- Volume 43, Issue 3, 1966

In a continued search for a more suitable generic location for the species tentatively designated Mycobacterium rhodochrous (Overbeck) Gordon & Mihm, strains of some species of Corynebacterium, both animal and plant pathogens, were found to have the same morphology and the same, or nearly the same, physiological properties as strains of the species provisionally labelled M. rhodochrous. Additional tests and observations applied to the strains of the species M. rhodochrous resulted in a pattern of approximately 30 different characteristics which distinguished the species. Among the newly studied properties was the ability to utilize glucose oxidatively. Because strains of the type species of the genus Corynebacterium ferment glucose, the assignment of the glucose-oxidizing strains tentatively named M. rhodochrous to the genus Corynebacterium is not proposed.

The influence of amino acids on respiration of Salmonella oranienburg was studied over a range of water activity (aw) values. Decrease of the aw value by adding NaCl or sucrose decreased the rate of glucose oxidation and induced a lag. At relatively low values (0.970 aw) but not at high values (0.998 aw), additions of certain amino acids (proline, aspartic acid, asparagine, glutamic acid, glutamine, cysteine) caused an appreciable synergistic increase in respiration rate. Proline was the most stimulatory amino acid tested. At 0–960 aw, only proline and its analogue azetidine-2-carboxylic acid gave appreciable stimulation. These were the only amino acids which decreased the lag, and lags were not affected by the presence of chloramphenicol. Proline was also stimulatory when glucose was replaced by pyruvate or succinate. The respiration of organisms grown at low aw values was not stimulated by proline unless the organisms were first subjected to osmotic shock. The proline effect was also observed with bacteria of four other genera. The data are discussed in relation to the stimulation by proline of bacterial growth at low water activities.

The standard methods of scoring for unselected markers the recombinant clones arising from crosses between various substrains of Escherichia coli K-12 do not easily allow recognition of types intermediate between or slightly deviating from the parental phenotypes. The theories of Hinshel-wood and his school suggest the general possibility of a continuous series of such intermediates, which have in fact been reported among the progeny of certain drug-resistant x drug-sensitive crosses. To examine whether similar types result with other kinds of unselected marker, crosses were performed in which the parents differed in their ability to utilize lactose (lac character) and/or to grow in the absence of proline (pro character), as well as in other characteristics utilizable as selected markers. Growth of recombinant and parental isolates on minimal media containing lactose as carbon source, or lacking proline, was then characterized quantitatively. No recombinants with intermediate pro character were found, and only one with intermediate lac character. However, in some cases the recombinants differed in lac or pro character from either parent in other ways. One donor strain, proline-requiring (pro -) and unable to utilize lactose (lac -), yielded pro - and lac - recombinants whose eventual growth in proline-less and lactose media respectively was greatly retarded by comparison with the donor parent. This donor strain was also crossed with a pro + lac + recipient which had been ‘trained’ to grow in minimal lactose medium with optimal growth rate. In lactose media, lac + recombinants all grew at the optimal rate, while lac - recombinants showed growth still retarded as compared to the lac - parent, but considerably more rapid than similar recombinants from the cross involving an untrained lac - recipient.

We have studied the effect of distamycin A on the synthesis of inducible enzymes in Escherichia coli strain K12. Distamycin A did not inhibit the growth of E. coli K12 when the micro-organism already contained constitutive or inducible enzymes for the metabolism of sugars. The growth of E. coli was prevented when distamycin A was present during the enzymic induction. Distamycin A inhibited the formation of inducible β-galactosidase; the activity of the antibiotic was directly related to its concentration, but independent of the concentration of the inducer. Thus it does not seem that distamycin A causes a decrease of the concentration of inducer. The enzyme synthesized in the presence of subinhibitory concentrations of distamycin A showed the same kinetic behaviour as β-galacto-sidase induced under normal conditions.

Serologically active fractions from 8 strains of Streptococcus faecalis, each with a different type antigen, were obtained by partial purification on a DEAE-cellulose column. By qualitative analysis of the hydrolysates of these fractions it was shown that glucose and glucosamine were always present, galactosamine and galactose were generally present. Arabinose was present as a minor component in some cases, whereas rhamnose was never found. Some evidence suggested that the glucose present in these hydrolysates was at least partly derived from the group D teichoic acid. N-acetylglucosamine or N-acetylgalactosamine and their corresponding hexoses were the main inhibitors of the quantitative precipitin reaction in 4 out of 8 type antigen systems investigated. In the type 19 system the two N-acetylhexosamines but no other sugars were inhibitory. No inhibition by simple sugars was observed with the remaining three systems. The conditions required to obtain serologically active extracts are discussed. Two kinds of inhibitors formed during HCl extraction were isolated.

The survival in air as a function of relative humidity (RH) and aerosol age is reported for Escherichia coli (strains B and Jepp) and for Serratia marcescens (strain UK 8) sprayed from suspensions in distilled water and in solutions of dextran and of raffinose. The survival in nitrogen (> 99.9%) of E. coli (strains B, Jepp and commune) is given as a function of RH, when sprayed from suspension in distilled water and in raffinose solution. The results show that E. coli in the aerosol was subject to at least three stresses, an air stress, an RH stress, and a collection stress. The air stress accounted for loss of viability at low RH and the RH stress occured at high RH and is expressed as RH ranges in which E. coli rapidly lost viability. The survival, especially at high RH, depended upon the composition of the collecting fluid. At high RH a range of RH was found in which the bacteria were unstable when collected in phosphate buffer. Addition of raffinose (chosen as a typical protecting agent to the spray fluid and the addition of M-sucrose to the collecting fluid eliminated the instability for E. coli (commune) and decreased it for the other two strains.

Several Gram-positive bacilli and Gram-negative bacteria were found to differ markedly in their ability to concentrate Mg2+ from simple chemically defined media. In cultures of the Gram-negative bacteria the uptake of Mg2+ was quantitative over the range where growth was limited by the concentration of Mg2+. The Gram-positive bacilli, however, were unable to utilize Mg2+ when the external concentration was low, and at higher concentrations sufficient to support about half maximal growth, assimilated less than 50% of the available Mg2+. Both Gram-negative and Gram-positive organisms initially liberated Mg2+ when suspended in Mg2+-free media, the Mg2+ being re-utilized rapidly by the Gram-negative organisms. In suspensions of Aerobacter aerogenes the liberation of Mg2+ was independent of temperature and the re-utilization of Mg2+ was dependent on temperature.

The heat resistance of the spores of six species of bacteria varied with water activity (aw) at which the spores were heated, although the magnitude of the changes differed greatly between species. At all aw values there was an approximately linear relation between the logarithm of the number of viable spores and the time of heating. The slopes of these straight lines were used to describe the observed death-rates as the time (D value) required to decrease the population by one log. unit. For all six species the greatest heat resistance was manifest at aw values of about 0.2–0.4, the maximum D values at 110° now varying from about 2 to 24 hr. At aw values less than 0.2 the heat resistance decreased; for spores rigorously dried over P2O5 (0.00 aw) the D values at 110° now varied between about 30 sec. and 30 min. When the spores were heated at aw values above 0.4 the resistance of 4 species decreased considerably, being lowest at 1.00 aw; with spores of Bacillus coagulans and B. stearothermo-philus the heat resistance decreased less at the high aw values; at 1.00 aw their D values were slightly greater than at 0.00 aw. At the high aw values the D values at 110° varied from less than 0.1 sec. for Clostridiuum botulinum type E to about 40 min. for B. coagulans and B. stearothermophilus. The Q10 for thermal death was about 10 at high aw values, decreasing to about 2 at aw values below 0.3. Under very moist conditions spores of B. stearothermophilus were about 50,000 times more heat resistant than were spores of C. botulinum type E; but at aw values less than about 0.5 this ratio fell to about 10. The convergence of this ratio resulted from more than a 100,000-fold increase in the resistance of the type E spores, and only a 20-fold increase in the resistance of the spores of B. stearothermophilus.

Vibrio fetus was examined in shadow-cast, negative-contrast and thin-sectioned preparations; Four morphological types were noted in shadowed specimens: comma, coccoid, S-shaped and filamentous. In negative-contrast preparations organisms were differentiated into two types on the appearance of their cytoplasmic mass. A large spherical cytoplasmic inclusion was frequently evident at the flagellated pole of freshly harvested organisms. Various morphological variants, ‘sports’, e.g. multiflagellate, were easily detected. A non-motile isolate was either devoid of flagella or exhibited only a short stub at the pole.

In thin-section preparations anatomical features noted were: a rugose loosely fitting membranous integument; a complex cytoplasmic membrane of honeycomb-like structure; a fibrillar nucleoplasm and fibrillar cytoplasmic ground substance with associated ribosomes; lamellated or cytoplasmic granules bounded by a smooth membrane. At the flagellated pole the cytoplasmic ground substance was generally rarefied and a cone-shaped flagellar basal granule was present. The flagella were enlarged at their proximal terminus. Mesosome-like elaborations of the cytoplasmic membrane were not observed. It was concluded that Vibrio fetus closely resembles members of the genus Spirillum.

Methods are described for the preparation of cell-wall material and flagella from Vibrio fetus. In common with certain other Gram-negative organisms V. fetus apparently possesses a three-layer wall which, it is proposed, consists of an outer lipoprotein layer, a middle lipo-poly-saccharide layer, and an inner mucopeptide layer. These layers were studied chemically and by electron microscopy after various extraction and enzymic degradation procedures with whole-wall material. None of the layers of the wall was found to possess a microscopically discernible substructure, although the extracted lipopolysaccharide agglomerated into a characteristic stringy array. The probable function, contribution to cellular rigidity, and composition of the wall layers is discussed.