Showing papers in "Journal of Bacteriology in 1973"

Solubilization of the Cytoplasmic Membrane of Escherichia coli by the Ionic Detergent Sodium-Lauryl Sarcosinate

Abstract: The sensitivity of the outer and cytoplasmic membranes of Escherichia coli to detergent was examined by isopycnic sucrose density gradient centrifugation. Sodium lauryl sarcosinate (Sarkosyl) was found to disrupt the cytoplasmic membrane selectively under conditions in which Triton X-100 and dodecyl sodium sulfate solubilized all membrane protein. These results were verified by gel electrophoresis; membrane proteins solubilized by Sarkosyl were identical to those of the cytoplasmic membrane. The presence of Mg2+ during treatment with Sarkosyl was found to afford partial protection of the cytoplasmic membrane from dissolution.

General Method for the Isolation of Plasmid Deoxyribonucleic Acid

Abstract: Plasmid deoxyribonucleic acid (DNA) ranging from 5 × 106 to 65 × 106 daltons may be isolated from chromosomal DNA by the preferential precipitation of the higher-molecular-weight chromosomal DNA in the presence of sodium lauryl sulfate and a high concentration of NaCl.

Ecology of Vibrio parahaemolyticus in Chesapeake Bay

Abstract: A study of the ecology of Vibrio parahaemolyticus and related vibrios in the Rhode River area of Chesapeake Bay was carried out over the period December 1970 through August 1971. The incidence of V. parahaemolyticus and related vibrios was found to be correlated with water temperature. The vibrios could not be detected in the water column during the winter months, although they were present in sediment. From late spring to early summer, when water temperatures were 14 ± 1 C, vibrios over-wintering in sediment were released from the bottom communities and attached to zooplankton, proliferating as the temperature rose. The number of vibrios in and on plankton was reflected in the water column bacterial population densities at water temperatures of ca. 19 C. Thus, temperature of the water column in the range of 14 to 19 C was found to be critical in the annual cycle of the vibrios. Interaction between sediment, water, and zooplankton was found to be essential in the natural estuarine ecosystem. Bacterial counts of zooplankton were found to be temperature dependent. The bacterial population associated with zooplankton was found to be predominantly on external surfaces and was specific, differing from that of the sediment. Vibrio spp. and related organisms comprised the total bacterial population associated with zooplankton in summer months. The ecological role of Vibrio spp., including V. parahaemolyticus, was found to be significant, with respect to their property of chitin digestion and in relation to the population dynamics of zooplankton in Chesapeake Bay.

Production and Characterization of the Slime Polysaccharide of Pseudomonas aeruginosa

Abstract: The slime polysaccharides produced by Pseudomonas aeruginosa isolated from a variety of human infections were investigated. Slime production in culture seemed optimal when adequate amounts of carbohydrate were present and under conditions of either high osmotic pressure or inadequate protein supply. The polysaccharides produced by the organisms were similar to each other, to the slime of Azotobacter vinelandii, and to seaweed alginic acids. They were composed of beta-1,4-linked d-mannuronic acid residues and variable amounts of its 5-epimer l-guluronic acid. All bacterial polymers contained o-acetyl groups which are absent in the alginates. The polysaccharides differed considerably in the ratio of mannuronic to guluronic acid content and in the number of o-acetyl groups. The particular composition of the slime was not found to be characteristic for the disease process from which the mucoid variants of P. aeruginosa were obtained.

Isolation of the Bacteriophage Lambda Receptor from Escherichia coli

Abstract: A factor which inactivates the phage lambda can be extracted from Escherichia coli. This factor is a protein and is located in the outer membrane of the bacterial envelope. It is found in extracts of strains which are sensitive to phage lambda, but not in extracts of strains specifically resistant to this phage. We conclude that this factor is the lambda receptor, responsible for the specific adsorption of the phage lambda to E. coli cells. A partial purification of the lambda receptor is described. Inactivation of the phage by purified receptor is shown to be accompanied by the release of deoxyribonucleic acid from the phage.

Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex.

Abstract: Colicinogenic factors ColE1 and ColE2 are bacterial plasmids that exist in Escherichia coli as supercoiled deoxyribonucleic acid (DNA) and as strand-specific, relaxation complexes of supercoiled DNA and protein. Newly replicated ColE1 DNA becomes complexed with protein after the replication event. This association of DNA and protein can take place under conditions in which DNA or protein synthesis is arrested. The addition of cyclic adenosine monophosphate (c-AMP) to normal cells growing in glucose medium results in a six- to tenfold stimulation in the rate of synthesis of the protein component(s) of the complex and a three- to fivefold stimulation in the rate of ColE1 DNA replication. Employing mutants deficient in catabolite gene activator protein or adenylate cyclase, it was shown that synthesis of both the plasmid-determined protein colicin E1 and the protein component(s) of the ColE1 relaxation complex is mediated through the c-AMP-catabolite gene activator protein system. Addition of c-AMP to ColE2-containing cells results in the stimulation of synthesis of ColE2 DNA and relaxation protein(s) as well as in the production of a protein component of the ColE2 relaxation complex that renders it sensitive to induced relaxation by heat treatment. In the case of ColE2, synthesis of the relaxation protein(s) is not dependent upon catabolite gene activator protein.

Transmissible Plasmid Coding Early Enzymes of Naphthalene Oxidation in Pseudomonas putida

Abstract: The capacity of Pseudomonas putida PpG7 (ATCC 17,485) to grow on naphthalene, phenotype Nah+, is lost spontaneously, and the frequency is increased by treatment with mitomycin C. The Nah+ growth character can be transferred to cured or heterologous fluorescent pseudomonads lacking this capacity by conjugation, or between phage pf16-sensitive strains by transduction. After mutagenesis, strains can be selected with increased donor capacity in conjugation. Clones which use naphthalene grow on salicylate and carry catechol 2,3-oxygenase, the initial enzyme of the aromatic α-keto acid pathway, whereas cured strains grow neither on salicylate nor naphthalene and lack catechol 2,3-oxygenase, but retain catechol 1,2-oxygenase and the aromatic β-keto adipate pathway enzymes.

Isolation of Deoxyribonucleic Acid Methylase Mutants of Escherichia coli K-12

Abstract: Fourteen deoxyribonucleic acid (DNA) and 10 ribonucleic acid (RNA) methylation mutants were isolated from Escherichia coli K-12 by examining the ability of nucleic acids prepared from clones of unselected mutagenized cells to accept methyl groups from wild-type crude extract. Eleven of the DNA methylation mutants were deficient in 5-methylcytosine (5-MeC) and were designated Dcm. Three DNA methylation mutants were deficient in N6-methyladenine (N6-MeA) and were designated Dam. Extracts of the mutants were tested for DNA-cytosine:S-adenosylmethionine and DNA-adenine:S-adenosylmethionine methyltransferase activities. With one exception, all of the mutants had reduced or absent activity. A representative Dcm mutation was located at 36 to 37 min and a representative Dam mutation was located in the 60-to 66-min region on the genetic map. The Dcm mutants had no obvious associated phenotypic abnormality. The Dam mutants were defective in their ability to restrict λ. None of the mutations had the effect of being lethal.

Use of a single-strand specific nuclease for analysis of bacterial and plasmid deoxyribonucleic acid homo- and heteroduplexes.

Abstract: Bacterial and plasmid homo- and heteroduplexes have been analyzed with a single-strand specific endonuclease, S1, of Aspergillus oryzae. Under appropriate assay conditions, there was a high degree of correlation between the degree of deoxyribonucleic acid (DNA)-DNA homoduplex formation assessed by the S1 endonuclease and by hydroxyapatite (HA). Heteroduplexes which contain extensive regions of polynucleotide sequences in common are similarly recognized by the S1 endonuclease and HA. In instances where there is little or imperfect complementarity between heterologous DNA strands, the S1 endonuclease and the HA method give slightly different estimates. From DNA duplex thermal stability experiments assayed with the S1 endonuclease, there is preliminary evidence that well-matched sequences identified by the enzyme are not similarly recognized by HA. The assay of homo- and heteroduplexes with the S1 endonuclease permits an accurate, reproducible and rapid determination of polynucleotide sequence relationships and may be seriously considered as a method of choice for survey work and for investigations which require a large number of DNA-DNA hybridization assays.

Rapid mapping of conditional and auxotrophic mutations in Escherichia coli K-12.

Abstract: The approximate genetic map locations of auxotrophic and conditional lethal mutations of Escherichia coli can be rapidly determined with replica plating techniques. A set of patches of 15 streptomycin-sensitive (Str(S)) Hfr strains with points of origin distributed around the map is replica plated onto a recombinant-selective plate with a lawn of Str(R) cells which carry an unmapped mutation. The map interval defined by the Hfr points of origin which are closest to the mutant locus is seen by the presence or absence of heavy patches of recombinants produced by transfer of early wild-type genes from the Hfrs. An alternative method is to replicate patches of different mutant strains (100 per plate) onto Hfr lawns; in this case more than 1,000 different mutants can be mapped in a single experiment in a few days. In this way, many types of mutations with similar phenotypes can be grouped as to approximate location on the genetic map. For ordering mutations within groups, the same replica plating methods can be used to cross F-prime derivatives of mutants with other mutants of the same group. Relative merits of these and other mapping methods of E. coli are discussed.

Induction of Superoxide Dismutase by Molecular Oxygen

Abstract: Oxygen induces superoxide dismutase in Streptococcus faecalis and in Escherichia coli B. S. faecalis grown under 20 atm of O2 had 16 times more of this enzyme than did anaerobically grown cells. In the case of E. coli, changing the conditions of growth from anaerobic to 5 atm of O2 caused a 25-fold increase in the level of superoxide dismutase. Induction of this enzyme was a response to O2 rather than to pressure, since 20 atm of N2 was without effect. Induction of superoxide dismutase was a rapid process, and half of the maximal level was reached within 90 min after N2-grown cells of S. faecalis were exposed to 20 atm of O2 at 37 C. S. faecalis did not contain perceptible levels of catalase under any of the growth conditions investigated by Stanier, Doudoroff, and Adelberg (23), and the concentration of catalase in E. coli was not affected by the presence of O2 during growth. S. faecalis, which had been grown under 100% O2 and which therefore contained an elevated level of superoxide dismutase, was more resistant of 46 atm of O2 than were cells which had been grown under N2. E. coli grown under N2 contained as much superoxide dismutase as did S. faecalis grown under 1 atm of O2. The E. coli which had been grown under N2 was as resistant to the deleterious effects of 50 atm of O2 as was S. faecalis which had been grown under 1 atm of O2. These results are consistent with the proposal that the peroxide radical is an important agent of the toxicity of oxygen and that superoxide dismutase may be a component of the systems which have been evolved to deal with this potential toxicity.

Chemotaxis Toward Sugars in Escherichia coli

Abstract: Using a quantitative assay for measuring chemotaxis, we tested a variety of sugars and sugar derivatives for their ability to attract Escherichia coli bacteria. The most effective attractants, i.e., those that have thresholds near 10(-5) M or below, are N-acetyl-d-glucosamine, 6-deoxy-d-glucose, d-fructose, d-fucose, 1-d-glycerol-beta-d-galactoside, galactitol, d-galactose, d-glucosamine, d-glucose, alpha-d-glucose-1-phosphate, lactose, maltose, d-mannitol, d-mannose, methyl-beta-d-galactoside, methyl-beta-d-glucoside, d-ribose, d-sorbitol, and trehalose. Lactose, and probably d-glucose-1-phosphate, are attractive only after conversion to the free monosaccharide, while the other attractants do not require breakdown for taxis. Nine different chemoreceptors are involved in detecting these various attractants. They are called the N-acetyl-glucosamine, fructose, galactose, glucose, maltose, mannitol, ribose, sorbitol, and trehalose chemoreceptors; the specificity of each was studied. The chemoreceptors, with the exception of the one for d-glucose, are inducible. The galactose-binding protein serves as the recognition component of the galactose chemoreceptor. E. coli also has osmotically shockable binding activities for maltose and d-ribose, and these appear to serve as the recognition components for the corresponding chemoreceptors.

Glucose Fermentation Products of Ruminococcus albus Grown in Continuous Culture with Vibrio succinogenes: Changes Caused by Interspecies Transfer of H2

Abstract: The influence of a H2-utilizing organism, Vibrio succinogenes, on the fermentation of limiting amounts of glucose by a carbohydrate-fermenting, H2-producing organism, Ruminococcus albus, was studied in continuous cultures. Growth of V. succinogenes depended on the production of H2 from glucose by R. albus. V. succinogenes used the H2 produced by R. albus to obtain energy for growth by reducing fumarate in the medium. Fumarate was not metabolized by R. albus alone. The only products detected in continuous cultures of R. albus alone were acetate, ethanol, and H2. CO2 was not measured. The only products detected in the mixed cultures were acetate and succinate. No free H2 was produced. No formate or any other volatile fatty acid, no succinate or other dicarboxylic acids, lactate, alcohols other than ethanol, pyruvate, or other keto-acids, acetoin, or diacetyl were detected in cultures of R. albus alone or in mixed cultures. The moles of product per 100 mol of glucose fermented were approximately 69 for ethanol, 74 for acetate, 237 for H2 for R. albus alone and 147 for acetate and 384 for succinate for the mixed culture. Each mole of succinate is equivalent to the production of 1 mol of H2 by R. albus. Thus, in the mixed cultures, ethanol production by R. albus is eliminated with a corresponding increase in acetate and H2 formation. The mixed-culture pattern is consistent with the hypothesis that nicotinamide adenine dinucleotide (reduced form), formed during glycolysis by R. albus, is reoxidized during ethanol formation when R. albus is grown alone and is reoxidized by conversion to nicotinamide adenine dinucleotide and H2 when R. albus is grown with V. succinogenes. The ecological significance of this interspecies transfer of H2 gas and the theoretical basis for its causing changes in fermentation patterns of R. albus are discussed.

Host Range and Properties of the Pseudomonas aeruginosa R Factor R1822

Abstract: R1822, a plasmid specifying multiple drug resistances, has been transferred to a variety of species representative of related and unrelated genera. The host range of the plasmid includes Enterobacteriaceae, soil saprophytes, Neisseria perflava, and photosynthetic bacteria. With the acquisition of drug resistance(s), these strains became sensitive to a small, ribonuclease-sensitive bacteriophage, designated PRR1, isolated by enrichment from sewage.

Oxygen Toxicity and the Superoxide Dismutase

Abstract: Oxygen caused an increase in the amount of superoxide dismutase in Escherichia coli B but not in Bacillus subtilis. E. coli B cells, induced by growth under 100% O2, were much more resistant to the lethal effects of 20 atm of O2 than were cells which contained the low uninduced level of this enzyme. In contrast, B. subtilis, which could not respond to O2 by increasing its content of superoxide dismutase, remained equally sensitive to hyperbaric O2 whether grown under 100% O2 or areobically. The catalase in these organisms exhibited a reciprocal response to oxygen. Thus, the catalase of E. coli B was not induced by O2, whereas that of B. subtilis was so induced. These results are consistent with the view that superoxide dismutase is an important component of the defenses of these organisms against the toxicity of oxygen, whereas their catalases are of secondary importance in this respect. The ability of streptonigrin to generate O2−, by a cycle of reduction followed by spontaneous reoxidation, has been verified in vitro. It is further observed that E. coli B which contain the high induced level of superoxide dismutase were more resistant to the lethality of this antibiotic, in the presence of oxygen, than were E. coli B which contained the low uninduced level of this enzyme. This difference between induced and uninduced cells was eliminated by the removal of O2. These results are consistent with the proposal that the enhanced lethality of streptonigrin under aerobic conditions may relate to its in vivo generation of O2− by a cycle of reduction and spontaneous reoxidation. In toto, these observations lend support to the hypothesis that O2− is an important agent of oxygen toxicity and that superoxide dismutase functions to blunt the threat posed by this reactive radical.

Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae.

Abstract: Deoxyribonucleic acid (DNA) synthesis was examined in asynchronous and synchronous cultures of a number of cdc (cell division cycle) temperature-sensitive mutant strains. The kinetics of DNA synthesis after a shift to the restrictive temperature was compared with that obtained after inhibition of protein synthesis at the permissive temperature, a condition that specifically blocks the initiation of new rounds of DNA replication, but does not block those in progress. Mutations in three genes (cdc 4, 7, and 28) appear to block a precondition for DNA synthesis since cells carrying these lesions cannot start new rounds of DNA replication after a shift from permissive to restrictive temperature, but can finish rounds that were in progress. These three genes are classified as having roles in the “initiation” of DNA synthesis. Mutations in two genes (cdc 8 and 21) block DNA synthesis, itself, since cells harboring these lesions that had started DNA synthesis at the permissive temperature arrest synthesis abruptly upon a shift to the restrictive temperature. Mutations in 13 other cdc genes do not impair DNA synthesis in the first cell cycle at the restrictive temperature.

Transport of Vitamin B12 in Escherichia coli: Common Receptor Sites for Vitamin B12 and the E Colicins on the Outer Membrane of the Cell Envelope

Abstract: The first step in the transport of cyanocobalamin (CN-B(12)) by cells of Escherichia coli was shown previously to consist of binding of the B(12) to specific receptor sites located on the outer membrane of the cell envelope. In this paper, evidence is presented that these B(12) receptor sites also function as the receptors for the E colicins, and that there is competition between B(12) and the E colicins for occupancy of these sites. The cell strains used were E. coli KBT001, a methionine/B(12) auxotroph, and B(12) transport mutants derived from strain KBT001. Colicins E1 and E3 inhibited binding of B(12) to the outer membrane B(12) receptor sites, and CN-B(12) protected cells against these colicins. Half-maximal protection was given by CN-B(12) concentrations in the range of 1 to 6 nM, depending upon the colicin concentration used. Colicin E1 competitively inhibited the binding of (57)Co-labeled CN-B(12) to isolated outer membrane particles. Functional colicin E receptor sites were found in cell envelopes from cells of only those strains that possessed intact B(12) receptors. Colicin K did not inhibit the binding of B(12) to the outer membrane receptor sites, and no evidence was found for any identity between the B(12) and colicin K receptors. However, both colicin K and colicin E1 inhibited the secondary phase of B(12) transport, which is believed to consist of the energy-coupled movement of B(12) across the inner membrane.

Specific inhibitors of ammonia oxidation in Nitrosomonas.

Abstract: The following compounds or treatments have been shown to inhibit the oxidation of ammonia, but not the oxidation of hydroxylamine in cells of Nitrosomonas: (i) metal-binding agents such as allylthiourea or potassium cyanide; (ii) compounds such as SKF 525 which interact with cytochrome P-450 of mammalian microsomes; (iii) carbon monoxide; (iv) inhibitors of catalase, peroxidase, and amine oxidases such as thiosemicarbazide, ethylxanthate, and iproniazid, respectively; (v) uncouplers of oxidative phosphorylation such as m-chlorocarbonylcyanidephenylhydrazone; (vi) electron acceptors such as phenazine methosulfate; (vii) compounds such as methanol or N2O which react with free radicals; and (viii) illumination with 420 lux (5,000 foot candles) of light.

Isolation and characterization of acid phosphatase mutants in Saccharomyces cerevisiae.

Abstract: Saccharomyces cerevisiae strain H-42 seems to have two kinds of acid phosphatase: one which is constitutive and one which is repressible by inorganic phosphate. The constitutive enzyme was significantly unstable to heat inactivation, and its Km of 9.1 × 10−4m for p-nitrophenylphosphate was higher than that of the repressible enzyme (2.4 × 10−4m). The constitutive and the repressible acid phosphatases are specified by the phoC gene and by the phoB, phoD, or phoE gene, respectively. Results of tetrad analysis suggested that the phoC and phoE genes are linked to the lys2 locus on chromosome II. Since both repressible acid and alkaline phosphatases were affected simultaneously in the phoR, phoD, and phoS mutants, it was concluded that these enzymes were under the same regulatory mechanism or that they shared a common polypeptide. The phoR mutant produced acid phosphatase constitutively, and the phoR mutant allele was recessive to its wild-type counterpart. The phoS mutant showed a phenotype similar to that of a mutant defective in one of the phoB, phoD, or phoE genes. However, the results of genetic analysis of the phoS mutant clearly indicated that the phoS gene is not a structural gene for either of the repressible acid and alkaline phosphatases, but is a kind of regulatory gene. According to the proposed model, the phoS gene controls the expression of the phoR gene, and inorganic phosphate would act primarily as an inducer for the formation of the phoR product which represses phosphatase synthesis.

Mode of Action of Glycine on the Biosynthesis of Peptidoglycan

Abstract: The mechanism of glycine action in growth inhibition was studied on eight different species of bacteria of various genera representing the four most common peptidoglycan types. To inhibit the growth of the different organisms to 80%, glycine concentrations from 0.05 to 1.33 M had to be applied. The inhibited cells showed morphological aberrations. It has been demonstrated that glycine is incorporated into the nucleotide-activated peptidoglycan precursors. The amount of incorporated glycine was equivalent to the decrease in the amount of alanine. With one exception glycine is also incorporated into the peptidoglycan. Studies on the primary structure of both the peptidoglycan precursors and the corresponding peptidoglycan have revealed that glycine can replace l-alanine in position 1 and d-alanine residues in positions 4 and 5 of the peptide subunit. Replacement of l-alanine in position 1 of the peptide subunit together with an accumulation of uridine diphosphate-muramic acid (UDP-MurNAc), indicating an inhibition of the UDP-MurNAc:l-Ala ligase, has been found in three bacteria (Staphylococcus aureus, Lactobacillus cellobiosus and L. plantarum). However, discrimination against precursors with glycine in position 1 in peptidoglycan synthesis has been observed only in S. aureus. Replacement of d-alanine residues was most common. It occurred in the peptidoglycan with one exception in all strains studied. In Corynebacterium sp., C. callunae, L. plantarum, and L. cellobiosus most of the d-alanine replacing glycine occurs C-terminal in position 4, and in C. insidiosum and S. aureus glycine is found C-terminal in position 5. It is suggested that the modified peptidoglycan precursors are accumulated by being poor substrates for some of the enzymes involved in peptidoglycan synthesis. Two mechanisms leading to a more loosely cross-linked peptidoglycan and to morphological changes of the cells are considered. First, the accumulation of glycine-containing precursors may lead to a disrupture of the normal balance between peptidoglycan synthesis and controlled enzymatic hydrolysis during growth. Second, the modified glycine-containing precursors may be incorporated. Since these are poor substrates in the transpeptidation reaction, a high percentage of muropeptides remains uncross-linked. The second mechanism may be the more significant in most cases.

Effect of Reversible Inhibition of Deoxyribonucleic Acid Synthesis on the Yeast Cell Cycle

Abstract: Hydroxyurea (HU) preferentially inhibited deoxyribonucleic acid (DNA) replication and division in Saccharomyces cerevisiae. Growth, ribonucleic acid synthesis, and protein synthesis were less sensitive to this drug. Upon addition of HU, cells underwent one cycle of budding and the nuclei migrated into the necks between the mother cells and buds. Neither the nucleus nor the cells divided. Removal of HU allowed immediate resumption of DNA synthesis. Nuclear division, budding, and cell division occurred 1.5, 2, and 4 hr, respectively, after HU was removed. If protein synthesis was blocked at the time HU was removed, budding and cell division did not occur. These results were interpreted to indicate that HU prevents accumulation of the potential to initiate a new cell cycle. Images

Formate Dehydrogenase of Clostridium thermoaceticum: Incorporation of Selenium-75, and the Effects of Selenite, Molybdate, and Tungstate on the Enzyme

Abstract: The formation of the nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase in Clostridium thermoaceticum is stimulated by the presence of molybdate and selenite in the growth medium. The highest formate dehydrogenase activity was obtained with 2.5 x 10(-4) M Na(2)MoO(4) and 5 x 10(-5) Na(2)SeO(3). Tungstate but not vanadate could replace molybdate and stimulate the formation of formate dehydrogenase. Tungstate stimulated activity more than molybdate, and in combination with molybdate the stimulation of formation of formate dehydrogenase was additive. Formate dehydrogenase was isolated from cells grown in the presence of Na(2) (75)SeO(2), and a correlation was observed between bound (75)Se and enzyme activity.

Molecular Relationships Among the Salmonelleae

Abstract: Polynucleotide sequence relatedness studies were carried out to determine the extent of divergence present in members of the tribe Salmonelleae and between salmonellae and other enteric bacteria. Typical Salmonella were 85 to 100% related. Two groups of biochemically atypical Salmonella showed somewhat lower binding to typical salmonellae and to each other. Arizona were 70 to 80% related to salmonellae. Two groups of Arizona were detected. These groups correlated with the presence of monophasic or diphasic flagellar antigens. Salmonella and Arizona were no more related to Citrobacter than to Escherichia coli (45-55%). Relatedness of Salmonella and Arizona to other enterobacteria ranged from 20 to 40% with klebsiellae and shigellae, to 20 to 25% with erwiniae, and to less than 20% with edwardsiellae and Proteus mirabilis.

Fermentation of glucose, fructose, and xylose by Clostridium thermoaceticum: effect of metals on growth yield, enzymes, and the synthesis of acetate from CO 2

Abstract: Clostridium thermoaceticum ferments xylose, fructose, and glucose with acetate as the only product. In fermentations with mixtures of the sugars, xylose is first fermented, then fructose, and last, glucose. Fructose inhibits the fermentation of glucose, and this inhibition appears to be due to a repression of the synthesis of an enzyme needed for glucose utilization. Addition of metals to the culture medium increases the cell yield drastically from about 7 to 18 g per liter, and Y(glucose) values between 40 and 50 are obtained. According to the postulated pathways of the fermentation of glucose and synthesis of acetate from CO(2) by C. thermoaceticum, 3 mol of ATP are available as energy for growth. Thus a Y(adenosine 5'-triphosphate) of 13 to 16 is obtained. Because the normal Y(ATP) value is 10.5, this could mean that an additional source of ATP is available by an unknown mechanism. The addition of metals also increases the nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase activity, the overall reaction ((14)CO(2) --> acetate), and the incorporation of the methyl group of 5-methyltetrahydrofolate into acetate. These reactions are catalyzed very efficiently by cells harvested in early growth, whereas cells obtained at the end of a fermentation have very low formate dehydrogenase activity and capacity to incorporate CO(2) into acetate. The following enzymes involved in the synthesis of acetate from CO(2) and in the metabolism of pyruvate are present in extracts of C. thermoaceticum: 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase, 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methylenetetrahydrofolate reductase, phosphate acetyltransferase, and acetate kinase. These enzymes are not or are very little affected by the addition of metals to the growth medium. The amount of corrinoids in cells from early growth is low, whereas it is high in cells harvested late in growth. The opposite is found for the activity of delta-aminolevulinate dehydratase, which is high at the beginning of growth and low at the end.

Cyclic adenosine 3',5'-monophosphate in Escherichia coli.

Abstract: The concentration of cyclic adenosine 3′,5′-monophosphate (c-AMP) in Escherichia coli growing on different sources of carbon was studied. Cultures utilizing a source of carbon that supported growth relatively poorly had consistently higher concentrations of c-AMP than did cultures utilizing sugars that supported rapid growth. This relationship was also observed in strains defective in c-AMP phosphodiesterase and simultaneously resistant to catabolite repression; in such strains the c-AMP concentration was slightly higher for several sources of carbon tested. Cultures continued to synthesize c-AMP and secreted it into the medium, under conditions that brought about an inhibition of the intracellular accumulation of the cyclic nucleotide. Transient repression of the synthesis of β-galactosidase was not associated with an abrupt decrease in the cellular concentration of c-AMP.

Minicells of Bacillus subtilis

Abstract: After nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine) mutagenesis, two Bacillus subtilis mutants (div IV-A1 and div IV-B1) were isolated that are defective in the location of division site along cell length. Both mutations were transferred into strain CU403 by transformation, and their properties were studied in the CU403 genetic background. Location of divisions in close proximity to cell pole regions in both mutants results in minicell production. Purified minicells contain a ratio of ribonucleic acid to protein comparable to that found in the parent cells. Autoradiographs of 3H-thymine incorporation into deoxyribonucleic acid (DNA), thymine-2-14C incorporation into DNA, electron micrographs, and chemical analyses for DNA all fail to demonstrate DNA in the minicells. Minicells produced by both mutants are highly motile, an indication of functional energy metabolism. Electron micrographs reveal that minicells are produced by a structurally normal division mechanism and that minicells contain a normal cell surface. The div IV-A1 mutation has been mapped by PBS1 transduction linked to ura. The div IV-B1 mutation is closely linked to pheA by both PBS1 transduction and by co-transformation.

Lipopolysaccharide Layer Protection of Gram-Negative Bacteria Against Inhibition by Long-Chain Fatty Acids

Abstract: Growth, amino acid transport, and oxygen consumption of Escherichia coli and Salmonella typhimurium are inhibited by short-chain (C(2)-C(6)) but not by medium or long-chain fatty acids (C(10)-C(18)) at concentrations at which these processes are completely inhibited in Bacillus subtilis. The resistance of gram-negative organisms is not correlated with their ability to metabolize fatty acids, since an E. coli mutant unable to transport oleic acid is still resistant. However, mutants of both E. coli and S. typhimurium in which the lipopolysaccharide layer does not contain the residues beyond the 2-keto-3-deoxyoctonate core are inhibited by medium (C(10)) but not by long-chain (C(18)) fatty acids. Furthermore, removal of a portion of the lipopolysaccharide layer by ethylenediaminetetraacetate treatment renders the organisms sensitive to medium and partially sensitive to long-chain fatty acids. The intact lipopolysaccharide layer of gram-negative organisms apparently screens the cells against medium and long-chain fatty acids and prevents their accumulation on the inner cell membrane (site of amino acid transport) at inhibitory concentrations. These results are relevant to the use of antimicrobial food additives, and they allow the characterization of gram-positive versus gram-negative bacteria and their lipopolysaccharide mutants.

Genetic Mutations Affecting the Respiratory Electron-Transport System of the Photosynthetic Bacterium Rhodopseudomonas capsulata

Abstract: Alternative energy-converting systems permit the nonsulfur purple photosynthetic bacterium Rhodopseudomonas capsulata to grow either with light or (dark) respiration as the source of energy. Respiratory mutants, unable to grow aerobically in darkness, can be readily isolated and the defective step(s) in their respiratory mechanisms can be identified by study of biochemical activities in membrane fragments derived from photosynthetically grown cells. Such analysis of appropriate mutants and revertants permits construction of a model for the respiratory electron-transport system of the wild type. The results obtained indicate differential channeling of electrons derived from succinate and reduced nicotinamide adenine dinucleotide, and are interpreted in terms of a branched electron-transport scheme. The scheme provides a guide for further, more refined analysis of the respiratory mechanisms through biochemical genetic approaches, and several of the mutants isolated can be exploited for investigation of unsolved problems relating to interactions between respiratory and photosynthetic electron transport and the mechanism of inhibition of bacteriochlorophyll synthesis by molecular oxygen.

Inorganic Phosphate Transport in Escherichia coli: Involvement of Two Genes Which Play a Role in Alkaline Phosphatase Regulation

Abstract: Two classes of alkaline phosphatase constitutive mutations which comprise the original phoS locus (genes phoS and phoT) on the Escherichia coli genome have been implicated in the regulation of alkaline phosphatase synthesis. When these mutations were introduced into a strain dependent on a single system, the pst system, for inorganic phosphate (Pi) transport, profound changes in Pi transport were observed. The phoT mutations led to a complete Pi− phenotype in this background, and no activity of the pst system could be detected. The introduction of the phoS mutations changed the specificity of the pst system so that arsenate became growth inhibitory. Changes in the phosphate source led to changes in the levels of constitutive alkaline phosphatase synthesis found in phoS and phoT mutants. When glucose-6-phosphate or l-α-glycerophosphate was supplied as the sole source of phosphate, phoT mutants showed a 3- to 15- fold reduction in constitutive alkaline phosphatase synthesis when compared to the maximal levels found in limiting Pi media. However, these levels were still 100 times greater than the basal level of alkaline phosphatase synthesized in wild-type strains under these conditions. The phoS mutants showed only a two- to threefold reduction when grown with organic phosphate sources. The properties of the phoT mutants selected on the basis of constitutive alkaline phosphatase synthesis were similar in many respects to those of pst mutants selected for resistance to growth inhibition caused by arsenate. It is suggested that the phoS and phoT genes are primarily involved in Pi transport and, as a result of this function, play a role in the regulation of alkaline phosphatase synthesis.

Molecular Studies of R Factor Compatibility Groups

Abstract: Molecular studies of R factors of six groups, F(II), I(1), I(2), N, B, and H, defined on the basis of compatibility, support the conclusions drawn from genetic studies. In general, R factors of a given compatibility group are similar in size. Deoxyribonucleic acid (DNA) reassociation occurs freely between members of the same group but is minimal between heterologous groups. An exception to this was found in group H, of which one factor showed minimal homology with the remaining plasmids of the group. A further exception was found with groups I(1) and B, which, although genetically distinct, show between 18 and 28% of DNA homology. Groups I(1) and I(2) are molecularly distinct, despite the fact that they both stimulate the synthesis of I-fimbriae.