- Volume 58, Issue 3, 1969

The polar carotenoid fraction from Sarcina flava does not not appear to be a free carotenoid, but a carotenoid associated with glucose and a peptide. The glucose seems to be glycosidically bound to the carotenoid through its reducing group. Presumably the peptide is linked to the glucose through either the primary or one of the secondary hydroxyl groups on the carbohydrate molecule. A model of the in vivo state of the carotenoid complex in the bacterial membrane has been proposed.

Carbenicillin, a 9-alpha carboxybenzyl penicillin, is bactericidal only for Escherichia coli strains which are resistant to less than 125 μg./ml. of ampicillin. All E. coli and Salmonella typhimurium strains in which penicillinase is a surface enzyme are resistant to carbenicillin. E. coli and S. typhimurium strains in which production of beta-lactamase is episomally mediated are resistant to both ampicillin and carbenicillin. A non-hydrolyzable penicillin (methicillin or dicloxacillin) does not allow carbenicillin to exert its antibacterial effect against resistant strains of E. coli, Pseudomonas, Klebsiella or Enterobacter. Carbenicillin shows no synergy with penicillinase-resistant penicillins.

Cell walls were prepared mechanically from both hyphae and conidia of Penicillium chrysogenum. The major carbohydrate components of cell wall hydrolysates were glucose, galactose and glucosamine, with lesser amounts of mannose and rhamnose. The conidia were richer in galactose and the hyphae contained more glucosamine. The distribution of the monomers in acid-soluble and alkali-soluble fractions was different in the two kinds of cell wall; conidial walls contained higher concentrations and a greater variety of amino acids than hyphal walls.

Proflavine in the post-aerosolization plating medium lowered by over 80% the number of viable Escherichia coli b/r organisms recovered from aerosols of 55% relative humidity (RH) and above, but had no effect on the recovery of viable E. coli wp2s organisms. Oxygen enhanced the lethal action of semi-dehydration on E. coli strain b/r, held at 50% RH and below, but had less effect on the survival of E. coli wp2s. In addition, E. coli b/r organisms displayed an increased rate of respiration, and discharged 260 mμ absorbing material, when held at intermediate values of RH. These phenomena were not observed with E. coli wp2s organisms. Thus E. coli b/r seems to possess an energy consuming mechanism by which it is able to survive semi-dehydration above 55% RH by a discharge of damaged cell components. This mechanism is very slowly destroyed when the organisms are semi-dehydrated in nitrogen but, below 50% RH, it is rapidly inactivated by oxygen. It is suggested that the mechanism is a membrane-bound system, possibly involving the cytochrome chain.

L-phase variants of Streptobacillus moniliformis and Proteus mirabilis resembled mycoplasmas in their ability to incorporate considerable amounts of exogenous cholesterol into their cell membranes. Much smaller quantities were incorporated by L-phase variants of Streptococcus pyogenes and Staphylococcus aureus, by the parent bacteria of all L-phase variants except S. moniliformis, and by a series of wall-covered bacteria, including their protoplasts and spheroplasts. Cholesterol-binding ability corresponded roughly to sensitivity to growth inhibition and lysis by digitonin. Like myco-plasma, the S. moniliformis L-phase had a non-enzymic cholesterol uptake mechanism, but its growth in a cholesterol-free medium was not improved by adding cholesterol. It is concluded that a bacterium, on transforming to its stable L-phase, does not necessarily develop the ability to bind cholesterol; if it does, the cholesterol bound is not essential for growth of the L-phase, unlike cholesterol-requiring mycoplasmas.

The hyphal cross-walls in Endomycopsis platypodis had dolipores and, depending on the conditions of growth, dolipores were sometimes present in the walls between budding cells also. The formation of walls, both in hyphae and between budding cells, was initiated by an electron-light primary wall. This was later thickened at both sides with electron-dense material, and the dolipore exhibited an extra swelling around the pore channel. Buds separated from their parents at the primary wall, which remained attached to the mother cell.

Streptococcus lactis organisms were grown in lactose-limited batch culture and transferred, after washing, to phosphate buffer at the growth temperature. Soluble protein was released from viable organisms into the suspending buffer and the intracellular free amino acid pool declined steadily with the components appearing in the suspending buffer; a net increase in the total amount of free amino acid indicated some protein hydrolysis. RNA was hydrolysed, resulting in the release of u.v.-absorbing bases and ribose from the organisms. Conditions which promoted rapid RNA breakdown also produced rapid death rates and long cell division lags in surviving organisms. After 28 hr starvation in buffer containing Mg2+, the bacterial dry wt decreased by 26 %; loss of RNA, protein and free amino acids accounted for 10·3 %, 7·3 % and 2·7 % of the total bacterial mass loss. The products of polymer hydrolysis appeared to be released in an undegraded form into the external buffer and there was no appreciable formation of lactate, ammonia or volatile fatty acids, possibly indicating the absence of any important endogenous energy sources. There was no appreciable degradation of DNA or carbohydrate but phospholipid was broken down on prolonged starvation. No polyglucose or poly-β-hydroxybutyrate was detected in the organisms.

Protein synthesis, as estimated from incorporation studies with [14C]valine, was barely detectable when organisms were starved in phosphate buffer containing Mg2+. The addition of an energy source promoted limited protein synthesis. In this respect glucose produced a much higher rate of [14C]valine uptake and incorporation than arginine. Although arginine prolonged survival, experiments with inhibitors showed that this was unlikely to be due to the protein synthesis which had been promoted. The ability of starved organisms to assimilate [14C]valine and to incorporate the isotope into protein in the presence of glucose appeared to be correlated with survival.

Synthesis of RNA in starved organisms, as determined from [14C|uracil incorporation experiments, depended on exogenous glucose. During brief exposures to [14C]uracil plus glucose at intervals during starvation, organisms incorporated decreasing amounts of isotope into RNA although incorporation by non-viable organisms occurred at approximately half the initial rate. Although resynthesis of both protein and RNA was demonstrated with added energy sources, no evidence was obtained to suggest that this favoured survival.

Arginine or glucose accelerated death of Streptococcus lactis starved in phosphate buffer. Both substrates were metabolized at linear but different rates and the rate of ATP production from glucose metabolism was about 7·5 times that for arginine metabolism. Mg2+ reduced glucose-accelerated death and abolished arginine-accelerated death to prolong survival. When glucose was fed continuously to organisms starved in phosphate buffer containing Mg2+, survival was related to the rate of glucose addition and hence to the rate of metabolism. By feeding low concentrations of glucose, the viability of most of the population was extended from about 1 to 4 days. The maintenance energy requirement appeared to be in the region of 0·045 to 0·09 mg. glucose/mg. dry wt bacteria/hr. The glycolytic activity of starved organisms declined at varying rates depending on the starvation environment but could not be correlated directly with loss of viability. Exogenous arginine substantially reduced the lethal effect of adverse pH values.

When Streptococcus lactis was starved in phosphate buffer the intracellular amino acid pool was rapidly released into the external medium from the onset of starvation whereas lactic dehydrogenase and DNA appeared in the suspending buffer only as organisms began to lose viability. Addition of spermine enhanced survival and suppressed the release of ultraviolet-absorbing material.

Thin sections of Streptococcus lactis were examined by electron microscopy at intervals during starvation in a number of environments. Ribosome particles rapidly disappeared from organisms starved in the absence of Mg2+ and non-viable organisms remained structurally intact; in the presence of Mg2+ ribo-somes were retained but, after prolonged starvation, some organisms autolysed soon after the death rate increased. Addition of a suitable energy source maintained cell structures intact for a much longer period but again lysis occurred as the death rate increased. Starvation led to unfolding or extrusion of the mesosomes and dislocation of the nuclear material. The death rate of starved S. lactis organisms in phosphate buffer partly depended on the presence of Mg2+, which probably acted by promoting polymer stability, particularly RNA. In this environment, a suitable exogenous energy source further enhanced survival which may ultimately be a function of cell wall and/or membrane stability.

Axenic combinations of Peronospora parasitica and callus of Brassica oleracea var. capitata, Drumhead cabbage, and other Brassica species were established. The fungus grew well in callus culture and sometimes had many of the characteristics of an aggressive parasite, in contrast with its growth habit in intact Brassica cotyledons. The susceptibility of different Brassica callus clones and root organ cultures to P. parasitica was not constant, and results obtained with callus were frequently at variance with results obtained using intact Brassica plant parts.

Toxin synthesis in restricted-iron batch cultures of Corynebacterium diphtheriae cn2000 (a pw8 s pd strain) was proportional to the bacterial mass above a minimum bacterial concentration. During glucose-limited growth 0·30 g. toxin was released/g. bacterial protein synthesized. In excess of glucose 0·06 g. toxin was released/g. bacterial protein synthesized. Toxin release in glucose-limited continuous cultures followed similar kinetics to the batch cultures.

Iron in the restricted-iron medium, 6 μg. atom/l., limited the quantities of iron taken up by the bacteria and of three iron-containing enzymes, but did not restrict bacterial concentration over the range 2·0–4·5 mg. bacterial protein/ml. The addition of 90 μg. atom Fe/l. to a restricted-iron culture rapidly inhibited toxin release.

In the presence of excess iron diphtherial toxin was not produced by Corynebacterium diphtheriae cn2000. 2·8 % of the intracellular iron was present in the soluble fraction of washed bacteria; the remainder was bound on the respiratory particles, 4 % as haeme and the rest as non-haeme iron. Succinate but not NADH reduced the cytochromes and a small proportion of the non-haeme iron.

Toxin was synthesized at a high rate by bacteria containing 6·5 % of the iron present in excess-iron bacteria. Under such iron-deficient conditions the iron-containing components of the respiratory particles were present in decreased quantities. NADH oxidation, which proceeded via a soluble flavoprotein, was not affected by iron-deficiency, nor could reduction of non-haeme iron by NADH be demonstrated. The transition between the iron-deficient and the excess-iron states was studied.