- Volume 128, Issue 12, 1982

Metronidazole (2-methyl-5-nitroimidazole-l-ethanol) was used to select nitrogen fixation (Nif−) mutants of Rhodopseudomonas capsulata, either by enrichment with metronidazole, or by direct selection on agar medium containing the drug. By the latter method, approximately 50% of the isolates obtained were Nif−. Several Nif− mutants showed the phenotype expected for mutants defective in electron transport to nitrogenase: their nitrogenase activity (determined as rate of acetylene reduction) relative to that of the wild-type strain was higher when assayed in toluene-treated cells in the presence of dithionite, than when assayed in resting cell suspensions with dl-malate as source of electrons. Glutamate-grown cells of one such mutant (RC5) resembled N-starved cells of the wild-type, and differed from glutamate-grown cells of the wild type in that nitrogenase activity in toluene-treated cells was unaffected by the addition of Mn2+ and the rate of H2 production by resting cell suspensions was significantly higher than the rate of acetylene reduction. These observations implied that glutamate-grown cells of the mutant contained predominantly nitrogenase A, the form of nitrogenase which is independent of the Mn2+-dependent membrane-bound activating factor, suggesting that the mutant may be affected in the interconversion of nitrogenase between the regulatory forms A and R.

Addition of tunicamycin to the culture medium of growing Saccharomyces cerevisiae protoplasts or cells resulted in the formation of a modified exo-1,3-β-d-glucanase which was detectable in both extracellular and intracellular fractions. This modified enzyme had a lower molecular weight than the native form and did not bind to concanavalin A. The activation energy and K m values of both enzyme forms were identical. Antibodies raised against the native protein readily precipitated the exo-1,3-β-d-glucanase produced after tunicamycin treatment. The latter enzyme was comparable, in terms of molecular size and lack of affinity for concanavalin A. to the β-d-glucanase obtained by treatment of the native form with endoglycosidase H; both lacked the carbohydrate moiety present in the native enzyme. The exo-1,3-β-d glucanase obtained in the presence of the antibiotic was more sensitive to variations in temperature and pH than both endoglycosidase H-treated and non-treated enzymes. Our results suggest that the carbohydrate moiety, if not necessary for exo-1,3-β-d-glucanase secretion, may play a role in the conformation of the protein and in stabilizing the enzymic activity.

At an external pH of 3·0, spheroplasts of the acidophilic bacterium Thiobacillus acidophilus possessed a ∆pH of 2·9 units, a membrane potential of +86 mV, and a protonmotive force of −88 mV. The ultrastructure of the whole cells or spheroplasts showed no unusual features. At given concentrations, SCN− was more effective in collapsing ∆pH in spheroplasts than in whole cells, indicating a lower permeability barrier in the former. However the ∆p values in spheroplasts were very similar to those in the whole cells, and respiratory chain inhibitors a protonophore, and an ATPase inhibitor, alone or in various combinations, were equally ineffective in collapsing ∆pH in whole cells and in spheroplasts. Thus, it is unlikely that the permeability barrier of the outer membrane accounted for the low values of measured ∆p or the ineffectiveness of various agents in collapsing ∆pH in whole cells of this organism; this confirms our previous suggestion that a sizeable ∆pH in T. acidophilus can be maintained by non-chemiosmotic means.

The sources of sulphide which appeared in the profundal zone of Blelham Tarn (English Lake District) were investigated. In experimental sediment cores, the absence of sulphate in the overlying water resulted in a drastic decrease in the release of sulphide, which suggested that sulphate reduction was a major contributor to the process. The quantity of suphide produced was much less than that of sulphate removed. This, and the inability to detect free sulphide in sediment interstitial water, was attributed to its rapid precipitation as FeS. In the absence of sulphate, production of sulphide would be from organic sources such as protein. Most probable number estimates indicated that the population of bacteria capable of producing sulphide from cysteine was much larger than that of sulphate reducers. Trace additions of 35S-labelled sulphate, cysteine and methionine were used to determine their turnover time to sulphide. Sulphate was turned over the fastest and methionine the slowest, and the turnover time was always shorter in profundal sediment. Poor recoveries of added label from littoral sediments were thought to be due to adsorption and higher rates of assimilation. Estimates of flux based on concentrations of sulphate and organic sulphur suggested that putrefaction was a more important source of sulphide in the littoral zone but contributed less than sulphate reduction in the profundal sediments.

The viabilities of planktonic bacteria from nine freshwaters and one raw sewage were compared. Viabilities estimated from microcolony formation in slide culture (18·4–48·7%) were higher than a conventional plate count method (1·8–14·9%). Microcolony viability was estimated in four ways; the one recommended was the ratio of the microcolony count and total count of microcolonies and single cells after 24 or 72 h incubation. Bacterial viability strongly correlated with the percentage of bacteria <0·2 μm3 in size and the concentration of soluble carbohydrate in the water.

A wide range of micro-organisms, including yeasts, was found to be inhibited by the primary form of Xenorhabdus spp., but not by the secondary form. Only one Xenorhabdus strain, the symbiont of Neoaplectana glaseri, did not inhibit any of the micro-organisms tested; it is suggested that this strain may not have been isolated in the primary form. Gram-positive bacteria were sensitive to all active isolates of Xenorhabdus; each of the yeasts and almost all of the Gram-negative bacteria were sensitive to some but not all Xenorhabdus isolates. Each Xenorhabdus isolate was sensitive to some other Xenorhabdus isolates. The antibiotic activity of X. nematophilus was unaffected by autoclaving but was lost after dialysis. Anaerobically incubated Xenorhabdus spp. did not exhibit antibiotic activity.

Pseudomonas putida mt-2, formerly known as Pseudomonas arvilla mt-2, which carries the wild-type TOL plasmid, and P. putida strain AC37 carrying TOL, were completely lysed by the pilus-adsorbing plasmid-specific bacteriophages PR4 and PRD1. Pseudomonas putida strain PpS388, also harbouring the plasmid, was not lysed. In a P. putida mt-2 host, TOL transferred 18-fold better on a surface (25 × 10−1 transconjugants per donor h−1) than in liquid; when P. putida PpS388 was the host, however, a frequency of only 23 × 10−4 transconjugants per donor h−1 was obtained. Thus, TOL was derepressed for transfer in P. putida mt-2 and P. putida AC37, but not in P. putida PpS388. Electron microscopy revealed that TOL determined thick (8·5–10 nm diameter) flexible pili in large numbers, suggesting constitutive expression in its derepressed state.

A new procedure has been developed for the isolation of the chromosome complex, termed chromatin, from Escherichia coli. The bacteria were subjected to low ionic strength and T4 lysozyme, followed by detergent treatment analogous to that employed for the isolation of eukaryotic chromosomes. The chromatin was an insoluble viscous material which contained approximately equal amounts of DNA and RNA. The protein content of the chromatin was almost three times greater than the nucleic acid content. Electron microscopy revealed that the chromatin was highly condensed, having multiple loops and beaded structures with various diameters. The chromatin could be completely solubilized by both micrococcal nuclease and DNAase I, whereas RNAase had no effect. The initial degradation by micrococcal nuclease resulted in the production of a DNA-protein particle, sedimentation coefficient 10S, and an RNA-protein complex of 24S. Further degradation led to a decrease in sedimentation coefficient of the DNA-protein complex, but not of the RNA-protein particle. The peak size of the DNA of the initial DNA-protein particle was approximately 2400 bp. The action of micrococcal nuclease also resulted in the production of several discrete RNA species of various sizes. Several low molecular weight proteins (12000–27000) were found in the DNA-protein complex. The DNA-binding protein HU was present in the undigested chromatin; varying amounts of HU were, however, detected in the DNA-protein and RNA-protein particles.

The determinant(s) of gonococcal resistance to killing by human polymorphonuclear (PMN) phagocytes appear to be present in outer membrane vesicles (OMV) purified from lithium chloride extracts of Neisseria gonorrhoeae strain BS4 (agar) by wheat germ agglutinin (WGA) affinity chromatography. OMV neutralized the ability of antisera raised against whole gonococci to drastically reduce the capacity of strain BS4 (agar) to survive within PMN phagocytes. Furthermore, analysis by SDS-PAGE of OMV/WGA precipitates from lithium chloride extracts of strain BS4 (agar) and strain BSSH, which was more susceptible to phagocyte killing than strain BS4 (agar) and yielded OMV with poor antiserum-neutralizing activity, suggested that three proteins were associated with resistance to phagocyte killing.

Strains of Escherichia coli were examined for their ability to grow in diffusion chambers implanted into the peritoneal cavities of mice and rabbits. In rabbit chambers E. coli K12 strains grew poorly, whereas isogenic strains harbouring ColV plasmids and wild-type isolates from extra-intestinal infections grew well. The difference was much less marked with strains grown in mouse chambers. Differences in sensitivity to the bactericidal action of human or rabbit serum were found in some cases between organisms of the same strain grown in vivo or in vitro. Host immunoglobulins were bound to the surface of all in vivo bacteria. Comparison of the polypeptide composition of bacterial cell envelope preparations on SDS-PAGE gels revealed differences between in vivo and in vitro grown E. coli. Thus E. coli growing in vivo may possess properties significantly different from the same organisms growing on laboratory medium.

The cell cycle of Escherichia coli contains a period of indeterminate length that reflects a stochastic reaction, beginning at some time after a round of chromosome replication, and ending before the cell divides. Although the chemical nature of this reaction is not known, the time of its onset and the single statistical half-life parameter required for its quantitative description have been measured previously. Here it is shown that this parameter implies the distribution of generation times and the age distribution, as well as the distributions of replication initiation and termination ages; these distributions are derived from this half-life parameter for exponentially growing populations of E. coli. It is also shown that the stochastic reaction affects the results and interpretation of any experiments involving synchronous growth of bacteria.

The duration of a morphological phase of the cell cycle is reflected in the steady state distribution of the sizes of cells in that phase. Relationships presented here provide a method for estimating the timing and variability of any cell cycle phase. It is shown that the mean size of cells initiating and finishing any phase can be estimated from (1) the frequency of cells exhibiting the distinguishing morphological or autoradiographic features of the phase; (2) the mean size of cells in the phase; and (3) their coefficient of variation. The calculations are based on a submodel of the Koch-Schaechter Growth Controlled Model which assumes that (i) the distribution of division sizes is Gaussian; (ii) there is no correlation in division sizes between successive generations; and (iii) every cell division gives rise to two daughter cells of equal size. The calculations should be useful for a wider range of models, however, because the extrapolation factors are not sensitive to the chosen model. Criteria are proposed to allow the user to check the method's applicability for any experimental case.

The method also provides a more efficient test of the dependence of growth on cell size than does the Collins-Richmond method. This is because the method uses the mean and coefficient of variation of the size of the total population, in conjunction with those of the cells in a final phase of the cell cycle, to test potential growth laws. For Escherichia coli populations studied by electron microscopy, an exponential growth model provided much better agreement than did a linear growth model.

The computer simulations were used to generate rules for three types of cell phases: those that end at cell division, those that start at cell division, and those totally contained within a single cell cycle. For the last type, additional criteria are proposed to establish if the phase is well enough contained for the formulae and graphs to be used. The most useful rule emerging from these computer studies is that the fraction of the cell cycle time occupied by a phase is the product of the frequency of the phase and the ratio of the mean size of cells in that phase to the mean size of all cells in the population.

A further advantage of the techniques presented here is that they use the extant distributions that were actually measured, and not hypothesized distributions nor the special distributions needed for the Collins-Richmond method that can only be calculated from the observed distributions of dividing or newborn cells on the basis of an assumed growth law.

The surface stress theory of the ontogeny of the bacterial rod depends critically on whether the old poles continue to incorporate new material into the stress-bearing murein. If insertion of peptidoglycan continues, then seemingly the shape must become gradually rounder due to the surface stress resulting from the internal hydrostatic pressure. We have reanalysed our earlier experimental data by classifying grains with respect to distance from the nearest pole, and not from the cell centre as was done previously, and conclude that old poles do incorporate new diaminopimelic acid residues. This eliminates the model we have proposed for Gram-positive rods, which assumed diffuse growth on the cylindrical sides and that poles once formed would be rigid. The new results are consistent with another model (presented elsewhere) in which insertion of new murein occurs all over the surface, although not equally. This new model leads to elongation and division if the energetics of wall expansion is altered by the cell in a control region at a particular point of the cycle by the cell.

The effects of exogenous thiamin on the growth yield and vitamin B6 content of 18 strains of yeasts and a few strains of bacteria were examined. The addition of thiamin hardly affected the growth yield of the yeasts tested, except for two strains Saccharomyces uvarum strain 4228 and Saccharomyces uvarum IFO 0751. In contrast, the vitamin B6 content of all the yeasts tested, except Pichia membranaefaciens IFO 0189, decreased markedly in the presence of thiamin. In S. uvarum IFO 1265, the synthesis of vitamin B6 was maximally inhibited by the addition of thiamin (1·5 nmol ml−1) to the growth medium without affecting cell growth, whereas the amounts of cellular vitamin B6 increased in the presence of the thiamin antagonist, pyrithiamin or oxythiamin, at concentrations that did not affect growth. When [4′-14C]pyridoxine. HCl (0·5 μg ml−1) was added to the growth medium at least 54% of the added isotope was incorporated into the cells during 24 h incubation. In the presence of thiamin (15 nmol ml−1), at least 32% of the added isotope was incorporated. The metabolism of [4′-14C]pyridoxine. HCl to inactive forms having no vitamin B6 activity was not stimulated by the addition of thiamin. Thus, vitamin B6 synthesis in many yeasts was affected by thiamin, whereas, in bacteria, growth yield and vitamin B6 content were not affected by thiamin.