- Volume 49, Issue 3, 1967

The development of the septa which cut off the gametangia of Rhizopus sexualis from the suspensor cells was studied by electron microscopy. The edge of the ingrowing septum extends by the incorporation of coalesced vesicles which were previously aligned in the plane of the developing septum. The membranes surrounding these vesicles fuse to form the plasmalemmas of the gametangium and suspensor cell and are eventually continuous with the plasmalemma of the original progametangium. New wall material is laid down between these plasmalemmas. Associated with this process is a considerable increase in complexity of the endoplasmic reticulum which forms broad, irregular bands on either side of the septum. Fine tubules, resembling plasmodesmata, pass through the wall at intervals. Thickening of the septum proceeds more rapidly on the gametangial side.

Cell-wall fractions from Mycobacterium lepraemurium were examined chemically and immunologically. The major sugar and amino acid components detected were arabinose and galactose, alanine, glutamic acid, a,e-diaminopimelic acid (DAP) and hexosamine, but substantial amounts of aspartic acid, glycine, valine, serine, threonine, leucine and wo-leucine were also present in preparations not extracted with neutral lipid solvents and alkaline ethanol. However, after such extraction and treatment with proteolytic enzymes, alanine, glutamic acid and DAP (1·5:1·0:1·0) were present in much larger amounts than any other amino acids (not more than 0·15), indicating that these three are the mucopeptide constituents. Cell-wall agglutination tests indicated the presence of an antigen shared with other mycobacteria.

When grown in sterilized soil inoculated with an effective Rhizobium strain which produced many large pigmented nodules on virus-free plants, plants infected with clover phyllody virus (CPV) produced mainly small white nodules characteristic of an ineffective reaction. Mainly small nodules were also given by virus-free plants exposed to mixtures of effective and naturally ineffective Rhizobium strains, which separately gave predominantly large and predominantly small nodules, respectively, indicating that the ineffective strain had a high competitive ability for invasion sites on the roots.

When the CPV-infected plants were removed and fresh seed sown in the soil, germination was poor and the seedlings produced predominantly small nodules. The rhizobia seemed modified to a less effective form which produced mainly small nodules and competed successfully with unmodified bacteria. Yields of clover in swards may therefore be decreased by CPV infection, not only through a direct effect on plant growth but also through effects on the soil Rhizobium population, and hence on the growth of infected plants and neighbouring virus-free plants.

A medium was developed for the more rapid culture of Pityrosporum ovale. [35S]Methionine was utilized preferentially as sole sulphur source from which cysteine/cystine were synthesized. Growth was less on cysteine, negligible on cystine and nil on inorganic sulphite and sulphate. Selenium disulphide, amorphous sulphur, stable colloidal (red) selenium and selenourea were extremely toxic, entering the organism by diffusion. Tellurium and seleno-methionine entered the organism, the latter by active transport, but were not toxic for up to i week of incubation although synthesis of [75Se]seleno-methionine was demonstrated. Selenite and metallic (black) selenium were not toxic and did not enter the organism. Possible mechanisms of toxicity are discussed.

Cultural characteristics and serological relationships of mycoplasmas from the bovine vagina, mastitic milk, and other sources were studied. Parasitic strains could be distinguished from saprophytic strains in that they required incorporation of horse serum into the PPLO agar for growth, produced surface crystallization on PPLO agar and did not ferment carbohydrates. All strains were haemolytic for red blood cells of the guinea pig, rabbit, sheep, cow and horse. Satellite growth enhancement was seen only with the strains associated with mastitis.

Rabbit antisera to each mycoplasma were tested against homologous and heterologous strains for precipitins and for growth inhibitory antibodies. Four serotypes were distinguished among the bovine isolates by agar-gel double-diffusion technique. Growth inhibitory antibodies were detected in homologous antisera against only four of the strains studied.

Whole organisms and soluble extracts of a Pseudomonas sp. were examined for their ability to oxidize suspected intermediates in the oxidation of p-isopropyltoluene. A comparison of the relative activities of glucose-grown and p-isopropyltoluene-grown bacteria and their extracts indicated that p-isopropylbenzyl alcohol, p-isopropylbenzaldehyde and p-isopropyl-benzoic acid are intermediates in the oxidation of the hydrocarbon, p-isopropylbenzoic acid was formed during growth on p-isopropyltoluene and this was also shown to be a product of the oxidation of p-isopropyltoluene, p-isopropylbenzyl alcohol and p-isopropylbenzaldehyde by soluble extracts prepared from p-isopropyltoluene-grown bacteria. The enzymes which catalyze the oxidation of p-isopropyltoluene were repressed by glucose and induced by growth on either p-isopropyltoluene or p-isopropylbenzoic acid.

The progress of secretion of the extracellular enzymes ribonuclease, α-amylase and proteinase by Bacillus subtilis, growing in a defined and in a complex medium, containing maltose, starch, glycerol or glucose as carbon source, was followed. In either medium and in the presence of any of the carbon sources the general characteristics of secretion of all three enzymes were the same. There was a low but definite production of exoenzyme from the moment the cells started to grow until the end of the logarithmic phase after which, when the rate of increase in cell mass was decreased, the rate of enzyme secretion increased to a high linear value which was maintained even into the stationary phase.

The results are discussed in relation to a possible regulatory mechanism which might account for the observed characteristics of extracellular enzyme secretion. A mechanism is proposed whereby exoenzyme m-RNA formation and hence enzyme production is limited during growth by a limitation of nucleic acid precursors caused by the depletion of the precursor pool during rapid ribosomal RNA synthesis. When the growth rate is decreased, ribo-some synthesis is also decreased; the nucleic acid precursor pool may then increase in size, thereby removing the limitation so that exoenzyme m-RNA and protein may be formed at the maximum rate.

By gel-diffusion precipitin and absorption tests six antigens were identified in group D streptococci of human oral origin that were common to Haemophilus influenzae; four of these were common also to Mycoplasma hominis, and three to the l-form of a group A streptococcus. No cross-reactions were obtained with Corynebacterium xerosis or C. hofmannii. These antibodies occurred in commercial Streptococcus and Haemophilus typing sera, but not in enterobacterial agglutinating sera.

It should be concluded that the genus Haemophilus and Mycoplasma hominis are more closely related to the genus Streptococcus than to the genus Corynebacterium.

Organisms of Escherichia coli k-12 strains sensitive and resistant to acri-flavine were plated on media with and without acriflavine after growth in media containing different concentrations of glucose. Proportionally more organisms produced colonies, in the presence of acriflavine, after growth in media containing a high concentration of glucose than in media with lower glucose contents. The final pH value of the growth medium was low with the high glucose media. With the resistant strain, the number of bacteria which survived acriflavine increased as the final pH value of the medium from which bacteria were harvested was decreased, but the initial glucose concentration rather than the final pH value of the culture was more influential in increasing survival with the sensitive strain. The acriflavine-binding capacity of the bacteria was affected by the initial glucose concentration of culture medium probably indirectly through a change of pH. Acriflavine sensitivity of the bacteria varied with the amount of acriflavine bound. The acriflavine-binding capacity of bacteria modified by the pH value of culture medium was stabilized in the course of several doublings of bacteria in that medium. The glucose concentration of the medium affected the acriflavine sensitivity of the sensitive strain through some mechanism other than the change of pH.

The ability of Pasteurella tularensis to grow from small inocula (less than 105 organisms) was found to be critically dependent upon the supply of materials produced by this organism (growth-initiating substance, GIS). In contrast, large inocula showed no requirement for added GIS. The growth-promoting activity of GIS was enhanced in the presence of blood. The evidence favours the conclusion that the inoculum-dependent growth characteristics of P. tularensis are attributable to a nutritional heterogeneity of the bacterial population. According to this interpretation, GIS is produced by a relatively small number of bacteria in the population and is excreted by them into the surrounding culture fluid. The excreted material enables growth of the GIS-requiring organisms which constitute a major proportion of the total population. This commensal relationship appears to account for the characteristic all-or-none type of growth response elicited by GIS.

Material produced by growing Pasteurella tularensis and required for the initiation of growth of P. tularensis from small inocula (growth-initiating substance, GIS) was purified by Sephadex gel filtration and ion-exchange resin chromatography. The purified material was characterized as an anionic compound of low molecular weight, heat-stable at neutral pH and in dilute alkali but destroyed by heating in dilute acid. Out of large variety of known nutrients tested, only iron salts and some iron-chelating compounds (sider-amines) replaced partially GIS in supporting growth of different P. tularensis strains from small inocula. GIS formed complexes with iron and copper ions. The production of GIS by P. tularensis in a chemically defined medium was enhanced by added ornithine. Isotopic evidence established the role of ornithine as a biosynthetic precursor of GIS.

The pyruvate-consuming system of Aerobacter aerogenes, which is inhibited by bacteriostatic concentrations of tetracyclines in an unaerated mineral salt medium, was isolated as a cell-free extract. The isolated system was inhibited by bacteriostatic concentrations of eight tetracyclines. The inhibitory molecular form of each of these tetracyclines, all of which were acids releasing either two or three protons successively in aqueous solution, was the first dissociation product. Quantitatively the inhibition was directly dependent on the concentration of the inhibitory form of the antibiotic present. The tetracyclines tested were derived structurally from tetracycline by changes of the group attached at position 5, 6 or 7 (type a), a de-(dimethyl-amino) tetracycline (type b) and anhydrotetracycline. None of the tetracyclines was inhibitory in the absence of free magnesium ions. In the presence of micromolar concentrations of these ions, the inhibitory form of the tetracycline competed with flavine mononucleotide: the effectiveness of different tetracyclines as inhibitors of unaerated cultures correlated with the stability constants of postulated tetracycline–magnesium–enzyme complexes. The intracellular concentrations of tetracyclines in organisms of sensitive and resistant strains were determined by measurements of inhibition of the pyruvate-consuming system. In medium containing bacteriostatic concentrations of types a and b tetracyclines, the intracellular concentrations of the sensitive organisms were the same as the extracellular concentrations. Anhydrotetracycline did not penetrate these organisms so readily. One of the resistant strains was insensitive because the tetracyclines did not penetrate into the organisms. There were no intracellular accumulations of tetracyclines from medium containing bacteriostatic concentrations. The bearing of these results on current theories to account for the bacteriostatic actions of tetracyclines is discussed.

The activity of 75 strains of Acinetobacter (16 strains of A. Iwoffii and 59 strains of A. anitratus) against carbohydrates is described. It is concluded that no firm distinction can be made between these species which represent extreme ends of a group of organisms possessing widespread but variable activity.

The rates of destruction of benzylpenicillin, cephaloridine, cephalothin and (in some cases) cloxacillin by cocci from induced cultures of 17 strains of Staphylococcus aureus were measured. Rates of destruction of cephaloridine, relative in each case to those of benzylpenicillin, by the 10 methicillin-resistant strains were not significantly different from those by the 7 methicillin-sensitive strains. V max for cephaloridine and cephalothin were about 0·2 % and 0·009 %, respectively, that for benzylpenicillin; cloxacillin was destroyed still more slowly. The inactivation of the cephalosporins is almost certainly brought about by the action of penicillinase. Notwithstanding its greater stability to staphylococcal penicillinase, cephalothin is less effective than cephaloridine at suppressing the growth of the methicillin-resistant strains, although there is cross-resistance between methicillin, cloxacillin, cephalothin and cephaloridine.

The architecture of the cell wall of Bacillus megaterium strain m was studied by using electron microscopy of shadowed or negatively stained preparations, ultrathin sections and simple chemical methods. No conspicuous visible structure was found on the surface of whole organisms, isolated cell walls or the ‘mucopeptide membranes’. Hot formamide treatment decreased the thickness of cell walls by about 50 %, leaving behind a rigid ‘mucopeptide membrane’. The removed material is assumed to correspond with the ‘teichoic acid’ bound presumably by chemical linkage to the rigid layer, thus forming a plastic layer of the cell wall. The total thickness of the cell wall ranged from 200 to 240 Å, that of the rigid membranes from 90 to 110 Å. Poststaining of glutaraldehyde-fixed organisms or cell walls with osmium tetroxide, potassium permanganate or lead citrate produced a ‘triple layered’ cell-wall profile. This was not the case with uranyl acetate or phosphomolybdate staining, where the cell wall was represented by one homogeneous and dense track.

The lytic action of lysozyme upon Bacillus subtilis walls was studied by following the disappearance of bacillary-colony-forming units and the appearance of L-colony-forming-units. The rapidity of cell wall removal by lysozyme fluctuated markedly during growth in a chemically defined medium, presumably because subtle changes in the cell wall were constantly occurring. When lysozyme-sensitive bacilli were grown with chloram-phenicol 10 μg./ml. for 3 hr they showed a notable increase in lysozyme resistance; at the same time, their walls almost doubled in thickness. As lysozyme attack proceeded in a given culture, the bacilli passed first through a rod-shaped osmotically sensitive stage, and then a spherical stage characterized by incomplete removal of cell wall before finally reaching the naked protoplast stage. The spherical forms with adherent wall residues formed L colonies on a medium containing the reversion inhibitor D-methionine and bacillary colonies on the same medium without D-methionine. Under the latter conditions, the cell wall residue served as a starting point for rebuilding of complete wall, much as residual wall permits reversion of Gram-negative spheroplasts to the bacillary state. In the presence of d-methionine, the feedback sequence required for wall formation was severed, resulting in heritable propagation of the protoplast state.