sulphate fractionation, DEAE-cellulose column chromatography and Sephadex G-200 gel filtration. The enzyme had a broad pH optimum at 6·0 to 7·0 and required Mn2+ or Mg2+, histone, and inorganic phosphate for activity. The Km for Mn-ATP was 0·53 mm, and for inorganic phosphate was 1·67 mm.

Free ATP concentrations greater than 8 μ m inhibited the enzyme. Free Mn2+ or Mg2+ concentrations greater than 2 mm or 6 mm, respectively, were also inhibitory. Activity was strongly inhibited by 4 mm-ADP, 1 mm-PPi or 20 mm-NaF. The effect of ADP might have resulted from reversing the equilibrium of the kinase reaction. The activation by phosphate ions might indicate a role for the enzyme in regulating intracellular phosphate levels or maintaining a phosphorus reserve. The level of enzymic activity in the bacteria responded to changes in inorganic phosphate concentration in the medium. Basic proteins, such as protamine, could substitute for histone as activator. Proteins such as casein or bovine serum albumin would also substitute for histone but only in the absence of inorganic phosphate. The presence of a protein might be necessary to form a complex with the product, thus preventing reversal of the reaction in vitro.

The reaction product was characterized, and found to be labile in hydroxylamine, base, and acid at 100 °C. It behaved as a long-chain-polyphosphate molecule on chromatography in an Ebel’s solvent. The enzymic activity was therefore not that of a protein kinase.

SUMMARY: Two bacteriocins were found in the supernatant fluid and in an extract of Streptococcus faecium strain E1. The small soluble enterocin E1A represented more than 90 % of the total activity in the supernatant fluid, and was purified 400-fold by ammonium sulphate fractionation, gel filtration on Sephadex G-75 and chromatography on DEAE-cellulose. Enterocin E1B, with a particle weight greater than 4 × 106, was the predominant type in the extract. It was released in appreciable quantities after breakage of the bacteria and was purified 100-fold by differential centrifugation, chromatography on Sepharose 4B and density gradient ultra-centrifugation. Enterocin E1A, a basic substance with a molecular weight of about 10000, was resistant to heat and was attacked by trypsin, whereas enterocin E1B was less thermostable and insensitive to proteolytic enzymes. The activity of enterocin E1B was unchanged by treatment with DNAase. Sensitivity to enterocin action was confined to certain strains of various enterococcus species, Streptococcus salivarius and Listeria monocytogenes; all the other Gram-positive and Gram-negative bacteria tested for sensitivity were unaffected by either enterocin.

SUMMARY: A study has been made of the intracellular changes occurring during thymineless death in Bacillus subtilis 2337. The kinetics of death were paralleled by the rate of breakdown of DNA. During thymineless death, single-strand breaks accumulated within DNA, breakdown of approximately 13% of the DNA to acid-soluble material occurred, deoxyribonuclease levels rose sharply, and yet double-strand breaks did not occur in the DNA. On restoration of thymine, however, double-strand breaks accumulated, though this could be prevented by the specific inhibition of DNA replication on restoration of thymine. The results probably indicate that during thymineless death, single-strand gaps accumulate within the DNA of cells. On restoration of thymine both DNA replication and repair of gaps are simultaneously initiated, and when a replication fork reaches a gap before it is repaired, double-strand breakage of the DNA occurs. The possible relevance of these events to the lethality of the cells is discussed.

SUMMARY: Lipopolysaccharides, extracted by phenol-water from five strains of Neisseria gonorrhoeae, were purified by treatment with ribonuclease followed by multiple washes. These preparations were fatal to mice when administered in submicrogram amounts with actinomycin D, the LD50 values varying from 4 to 16 μg/kg. Analyses showed that all preparations contained glucose, galactose, glucosamine, heptose, 2-keto-3-deoxyoctonic acid and phosphate. All the lipopolysaccharides contained the same fatty acids, namely β-OH-10:0, β-OH-12:0,β-OH-14:0, 12:0, 14:0 16:0, 16:1, 18:0 and 18:1. We were unable to detect significant differences between the lipopolysaccharides of virulent and avirulent gonococci or between penicillin-sensitive and resistant strains. Gonococcal lipopolysaccharides appeared to lack O-antigen side chains.

SUMMARY: A soluble extract from Corynebacterium poinsettiae able to synthesize the nucleotide precursor of its peptidoglycan was prepared. This extract contained all the enzymes necessary for the synthesis of the peptide side-chain. The specificity of these enzymes was determined and compared with the specificity of similar enzymes extracted from the closely related Corynebacterium insidiosum. In both organisms, addition of the third amino acid of the peptide side-chain was specific for the amino acid and nucleotide dipeptide involved in peptidoglycan synthesis in the parent organism. l-Diaminobutyric acid, which is found as the acetyl derivative in the precursor nucleotide and in the completed peptidoglycan of C. insidiosum, was added as the free amino acid and not as the acetylated compound.

SUMMARY: β-Lactamases (EC. 3.5.2.6) from strains of Gram-negative bacteria have been studied using analytical isoelectric focusing. This permits a visual comparison of the patterns of β-lactamase bands produced by enzymes from different organisms. Purification of crude intracellular preparations is unnecessary and the technique is sufficiently sensitive to demonstrate β-lactamase in mutants previously reported to lack the enzyme. R factor RTEM and RP1 β-lactamases that have not been distinguished from one another biochemically or immunologically can be differentiated by isoelectric focusing. Conversely, the enzymes specified by the R factors RTEM, R1 and RGN14, with identical isoelectric focusing patterns, have the same biochemical properties. Chromosomal and R factor-mediated β-lactamases from single strains have been separated and their identities confirmed by immunoisoelectric focusing. R factor-mediated enzymes gave identical isoelectric focusing patterns irrespective of the host strain. Isoelectric focusing can therefore be used to observe the transfer of β-lactamases carried by R factors.

The wide host-range antibiotic resistance plasmid RP1 was transferred from Pseudomonas aeruginosa via Escherichia coli into Pseudomonas glycinea. The plasmid was then acquired by Pseudomonas phaseolicola both in vitro and in planta in Phaseolus limensis leaves and pods. This was the first step in the design of a model system to determine the possible epidemiological significance of antibiotic resistance plasmids in the control of plant disease.

SUMMARY: Six auxotrophic markers of a halotolerant collagenolytic strain of Achromobacter were transduced by four α phages. Abortive transduction was also demonstrated. The generalized transduction system is unusual as the transductants were unstable, characteristic of transduction by lysogeny. The Achromobacter strain is a cryptic lysogen for α and purified transductants were either sensitive or resistant to α. Purified clones from four resistant transductants and one sensitive transductant liberated phage spontaneously. The host ranges of these spontaneous phages differed from that of the α phage used for the transduction experiment. Some initially resistant transductants became semi-sensitive to α (efficiency of plating [e.o.p.] 10−1 to 10−2) after repeated cloning.

SUMMARY: Eighty-one strains of Staphylococcus aureus that appeared to be tetracycline resistant on the basis of a preliminary disc-diffusion test were examined for resistance to tetracycline and to the semi-synthetic tetracycline, minocycline. Minimum inhibitory concentration (m.i.c.) values for both drugs were determined after induction of the strains by growth for 2 h in sub-inhibitory concentrations of tetracycline. Forty-seven strains (58%) had m.i.c. values for minocycline of 12·5 μg/ml or greater, and were considered to be minocycline resistant. An additional ten strains had m.i.c. values for minocycline of 3·125 to 6·25 μg/ml and were classified as low-level resistant strains. It appears, therefore, that a fairly high proportion of tetracycline-resistant strains isolated at the present time are resistant to concentrations of minocycline unattainable in vivo with the recommended dosage for this antibiotic (Frisk & Tunevall, 1969).

Transductional analysis of the genetic determinants for tetracycline resistance revealed the existence of two types of resistance to high concentrations of tetracycline. Strains in the first category (A) were inducibly resistant to tetracycline but sensitive to minocycline; in these strains the resistance determinant was plasmid-borne. Strains in the second category (B) were resistant to both tetracycline and minocycline and had low induction ratios for tetracycline resistance; the genetic determinant for resistance in these strains was chromosomal. In addition, certain strains in category A were found to carry a chromosomal gene controlling low-level resistance to tetracycline and minocycline. This low-level resistance to tetracycline was masked in the presence of the tetracycline plasmid but could be demonstrated after loss of the plasmid. The results suggest that more than one mechanism of resistance to tetracyclines may exist in staphylococci.

SUMMARY: The molar growth yields of Pseudomonas denitrificans, for nitrate, nitrite and nitrous oxide, were determined in chemostat culture under electron acceptor-limited conditions. Glutamate was used as the source of energy, carbon and nitrogen. The catabolic pattern was identical, irrespective of the terminal electron acceptors.

The molar growth yields, corrected for maintenance energy, were 28·6 g/mol nitrate, 16·9 g/mol nitrite and 8·8 g/mol nitrous oxide. The energy yield, expressed on an electron basis, was proportional to the oxidation number of the nitrogen: nitrate (+5), nitrite (+3) and nitrous oxide (+1). It was concluded that oxidative phosphorylation occurs to a similar extent in each of the electron transport chains associated with the reduction of nitrate to nitrite, nitrite to nitrous oxide and nitrous oxide to nitrogen.

SUMMARY: An obligate osmophilic mutant (strain B1/4) of Saccharomyces rouxii has been isolated that fails to grow at osmotic pressures corresponding to 20% (w/v) sucrose or less. In 30% sucrose the yeast is filamentous and grows slowly. In 40% sucrose it is mainly filamentous and has over twice the normal diameter. In 60% sucrose it grows in the yeast form with a growth rate twice that of the culture in 40% sucrose. This mutant is lysed by a sudden drop in the osmotic pressure of the environment. Cell envelopes of the parent strain contained glucose and mannose in the ratio 1.2:1 and contained 3·8% (w/v) hexosamine, whereas the envelopes of the mutant contained 0·8% hexosamine. Cell envelopes of the mutant grown in 40% sucrose contained glucose and mannose in the ratio 1·9:1, whereas for envelopes of the yeast grown in 60% sucrose the ratio was 1·2:1. Neutral lipids from whole cells and those from the envelopes of the mutant strain generally contained more unsaturated fatty acids than the corresponding fractions from the parent strain.

SUMMARY: Large-scale synchronous cultures of Crithidia fasciculata were prepared by a sedimentationvelocity-size-selection method using a non-osmotic gradient in a zonal rotor. Synchrony indices of up to 0·68 were obtained (average length of cell-cycle, 5·1 h). Dry weight, protein and RNA increased continuously so as to double in one cell-cycle. Rates of oxygen uptake/ml culture increased overall so as to double over a cell-cycle, but rose to maxima at five periods in the cell-cycle. KCN gave a similar degree of inhibition throughout, and so did not alter the periodicity or amplitude of the oscillations. Total adenylates doubled over a cycle, and complex changes in pool sizes of ATP, ADP and AMP were temporally interrelated and were correlated with changes of oxygen uptake rates rather than with changes in biosynthetic requirements. Adenylate charge varied between 0·47 and 0·66. Discontinuous respiratory activity of mitochondria through the cell-cycle and possible mechanisms for its control are discussed with reference to previous data.

SUMMARY: The separation of the smallest cells from an exponentially growing culture by passage through a continuous flow centrifuge rotor under controlled conditions of rotor speed and flow rate, provides a simple method for the rapid production of large-scale synchronous cultures. The effluent from the rotor, containing the smallest-sized class of organisms, still suspended in their growth medium and still growing undisturbed throughout this rapid procedure, provides a culture which goes on to exhibit synchronous cell division. Successfully applied to budding and fission yeasts and to amoeboid and ciliated protozoa, this procedure is potentially applicable to any non-filamentous, non-aggregating, unicellular organism or to cells of higher plants or animals growing in liquid tissue-cultures.