- Volume 47, Issue 1, 1967

The taxonomic position of Thiobacillus denitrificans was investigated by using six newly isolated strains which were compared with other species of this genus, under aerobic and anaerobic growth conditions. The validity of T. denitrificans is shown. Thiobacillus intermedius and T. thermophilica, two species recently described by other authors, were also investigated.

To examine the role of bacteria in the life history of a temporary pond, nine physiological groups of bacteria were determined by plate counts and enrichment culture techniques in pond water, in soil from the pond basin, and in soil from the pinewoods area surrounding the basin. Sampling was begun when the pond was dry and continued through the period when it contained water, into the next dry period. Low counts were obtained for sulphur-, ammonia-, and nitrite-oxidizing autotrophs; iron-oxidizing autotrophs were not detected. This indicates that bacteria did not make a major contribution to the ecosystem as primary producers. Soil samples contained millions to hundreds of millions (106-108) of aerobic nitrogen-fixers and millions to tens of millions (106-107) of urea-utilizing organisms per gramme, suggesting that they may be of significance in the nitrogen cycle in the ecosystem. Hundreds of thousands to tens of millions (105-107) of cellulose-decomposers, and tens of millions to hundreds of millions (107-108) of aerobic and of anaerobic heterotrophs were present per gramme in soil samples. The several heterotrophic types were also present in pond water in maximum numbers ranging from tens of thousands per millitre for nitrogen-fixers to millions per millilitre for anaerobic heterotrophs. As the pond dried the numbers of bacteria in its water decreased. These data suggest that bacteria in the pond ecosystem play a role in the nitrogen, carbon and energy cycles as decomposers and transformers, as a source of nutrilites and as members of the food chain.

The resistance to nisin was examined for the vegetative forms and the endospores of 14 strains of 9 species of the genus Bacillus. Bacilli, endospores and culture filtrates were tested for ability to inactivate nisin. Marked antinisin activity was detected in extracts of bacilli and endospores of organisms which have a lytic mechanism for rupture of the spore coat (type L); little or no activity was observed in extracts from organisms which rupture the spore coat mechanically (type M). No significant extracellular activity was detected, except after the autolysis of bacilli. When organisms were cultivated in the presence of nisin (50 Reading units/ml.; Berridge, 1949) the yield of organisms and the specific activity of the extracts was decreased; evidence to explain these observations is presented. No quantitative correlation was observed between the production of anti-nisin activity and the resistance of vegetative forms.

The anti-nisin activity of cell-free extracts of bacilli of two species (Bacillus cereus and B. polymyxa) was studied further; there was evidence that the activity was probably enzymic. A preliminary study of the properties of the enzymes from these organisms was made. It was found that the anti-nisin enzymes of two strains of B. cereus differed from the lytic enzymes previously described (Strange & Dark, 1957 a, b). The anti-nisin enzymes had no effect on polymyxin, gramicidin or bactracin but inactivated subtilin. Proteolytic activity was not observed in the preparations.

Although the most reliable techniques for determining total bacterial numbers in populations recovered from aerosols are based on radioactive tracers, non-isotopic methods must be used for certain purposes. A tracer technique based on the enzyme galactosidase was developed for the determination of bacterial numbers in samples containing Escherichia coli strain B organisms 1 to 2 × 107/ml. By using the [14C]-tracer technique as a reference standard, the enzyme method was shown to be unaffected by the relative humidity at which the aerosol was stored and by the viability of the recovered population and to be scarcely affected by the age of the bacterial cloud. The principle of this method may be applied to other organisms and other suitable enzymes.

Modifications to an anaerobic continuous culture apparatus to allow pH control, and pH and Eh measurements, are described. Two anaerobic rumen bacteria were grown under different conditions, but as carbohydrate-limited cultures. The effects of growth rate, pH value and Eh value on yields of bacteria, enzyme activities and fermentation products are described. Optimum bacterial yields per mole of substrate fermented and per mole of ATP presumably formed in the fermentation were variable with the particular bacterium and the substrate, and were high for hexose fermentations. Yields of bacteria varied with growth rate, being lowest at low growth rates. Fermentation products also varied with growth rate and the pH value of the culture in some cases, as did the production of enzymes. Maximum growth rates calculated from batch cultures were in agreement with those found in the continuous cultures.

Forty-one strains representing 21 nomen species of the genus Pseudomonas and 2 of the genus Alcaligenes were screened for sensitivity to ethylene-diaminetetra-acetic acid (EDTA). Sensitivity of organisms was assessed by determining the release of intracellular solutes and the loss of viability under the action of EDTA. The latter criterion was found to be the more useful for the differentiation of sensitive and resistant organisms. Strains of Pseudomonas diminuta, P. geniculata, P. iodinum, P. maltophilia, P. pavonacea and possibly P. rubescens were considered as resistant to EDTA. Strains of P. alcaligenes were clearly differentiated from strains of Alcaligenes faecalis, which were highly resistant to EDTA. The practical value and possible taxonomic significance of the results are discussed.

Effects of mutations in three independent proline suppressor loci were studied. Mutants of the su-6 locus were almost completely deficient in ornithine transcarbamylase activity and mutants of the su-19 locus produced ornithine δ-transaminase constitutively or semiconstitutively; this latter enzyme was strongly induced by arginine in the wild type. The su-6 and su-19 suppressor mutations were all recessive. Dominant and recessive mutants of the third suppressor locus, su-2, showed higher activities of arginase and ornithine δ-transaminase than did the wild type. It is suggested that in all three cases the enzyme alterations caused by the suppressor mutations allow the synthesis of proline by an alternative route, replacing the blocked major pathway.

Mutants of Pseudomonas aeruginosa strain 8602 were isolated which were unable to produce an aliphatic amidase (acylamide amidohydrolase, EC 3.5.1.4) and could not grow on acetamide as a carbon or nitrogen source. Amidase-constitutive mutants, producing amidase in the absence of inducing amides, were isolated by selection on succinate+formamide agar. Sixteen mutants were magno-constitutive non-inducible mutants producing amidase at about the same rate or greater than the fully induced wild-type strain. Amidase synthesis in one magno-constitutive mutant was repressed by the non-substrate inducer N-acetylacetamide, but the others were not affected in any way. Six mutants were semi-constitutive, producing amidase at 10–50% of the rate of the magno-constitutive mutants and were induced by N-acetylacetamide. Most of the constitutive mutants were very sensitive to catabolite repression by succinate in pyruvate medium, but succinate produced only partial repression of one magno-constitutive mutant and three semi-constitutive mutants; one semi-constitutive mutant was not repressed except in the presence of inducer.

Six mutants isolated from succinate + formamide agar had altered inducer specificity and were induced to form amidase by formamide, which is a very poor inducer for the wild-type strain. The formamide-inducible mutants were also sensitive to catabolite repression by succinate although one mutant was only partially repressed.

Phage F 116 was used to transduce the amidase structural and regulator genes. In crosses between constitutive mutants of Pseudomonas aeruginosa as donors and amidase-negative mutants as recipients, the two characters were co-transduced with frequencies of 80–100%. Similarly, in crosses between formamide-inducible and amidase-negative mutants these two characters were co-transduced with frequencies of 89–96%. The amidase structural and regulator genes are considered to be closely linked.

A strain of Mycoplasma bovigenitalium, designated M 120, was grown, and produced a marked cytopathic effect (c.p.e.) in calf-, pig-, and monkey-kidney cell cultures. The c.p.e. was characterized by enlargement of the cells, the appearance of intracytoplasmic inclusions and partial destruction of the cell monolayers. A similar c.p.e. was produced in tissue-culture cells following the inoculation of mycoplasma ‘toxins’.

Comparative growth studies of strain M120 in calf-kidney cell cultures and in tissue-culture medium alone showed that the organism grew more readily in the presence of cells. The rate of virus production and appearance of c.p.e. of infectious bovine rhinotracheitis (I.B.R.) virus was delayed in cultures previously infected with the M 120 strain of mycoplasma as compared with normal cultures.

Comparative studies of growth, yield of organism and synthesis of nucleic acids were made on a strain of Mycobacterium smegmatis grown with and without sufficient iron. Although the bacteria grew exponentially at the same rate in the iron-deficient medium as in the iron-replete medium, the content of RNA and DNA was lower in the iron-deficient bacteria. In the iron-deficient cultures, growth ceased abruptly after an exponential phase characterized by an inhibition of DNA synthesis. The pH value of the medium became increasingly acidic as iron-deficient growth proceeded.

During germination of Bacillus cereus spores, the cortex was lost completely, but with B. polymyxa spores there was no apparent alteration in the cortex structure. On the other hand, the quantities of dipicolinic acid, calcium and mucopeptide, measured as hexosamine, released from germinating B. polymyxa spores were similar to those released from germinating B. cereus spores. It appears that only about 30% of the mucopeptide of these species is involved in the maintenance of spore dormancy. The solubilization of the spore dipicolinic acid, calcium and mucopeptide during germination only accounted for about half of the loss of dry weight from the spores. During the outgrowth of B. cereus, the spore coats dissolved away at one pole and the vegetative form grew out, leaving only fragments of the spore integument free in the medium. Bacillus polymyxa grew when the largely unaltered coat and cortex layers of the spore split open at the organism's equator.

Conditions necessary for the production of thymineless mutants in Gramnegative bacteria by aminopterin treatment were examined. Although Escherichia coli and Proteus strains were readily made ‘thymineless’ (i.e. requiring added thymine) no such mutants were obtained with the majority of Aerobacter strains. However, two methods which produced thymineless mutants in all the Aerobacter strains were found; these were: treatment with alkaline EDTA before, or temperature shift down during, incubation with aminopterin. In every case where temperature shift alone resulted in the production of thymineless mutants more were found when it was used in conjunction with EDTA pretreatment. It was also found that the phase and size of inoculum, the method used to sterilize aminopterin and the concentration of thymine in the plating media had profound effects on the production and detection of thymineless mutants.

When inoculated with a clone of Rhizobium that produced large pink effective root nodules in virus-free plants, white clover plants infected with clover phyllody virus (CPV) produced mainly small white nodules characteristic of reduced effectiveness in nitrogen fixation. Bacteria from nodules borne on these CPV-infected plants again produced mainly small white nodules in virus-free clover cuttings and seedlings, but did not transmit CPV. Either exposure to the virus, or culture in CPV-infected tissues, seemed to induce a change in the Rhizobium.

In mixed cultures, a genetic factor (designated Hly factor) responsible for α-haemolysin production in 10 of 53 strains of Escherichia coli was transmitted at a relatively high rate to other organisms, including shigellas and salmonellas as well as E. coli. Transmission was evidently by conjugation for it was not achieved by bacteria-free culture fluids. Although the factor was not eliminated by acriflavine or ultraviolet irradiation, it was probably a plasmid. Salmonella recipients were very unstable, and during serial subculture in broth the Hly factor was lost from most of these organisms. The factor was easily reintroduced into these segregants, but with difficulty or not at all into the rare E. coli organisms which had lost it. Hly factor was transmitted independently of F, R and coli factors and phages were not involved in its transmission. Strains of E. coli K12 F+ into which Hly factor had been introduced became resistant to the F-specific phage. From this it appeared that the factor has the fi + character observed in certain R factors. The illness produced in mice by the intravenous injection of culture fluids of α-haemolytic strains of E. coli was shown to be caused by the α-haemolysin itself.