J. R. Postgate

Viability Measurements and the Survival of Microbes Under Minimum Stress

Publisher Summary The study of the survival of vegetative microbes under minimum stress generally provides some practical consequences. Microbes, being uni- or non-cellular, have no simple analog of the “natural” senescence and death undergone by multicellular organisms. Most studies on microbial survival are concerned with the viability of populations after they have been subjected to some kind of stress such as a population is largely examined after it has been heated, frozen, dried, exposed to adverse pH, pressure, salinity, and exposed to toxic chemicals or lethal radiation or to biological antagonists. It is necessary to adopt a form of treatment in which as mild and simple stress as possible is imposed on the microbial population. Three conditions are generally considered: (1) exposure of microbial populations to non-nutrient environments in conditions in which growth is in principle possible, but is prevented by withholding one or more essential nutrients, (2) allowing a culture to age until multiplication has ceased, and (3) growing a continuous culture at so slow a rate that the spontaneous death-rate of the population makes a contribution to the dynamics of the steady state. The chapter considers the techniques that are used to assess microbial survival.

Effects of Oxygen on Acetylene Reduction, Cytochrome Content and Respiratory Activity of Azotobacter chroococcum

SUMMARY The respiratory activities and cytochrome a 2 contents of nitrogen-fixing continuous cultures of Azotobacter chroococcum (NCIB 8003) increased with the partial pressure of oxygen encountered during growth. Above 0.6 atm., wash-out of the culture occurred. Acetylene reduction by culture samples of low respiratory activity was far more easily inhibited by oxygenation than was that of samples of high respiratory activity, though their maximum acetylene-reducing activities at their optimal pO2 values were similar. Inhibition by oxygen was reversible after mild oxygenation: 70 to 100% of the original activity returned immediately when the degree of oxygenation was decreased. Irreversible inhibition occurred after vigorous oxygenation and was associated with a loss of activity in cell-free extracts, which was restored by adding the oxygen-sensitive protein component of Azotobacter nitrogenase. These observations support earlier proposals that augmented respiration can scavenge oxygen from the nitrogen-fixing site and that a conformational change in the state of nitrogenase can prevent damage to the enzyme by oxygen. Vigorous aeration, however, may overcome these protective mechanisms.

Failure of Putative Nitrogen-fixing Bacteria to Fix Nitrogen

SUMMARY: Analyses, tests with isotopic nitrogen and tests for acetylene and isocyanide reduction, using both continuous and batch cultures, were made with seven strains of putative nitrogen-fixing bacteria and three local isolates. Only two (single strains of Mycobacterium flavum and Pseudomonas azotogensis) fixed nitrogen; the active pseudomonad differed in several respects from the organism originally reported. Other Pseudomonas, Nocardia and Azotomonas species and the three local isolates did not fix; some simulated nitrogen fixation in cultural tests most impressively, but proved simply to be very efficient scavengers of traces of fixed nitrogen.

Chromosomal integration of Klebsiella nitrogen fixation genes in Escherichia coli.

SUMMARY Escherichia coli, C-M7, a His+Nif+ hybrid obtained by intergeneric mating with a Klebsiella pneumoniae donor strain, also inherited the unselected markers gnd and rfb. The R factor, R144drd3, which had been used to confer fertility on the donor, was present, detectable as covalently closed circular DNA of molecular weight 69 x lo6 daltons. No other species of supercoiled DNA were isolated and the elimination of R144drd3 did not result in the loss of Klebsiella genes. Segregation analysis of donor markers indicated that the Klebsiella DNA was integrated at the his region of the E. coli chromosome in the probable order his-gnd-nif-rfb. Strain C-M7 produced a nitrogenase physiologically identical to that of K. pneumoniae, but synthesized a heteromeric species of gluconate-6-phosphate dehydrogenase.

The Physiology of Sulphate-Reducing Bacteria

Publisher Summary The chapter discusses the physiology of sulphate-reducing bacteria. The chapter presents an account of their ecology and economic activities as relevant to the subject as their multiplication can have considerable ecological and economic consequences, and since these are due to their special physiology, in particular, the production of hydrogen sulphide. The sulphate-reducing bacteria form a physiologically distinctive group of anaerobic bacteria, their oxidative metabolism being based, not on fermentation, but on the reduction of sulphate or certain other inorganic sulphur compounds. Their physiology has broad analogies with that of the nitrate-reducing bacteria (denitrifying bacteria), but they are all exacting anaerobes and no examples of facultative aerobes are known. Some representatives of the group are capable of growth by non-respiratory processes involving dismutation of substrates, such as pyruvate, fumarate, or choline. Even when reducing sulphate, these organisms are completely unable to oxidize their carbon compounds, such as fatty acids; usually acetic acid plus carbon dioxide are the normal end products of carbon metabolism. The present chapter brings up to date all those publications on this group of bacteria, which had come to the authors attention by early 1972. Reviews of related subjects that have been published during this period, and which makes reference to these bacteria reviewing the general metabolism of sulphur bacteria. The chapter also discusses the chemical and biochemical activities of these bacteria.

Genome size and complexity in Azotobacter chroococcum

All of eight strains of Azotobacter chroococcum examined contained between two and six plasmids ranging from 7 to more than 200 MDal in size. Strain MCC-1, a derivative of NCIMB 8003, was cured of various of the four largest of its five plasmids and the phenotypes of the strains compared. all fixed nitrogen and exhibited uptake hydrogenase activity. No differences were observed in carbon source utilization or antibiotic, heavy metal or UV resistance. The genome sizes of two strains of A. chroococcum were determined by two-dimensional electrophoresis. Strain CW8, an isolate from local soil containing two small plasmids of 6 and 6.5 MDAl contained unique DNA sequences equivalent to 1.78 x 10(6) (+/- 20%) bp (1.2 x 10(9) Dal). In strain MDC-1, a derivative of MCC-1, containing a 190 MDal and 7 MDal plasmid, the genome size was 1.94 x 10(6) (+/- 20%) bp. In exponential batch cultures, both contained 20 to 25 genome equivalents per cell. MCD-1 exhibited complex UV kill kinetics with a marked plateau of resistance; CW8 showed a simple response inconsistent with the possibility of organization of its DNA into identical chromosome copies capable of independent segregation.

Control of Nitrogenase Synthesis in Klebsiella pneumoniae

Summary: Sulphate-limited continuous cultures of Klebsiella pneumoniae showed a proportional repression of nitrogenase activity with increasing concentrations of ammonium ion in the influent medium. A fully repressed population had a 50 % greater bacterial density than a fully derepressed one. On derepression, synthesis of nitrogenase lagged for 90 min after exhaustion of NH4+ from the medium but was complete within one doubling time. Casamino acids did not decrease the pre-fixation lag obtained under sulphate-limited conditions in the chemostat. Longer lags were obtained when NH4+ was exhausted under N-limited conditions. Such lags were decreased by Casamino acids, yeast extract, or L-aspartate. Aspartate-N completely repressed nitrogenase under sulphate- or carbon-limited conditions but not under nitrogen limitation. In the initial stages of repression of nitrogenase synthesis by NH4+, apparent production of active enzyme continued for a short time in the absence of protein synthesis de novo; nitrogenase was then diluted out as the organisms multiplied. Repression by NH4+ was at the level of mRNA transcription according to studies with rifampicin and chloramphenicol. ‘Coding capacity’ for nitrogenase synthesis declined with a half-life of about 4.5 min following inhibition of RNA synthesis with rifampicin. NH4+ did not influence the decay rate but stimulated translation of such nitrogenase-specifying mRNA as had been initiated.

A chemostat study of the effect of fixed nitrogen sources on nitrogen fixation, membranes and free amino acids in Azotobacter chroococcum.

SUMMARY: Increasing concentrations of ammonium ions in the medium of nitrogen-fixing, sulphate-limited continuous cultures of Azotobacter chroococcum caused a proportionate repression of nitrogenase activity; free NH4 + could be detected in the extracellular culture fluid only when nitrogenase activity was wholly repressed. The NH4 + concentrations giving 50% or 100% repression were proportional to the population density. Nitrate ions repressed with similar stoichiometry; glutamate, glutamine and aspartate did not repress and were not metabolized; repressed and derepressed populations contained equal amounts and proportions of glutamate-forming enzymes. Repressed populations lacked both enzymatic components of nitrogenase. The intracellular free amino acid pools were typical of Gram-negative bacteria; an increase in the degree of repression was associated with an increase in the pool levels of ammonia, aspartate and glutamate. Nitrogen-fixing populations possessed a convoluted intracytoplasmic membrane system which was absent from ammonia-assimilating organisms, but the phospholipid contents of the two types of population were similar. All members of a half-repressed population possessed these membranes, but to a lesser extent that fully derepressed populations. When N2-fixing chemostat populations were abruptly exposed to repressive concentrations of ammonium succinate. repression occurred exponentially and nitrogenase activity disappeared from the culture faster than wash-out of stable enzyme. Repression was not alleviated by exogenous cyclic AMP. Derepression was complete, according to the acetylene test, within half a doubling time of disappearance of free ammonium ions from the culture.

Derivation and properties of F-prime factors in Escherichia coli carrying nitrogen fixation genes from Klebsiella pneumoniae.

A His+ Nif+ Escherichia coli K12, Hfr strain (UNF43) was constructed by an intergeneric mating between a Klebsiella pneumoniae donor strain (HF3) and a his-HFR E. coli strain (SBI824) which transfers his as an early marker. An F-prime nif plasmid, FN39, carrying genes which correspond to the E. coli chromosomal region, metG gnd his shiA, but excluding purF and aroD, was isolated from UNF43. Translocation of carbenicillin resistance genes from a P-type R-factor, R68, to FN39 increased the stability of his and nif on the derivative F-prime, FN68. Sedimentation analysis of both F-primes in sucrose gradients revealed our covalently closed circular(CCC) DNA species of molecular weights 279 +/- 9, 136 +/- 3, 90 +/- 1 and 44 +/- 1 megadaltons. It is suggested that the two smallest CCC-DNA species are component replicons of the composite F-primes of molecular weight 136 +/- 3 megadaltons, and that the molecules of 279 +/- 9 megadaltons are CCC-dimers. FN68 was transferable in intergeneric matings to Klebsiella aerogenes, K. pneumoniae and Salmonella typhimurium but not to Proteus mirabilis; only carbenicillin resistance and sex factor activity were transferred to Erwinia herbicola. nif genes on FN68 were expressed in a Nif- mutant of K. pneumoniae and also in S. typhimurium, which in conventional tests is naturally non-nitrogen-fixing; expression of the his determinant of FN68 became temperature-sensitive in S. typhimurium.

The Genomes of Desulfovibrio gigas and D. vulgaris

Two-dimensional electrophoresis of sequential double-restriction digests showed that the genome of Desulfovibrio gigas compromised 1.63 x 10(6) bp (1.09 x 10(9) Dal) of DNA; an ammonia-limited chemostat population possessed an average of nine genomes per cell and a multiplying batch culture possessed approximately 17 genomes per cell. The genome size of D. vulgaris (Hildenborough) was 1.72 x 10(6) bp (1.14 x 10(9) Dal); a population from an ammonia-limited batch culture contained four genomes per cell. Control digestions and analyses with Escherichia coli GM4 agreed reasonably with published values: a genome size of 3.95 x 10(6) bp and approximately two genomes per cell from a stationary batch culture in glucose minimal medium. Desulfovibrio gigas carried two plasmids of approximately 70 MDal (1.05 x 10(5) bp) and approximately 40 MDal (6 x 10(4) bp); D. vulgaris (Hildenborough) contained one of approximately 130 MDal (1.95 x 10(5) bp). Single plasmids were also detected in a second strain of D. vulgaris and in strain Berre sol of D. desulfuricans but not in 10 other desulfovibrios including representatives of D. desulfuricans, D. vulgaris, D. salexigens and D. africanus.