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PubMed1963

Comparative study of lethal photosensitization of Sarcina lutea by 8-methoxypsoralen and by toluidine blue.

Mathews, Micheline M. (Department of Bacteriology, University of California, Berkeley). Comparative study of lethal photosensitization of Sarcina lutea by 8-methoxypsoralen and by toluidine blue. J. Bacteriol. 85:322-328. 1963.-A comparative study has been made of the photo-killing of Sarcina lutea by 8-methoxypsoralen (8-MOP) and by toluidine blue. It has been found that photosensitization by 8-MOP differs from photosensitization by toluidine blue, in that it has a temperature coefficient of less than one, and that the presence of oxygen is not necessary for, and even is deleterious to, the photosensitization, the psoralen being destroyed by its presence. It has previously been shown that the presence of carotenoid pigments protects the cells of S. lutea from lethal photosensitization by toluidine blue; it was found that the presence of these pigments has no protective effect in photosensitization with 8-MOP. Studies on the lethal photosensitization of S. lutea with toluidine blue suggested that the primary sensitive site of the photokilling was the protein of the cell membrane, as manifested by the destruction of membrane enzyme activity and the regulation of permeability. It has been found that photokilling by 8-MOP has no effect on these functions. A study was made on the effect of photokilling by 8-MOP on the production of penicillin-resistant mutants as an indication of an alteration in the cellular deoxyribonucleic acid (DNA) by the psoralen. Psoralen photosensitization resulted in the development of many penicillin-resistant mutants. On the basis of the findings reported in this paper, it is suggested that photosensitization of S. lutea by 8-MOP does not reflect damage to cellular protein, as does toluidine blue, but rather damage to cellular DNA.

Cell Membrane↗
PubMed1963

NATURE OF THE LIPIDS OF SOME LACTIC ACID BACTERIA.

Ikawa, Miyoshi (University of California, Berkeley). Nature of the lipids of some lactic acid bacteria. J. Bacteriol. 85:772-781. 1963.-Lactobacillus casei, L. plantarum, Leuconostoc mesenteroides, Pediococcus cerevisiae, and Streptococcus faecalis were grown on a lipid-free medium, and the amounts of acetone-soluble lipids, acetone-insoluble phospholipids, and unextracted or bound lipid in the cells were estimated. Neutral glycerides appeared to be absent, or present only in traces, in L. casei and S. faecalis. L. plantarum, L. mesenteroides, and P. cerevisiae, in addition to containing glycerides, appeared to contain glycolipids of glucose and galactose. Glycerol was present in the phospholipid fraction of all the organisms, and, in addition, galactose was present in L. mesenteroides. Paper chromatography of hydrolysates of the phospholipid fractions showed the absence of the usual phospholipid nitrogen bases, serine, ethanolamine, and choline, in these organisms. Microbiological assays of lyophilized unextracted cells for myo-inositol and choline also showed that lipids containing these components occurred in very small amounts at most. The principal ninhydrin-reacting substance in the phospholipid fraction of L. casei, L. plantarum, P. cerevisiae, and S. faecalis was lysine, which seemed to be exclusively of the l configuration. The principal ninhydrin-reacting substance in the L. mesenteroides phospholipid fraction was d-alanine. These amino acids appeared to be bound in components of the phospholipid fraction and not free.

Enterococcus faecalis↗
PubMed1963

FREE RADICAL FORMATION AND SURVIVAL OF LYOPHILIZED MICROORGANISMS.

Heckly, Robert J. (University of California, Berkeley), R. L. Dimmick, and J. J. Windle. Free radical formation and survival of lyophilized microorganisms. J. Bacteriol. 85:961-966. 1963.-A correlation between death and spontaneous free radical production, measured by an increase in the relative electron paramagnetic resonance (EPR) signal, was shown to exist for several species of microorganisms stored in the freezedried state, but the relationship between the free radical concentration and number of dead cells was not a simple proportion. Lactose added to Sarcina lutea reduced radical production and increased stability when dry preparations were stored in air. Death and free radical formation were more extensive when lyophilized Streptococcus lactis cultures were stored in air than in vacuum. Free radicals were also produced by dry yeast. Few, if any, free radicals were produced by bacteria or yeast stored in vacuum. It was shown that the observed free radical production was not caused by exposure to light. The EPR signal produced by dry Serratia marcescens decreased rapidly when cells were exposed to a humid atmosphere but the EPR signal slowly increased after cells were redried and exposed to oxygen.

Bacteria↗
PubMed1963

METABOLIC ROLE OF THE BR FACTOR IN BUTYRIBACTERIUM RETTGERI.

Kline, Leo (University of California, Berkeley), L. Pine, and H. A. Barker. Metabolic role of the BR factor in Butyribacterium rettgeri. J. Bacteriol. 85:967-975. 1963.-The BR factor, which is replaceable by lipoic acid, was shown to be required for the decomposition of lactate by Butyribacterium rettgeri. Since the factor was not required for the fermentation of pyruvate or glucose by this organism, the results indicate that BR factor is essential only for some reaction involved in the oxidation of lactate to pyruvate, possibly the transport of electrons from lactate to the ultimate anaerobic electron-acceptor systems. The same products, namely, acetate, butyrate, and carbon dioxide, are formed from glucose or pyruvate in the presence or absence of BR factor and from lactate in the presence of the factor. Thus the factor is not required for the conversion of glucose or pyruvate to acetate, butyrate, or carbon dioxide. Although B. rettgeri does not require the BR factor for growth on glucose, growth of lactate-adapted cells on glucose is slow and is markedly stimulated by addition of the factor. Such cells growing on glucose do not show any ability to decompose lactate; consequently, an additional function of the BR factor, probably associated with the conversion of glucose to pyruvate, is indicated. Cells fully adapted to glucose grow rapidly on this substrate and do not respond to the factor. Lactate is formed during growth of glucose-adapted cells on glucose, although such cells cannot decompose lactate.

Acetates↗
PubMed1963

STAPHYLOCOCCAL SENSITIZATION: SPECIFIC BIOLOGICAL EFFECTS OF PHAGE K ON THE BACTERIAL CELL WALL IN LYSIS-FROM-WITHOUT.

Ralston, Doris J. (University of California, Berkeley). Staphylococcal sensitization: specific biological effects of phage K on the bacterial cell wall in lysis-from-without. J. Bacteriol. 85:1185-1193. 1963.-Phage K, shown previously to sensitize staphylococcal-wall mucopeptide to the action of a phage-induced enzyme, virolysin, was found to act in a specific manner in that its sensitizing effects were restricted to chemical linkages affected by three staphylococcal lysins. These caused an immediate lysis, whereas egg-white lysozyme, which could also digest the wall mucopeptide, exerted variable effects, even when in the absence of phage it produced some lysis. Evidence was presented that the K(1) normal cell autolysin and the K phage virolysin could act synergistically with lysozyme on phage-sensitized cells, and that any effects observed with lysozyme were due to the simultaneous presence of trace amounts of these staphylococcal lysins. None of a series of lysozymelike agents from sea urchins, marine sepunculids, and from rabbit peritoneal histiocytes caused accelerated lysis of phage-sensitized cells, although like lysozyme they showed a slow lysis of phage-free living cells. Other enzymes which did not reduce the turbidity of sensitized cells included agents specific for intracellular components (proteins, lipids, nucleic acids), and enzymes, as decarboxylase, alkaline phosphatase, d-amino oxidase, and hyaluronidase. These results suggested that the main effects of the phage in sensitization were limited to areas of the cell wall involved in protection against the action of the staphylococcal lysins.

Animals↗
PubMed1963

INHIBITORY ACTION OF PHAGE K ON STAPHYLOCOCCAL DEHYDROGENASES. I. EFFECT ON VARIOUS STRAINS OF STAPHYLOCOCCUS AUREUS, INCLUDING MEMBERS OF THE PHAGE-TYPING SERIES.

Ralston, D. J. (University of California, Berkeley) and B. S. Baer. Inhibitory action of phage K on staphylococcal dehydrogenases. I. Effect on various strains of Staphylococcus aureus, including members of the phage-typing series. J. Bacteriol. 86:666-672. 1963.-The polyvalent phage K was found to depress the dehydrogenase activities of a large number of Staphylococcus aureus strains, as measured by the reduction of triphenyl tetrazolium chloride to the insoluble red formazan. The inhibition occurred immediately after the absorption of a multiplicity of phage particles, and was independent of the infectibility of the strains and of the killing ability of the phage. It appeared to be closely associated with the phenomenon of sensitization-a change in the cell surface which increased the susceptibility of the wall to digestion by soluble staphylococcal lysins and simultaneously abolished the capacity to synthesize phage. The inhibitory effect occurred both in nutrient media and in a nongrowth glucose - phosphate buffer supplemented with cysteine.

Bacteriophage Typing↗
PubMed1963

INHIBITORY ACTION OF PHAGE K ON STAPHYLOCOCCAL DEHYDROGENASES. II. ITS POSSIBLE RELATIONSHIP TO SENSITIZATION AND CELL LYSIS.

Ralston, D. J. (University of California, Berkeley) and M. D. Perry. Inhibitory action of phage K on staphylococcal dehydrogenases. II. Its possible relationship to sensitization and cell lysis. J. Bacteriol. 86:740-748. 1963.-By measuring the reduction of the dye triphenyl tetrazolium chloride to the insoluble red formazan, an analysis was made of the inhibition by phage K of the dehydrogenase capacity of populations of Staphylococcus aureus K(1), to determine to what extent this might be associated with the ability of phage K to sensitize the cells-a reaction characterized by the conversion of the cell wall to susceptibility to lysis by the staphylococcal enzyme, virolysin. Increasing multiplicities of phage progressively increased the fractions of sensitized cells and also caused increasing inhibition of the dehydrogenase activity of the populations. The dehydrogenase activities of the sensitized fractions were compared with those of the nonsensitized fractions. Over a wide range of phage-bacterium ratios, the dehydrogenase activities of the sensitized fractions were found to be lower than those of the nonsensitized fractions. Microscopically, this was reflected by the appearance of large numbers of cells with a reduced amount of visible formazan granules. When lysin was added to the phage-treated cells, lysis occurred mainly from cell fractions which possessed little or no tetrazolium-reducing capacity. The data indicated that sensitization by phage was accompanied by a marked decrease in cellular dehydrogenase activity but was not associated with a complete inhibition of these enzymes. A comparison was made between the dehydrogenases of phage-sensitized cells and cells found to be "spontaneously" sensitive to virolysin, i.e., lysed by the enzyme without any prior exposure to phage. Like phage-sensitized cells, the spontaneously sensitized staphylococci possessed low dehydrogenase activity and lacked the capacity to support phage synthesis. In tests of a given cell preparation, the dehydrogenase levels of the phage-sensitized fractions were found to be close to, or even lower than, the level of the spontaneously sensitized fraction, suggesting that in S. aureus K(1) the sensitized state is associated in some manner with a reduction of the dehydrogenase activity to a critical level. There is as yet no evidence for any direct causal relationship between sensitization and dehydrogenase inhibition.

Bacteriophages↗
PubMed1963

FINE STRUCTURE OF MYCOTA. XI. OCCURRENCE OF THE GOLGI DICTYOSOME IN THE HETEROBASIDIOMYCETE PUCCINIA PODOPHYLLI.

Moore, Royall T. (University of California, Berkeley). Fine structure of mycota. XI. Occurrence of the Golgi dictyosome in the heterobasidiomycete Puccinia podophylli. J. Bacteriol. 86:866-871. 1963.-The Golgi dictyosome is reported for the first time in the subclass Heterobasidiomycetidae in the rust Puccinia podophylli Schw. It is observed that the dictyosome appears to be limited to primordial cells, particularly those nearest the host, and does not occur in the aeciospores. In comparison with the Golgi systems of plant and animal cells, the Golgi complex of P. podophylli is found to be more similar to those of the latter. Also discussed is the distribution of the Golgi complex in fungi described in published reports and the need for more such reports so that the range of its variation and distribution may be established.

Animals↗
PubMed1964

NUTRITIONAL REQUIREMENTS OF LACTOBACILLUS 30A FOR GROWTH AND HISTIDINE DECARBOXYLASE PRODUCTION.

Guirard, Beverly M. (University of California, Berkeley), and Esmond E. Snell. Nutritional requirements of Lactobacillus 30a for growth and histidine decarboxylase production. J. Bacteriol. 87:370-376. 1964.-The nutritional requirements of Lactobacillus 30a include each of the naturally occurring amino acids, several B vitamins, ascorbic acid, glucose, acetate, and oleate. The nutritional requirements for optimal histidine decarboxylase production (up to 900 muliters of CO(2) per hr per mg of cells) differ to some extent from those for optimal growth. Wholly synthetic and partially defined media are described which produce high enzyme activity, together with rapid and luxuriant growth.

Amino Acids↗
PubMed1964

FACTORS AFFECTING THE FORMATION OF COBAMIDE COENZYMES IN CLOSTRIDIUM TETANOMORPHUM.

Toohey, J. I. (University of California, Berkeley), and H. A. Barker. Factors affecting the formation of cobamide coenzymes in Clostridium tetanomorphum. J. Bacteriol. 87:504-509. 1964.-Tests were carried out to determine the optimal culture conditions for the production of cobamide coenzymes in Clostridium tetanomorphum strain H1. A method is described for carrying out coenzyme determinations on the cells from 10-ml cultures of the bacterium. In a basal medium containing magnesium sulfate, ferrous sulfate, manganese sulfate, sodium molybdate, calcium chloride, and potassium phosphate, the optimal concentration of monosodium glutamate was 0.1 m and of yeast extract was 3 g per liter. Addition of glucose at a concentration of 0.05 m was found to double the yield of cells and to increase tenfold the specific coenzyme yield. Addition of cobaltous chloride (2 x 10(-5)m) also increased coenzyme production. Addition of benzimidazole caused an apparent increase in coenzyme production by causing the synthesis of the highly active benzimidazole analogue. Addition of methionine (5 x 10(-6)m) appeared to inhibit coenzyme production.

Bacteriological Techniques↗
PubMed1964

PLAQUE ASSAY PROCEDURE FOR COLORADO TICK FEVER VIRUS.

Deig, E. Frank (University of California, Berkeley), and H. M. S. Watkins. Plaque assay procedure for Colorado tick fever virus. J. Bacteriol. 88:42-47. 1964.-A reproducible plaque assay procedure is described for the quantitation of Colorado tick fever virus in a cell line established from embryonic hamster tissue. Under the best conditions, plaques approximately 4 mm in diameter were formed after incubation at 37 C of 4 to 6 days. Several environmental variables in the procedure were studied. Efficiency was increased markedly by combining the virus during adsorption with serum proteins, and by carrying out this step at 25 C rather than at 37 C. The overlay medium used contained metabolites which promoted cell viability for periods greater than 1 week, and allowed plaques to develop. Plaque formation was relatively insensitive to a variation in pH between 7.1 and 8.1 (with optimal concentrations of bicarbonate). However, plaque development was inhibited with medium containing greater than 0.22% bicarbonate (at optimal pH), or when the initial pH was less than 7.0.

Adsorption↗
PubMed1964

EFFECT OF POLYAMINE STRUCTURE ON GROWTH STIMULATION AND SPERMINE AND SPERMIDINE CONTENT OF LACTIC ACID BACTERIA.

Guirard, Beverly M. (University of California, Berkeley), and Esmond E. Snell. Effect of polyamine structure on growth and spermine and spermidine content of lactic acid bacteria. J. Bacteriol. 88:72-80. 1964.-Growth from small inocula of six species of lactobacilli was stimulated by addition of spermine or spermidine to a defined medium; none of four streptococcal species showed this effect. Lactobacillus casei was stimulated to the greatest extent. Several homologues and analogues partially duplicated the growth-promoting effects of spermidine; one inactive homologue, N-(3-aminopropyl)-1,6-hexanediamine, competitively inhibited the growth response to spermidine and spermine, and reduced or eliminated the response to several weakly active compounds. A procedure for separation of spermine and spermidine, and their estimation by bioassay with L. casei, was developed and applied to the estimation of these compounds in bacterial cells. Both compounds are present in lactic acid bacteria in amounts much smaller (1 to 5%) than those found in Escherichia coli. Addition of spermine or spermidine to the growth medium results in large accumulations in the cells, and the two amines show limited interconvertibility. Putrescine does not increase the cell content of either spermine or spermidine. Presence of the inhibitor prevents accumulation of the growth-stimulating amines. The polyamines appear to fill at least two valuable roles in the cell, one relatively nonspecific in its structural requirements, and one filled only by spermine and spermidine or their nearest homologues. N-(3-aminopropyl)-1,6-hexanediamine appears to prevent the latter function by competition for an appropriate cellular receptor.

Amines↗
PubMed1964

CELL-WALL LYSINS OF STAPHYLOCOCCUS AUREUS STRAINS INDUCED BY SPECIFIC TYPING PHAGES.

Ralston, Doris J. (University of California, Berkeley), and Mary McIvor. Cell-wall lysins of Staphylococcus aureus strains induced by specific typing phages. J. Bacteriol. 88:667-675. 1964.-At least 12 different phages induced the formation of soluble lysins (separable from the phages by ultracentrifugation). The lysins caused rapid clearing of heat-killed cells of all strains of Staphylococcus aureus tested, irrespective of the capacity of the phage to form plaques on living cells of the strain. The uninfected cells of the 12 strains contained a second lysin, an autolysin, released upon cellular autolysis. The autolysin preparations differed from the phage-induced lysins, in that they exhibited relatively high activity for lysing Micrococcus lysodeikticus and low activity for strain S. aureus K(1N), and were each specifically inhibited by antiserum prepared against purified autolysin from strain K(1). A third kind of lysin, virolysin, induced by polyvalent phage K, was differentiated from the lysins of the specific phages on the basis of its antigenic specificity and lack of action on M. lysodeikticus. All three kinds of lysins digested the mucopeptide portion of staphylococcal cell walls. No evidence was found that any of these lysins possessed specific host ranges which could be correlated with the lytic host range of the inducing phage.

Animals↗
PubMed1964

LYSIS-FROM-WITHOUT OF STAPHYLOCOCCUS AUREUS STRAINS BY COMBINATIONS OF SPECIFIC PHAGES AND PHAGE-INDUCED LYTIC ENZYMES.

Ralston, Doris J. (University of California, Berkeley) and Mary McIvor. Lysis-from-without of Staphylococcus aureus strains by combinations of specific phages and phage-induced lytic enzymes. J. Bacteriol. 88:676-681. 1964-Several typing phages, adsorbed in sufficient concentrations to their homologous propagating strains, altered the cell surface so as to render the cells sensitive to rapid and synergistic lysis by extra-cellular additions of wall lysins. Lysis was effected both by lysins induced by the individual phages and by phage K(1) virolysin. Phage K(1) also rendered cells sensitive to the lysins of the typing phages. With the exception of lysins from PS 53, 70, and 77, none of the lysins nor purified phages tested separately caused significant lysis of living cells. Lysis-from-without in Staphylococcus aureus appears to be a stepwise process: sensitization by phage followed by digestion of the wall by lysin.

Bacteriolysis↗
PubMed1964

ENERGY REQUIREMENT FOR THYMINELESS DEATH IN CELLS OF ESCHERICHIA COLI.

Freifelder, David (University of California, Berkeley), and Ole Maaløe. Energy requirement for thymineless death in cells of Escherichia coli. J. Bacteriol. 88:987-990. 1964.-Thymineless death in thymine-requiring Escherichia coli is arrested immediately and reversibly by nitrogenation if the bacterial population is growing in a medium containing a carbon source that can only be metabolized aerobically. The mechanism of death, therefore, involves a metabolic process.

Carbon↗
PubMed1964

DIFFERENTIATION OF EXOTOXIN AND OTHER BIOLOGICALLY ACTIVE SUBSTANCES IN PSEUDOMONAS PSEUDOMALLEI FILTRATES.

Heckly, Robert J. (University of California, Berkeley). Differentiation of exotoxin and other biologically active substances in Pseudomonas pseudomallei filtrates. J. Bacteriol. 88:1730-1736. 1964.-Denaturing agents such as phenol, formaldehyde, and urea reduced lethal toxicity and proteolytic activity of partially purified preparations from Pseudomonas pseudomallei at about the same rate. Neither toxin nor enzyme was stable at pH 11, when the solution was adjusted with sodium hydroxide, but there was a slight difference in their rates of inactivation. However, under certain conditions, ammonium hydroxide destroyed most of the enzymatic activity with only a slight effect on lethality. Conversely, toxin was less stable in acid solutions than was the enzyme. Thus, treatment with ammonium hydroxide or acetic acid yielded preparations with either a low or a high enzyme-to-toxin ratio, indicating that lethality was not dependent on enzyme activity. Although proteolysis of any one of the essential factors in the blood coagulation system can inhibit clotting of blood, the potent anticoagulant activity of culture filtrates was not associated with its proteolytic activity, but was directly correlated with lethal toxicity. It is of considerable interest that the necrotoxicity was, however, associated with enzymatic activity and not with lethality. Serological reactivity of the enzyme, as well as its proteolytic activity, was altered by ammonium hydroxide. Similarly, antigenicity and toxicity of the lethal toxin were reduced by acidification. Each acid- or alkali-treated preparation produced a single precipitin line in double diffusion in agar when reacted with antisera produced by injection of crude filtrate. Partially purified preparations, having both lethal and enzymatic activity, produced two lines, one identifiable with the enzyme preparation, and one with the toxin. Furthermore, specific precipitation with the respective antisera removed either enzyme or toxin from crude preparations. Therefore, the lethal exotoxin and proteolytic enzyme are separable entities.

Animals↗
PubMed1965

FORMATION AND INTERRELATIONSHIPS OF TRYPTOPHANASE AND TRYPTOPHAN SYNTHETASES IN ESCHERICHIA COLI.

Newton, W. Austin (University of California, Berkeley), and Esmond E. Snell. Formation and interrelationships of tryptophanase and tryptophan synthetases in Escherichia coli. J. Bacteriol. 89:355-364. 1965.-In addition to the classical tryptophan-repressible tryptophan synthetase (TSase-tr), tryptophan auxotrophs of Escherichia coli contain another distinct tryptophan synthetase (TSase-ti) which is induced by tryptophan and is identical with tryptophanase (TPase). Escherichia coli B (wild type) forms only TSase-tr when the growth medium lacks tryptophan. When tryptophan is supplied, parallel induction of TPase and TSase-ti occurs while TSase-tr is repressed. Antiserum prepared against purified TPase neutralized TPase and TSase-ti equally, but not TSase-tr. TPase-negative strains of E. coli do not form TSase-ti. Unlike TSase-tr, TSase-ti is not readily detected by whole-cell assays. In the tryptophan auxotroph, E. coli B/1t7, a direct correlation exists between the effectiveness of 4-, 5-, and 6-methyl-tryptophan in inducing TPase and in promoting growth in the presence of indole. In a mutant of this organism, E. coli B/1t7-A, which is constitutive for TPase, 5-methyl-tryptophan and other substrates of TPase increased the rate of growth on limiting indole, a result ascribed to their ability to inhibit degradation of tryptophan and to supply the 3-carbon side chain for synthesis of tryptophan by TPase. This organism produced maximal amounts of TPase when inocula from log-phase cells grown in tryptophan-supplemented minimal medium were allowed to undergo two cell generations in an enriched broth medium.

Culture Media↗
PubMed1965

ENZYMATIC ENHANCEMENT OF INFECTIVITY OF REOVIRUS.

Spendlove, R. S. (California State Department of Public Health, Berkeley), and F. L. Schaffer. Enzymatic enhancement of infectivity of reovirus. J. Bacteriol. 89:597-602. 1965.-Enhancement of infectivity by chymotrypsin treatment has been demonstrated with all three types of reovirus, although not in all viral preparations. Enzyme treatment did not produce a simultaneous increase in the hemagglutinating activity of reovirus type 1 (the only type tested). The infectivity of reovirus type 1 (Lang strain) was increased by treatment with chymotrypsin, trypsin, papain, and a filtrate from a culture of a Pseudomonas sp. but not by treatment with pepsin. Sedimentation experiments showed that the property of enhanceability was closely associated with the virus particles themselves. Results of studies involving various sequential treatments with chymotrypsin, and with heat in the presence or absence of 2 m MgCl(2), were compatible with the interpretation that inhibited virus is resistant to exposure to a temperature of 56 C in the absence of MgCl(2), whereas activated virus is thermolabile. Activation of reovirus infectivity by heat in the presence of MgCl(2) and by chymotrypsin was not additive.

California↗