Showing papers in "Journal of Bacteriology in 1975"

Plasmid required for virulence of Agrobacterium tumefaciens.

Abstract: The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37 C is shown to be due to loss of a large plasmid (1.2 X 10-8 daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6 is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 X A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid.

Abstract: Pseudomonas putida (arvilla) mt-2 carries genes for the catabolism of toluene, m-xylene, and p-xylene on a transmissible plasmid, TOL. These compounds are degraded by oxidation of one of the methyl substituents via the corresponding alcohols and aldehydes to benzoate and m- and p-toluates, respectively, which are then further metabolised by the meta pathway, also coded for by the TOL plasmid. The specificities of the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase for their three respective substrates are independent of the carbon source used for growth, suggesting that a single set of nonspecific enzymes is responsible for the dissimilation of the breakdown products of toluene and m- and p-xylene. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are coincidently and possible coordinately induced by toluene and the xylenes, and by the corresponding alcohols and aldehydes. They are not induced in cells grown on m-toluate but catechol 2,3-oxygenase can be induced by m-xylene.

Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae.

Abstract: The interdependence of spindle plaque with other aspects of cell division and conjugation in Saccharomyces cerevisiae has been investigated. Three forms of the spindle plaque appear sequentially before the formation of the complete, intranuclear spindle. The single plaque is present initially in the mitotic cycle; it becomes transformed into a satellite-bearing single plaque during the latter part of G1. Subsequently, plaque duplication yields the double plaque characteristic of the early phase of budding, which coincides with the period of chromosome replication (S). The eventual separation of these plaques to form a complete spindle, with a single plaque at each pole, is nearly coincident with the completion of S. The form of the plaque differs in two independent cases of G1 arrest: the single plaque is found in a cell in stationary arrest of growth, whereas a cell arrested by mating factors in preparation for conjugation contains a satellite-bearing single plaque. The latter form is retained during zygote formation, where it serves as the initial site of fusion of each prezygotic nuceus with the other. This fusion results in the formation of a single zygotic nucleus with a satellite-bearing single plaque, which is subsequently transformed into a double plaque as the zygote buds. The double plaque is situated adjacent to the site of bud emergence in both vegetative cells and zygotes. Images

Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid.

Abstract: A composite plasmid has been constructed in vitro from colicin E1 factor (mass of 4.2 megadaltons [Md]) and nontransmissible resistance factor RSF 1010 (mass, 5.5. Md) deoxyribonucleic acids (DNAs) by the sequential action of Escherichia coli endonuclease (RI (Eco RI) and T4 phage DNA ligase on the covalently closed circular forms of the constituents. The composite plasmid was selected and amplified in vivo by sequential transformation of E. coli C600 with the ligated mixture and selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-cesium chloride solution. Treatment of the composite plasmid with Eco RI yielded two fragments with mobilities corresponding to the linear forms of the parental plasmids, whereas Serratia marscesens endonuclease R (SmaR), which introduces a single scission in the colicin E1 factor but not in RSF 1010, convErted the composite plasmid to a single linear molecule (mass, 9.7 Md). Sequential degradation of colicin E1 factor with Sma R and Eco RI produced two fragments with masses of 3.5 and 0.7 Md; sequential degradation of RSF 1010 produced only one fragment (due to the cleavage with Eco RI), and sequential degradation of the composite plasmid produced the expected three fragments--an RSF 1010 Eco RI linear and the two expected products from the colicin E1 factor moiety. The composite plasmid conferred on the host cell resistance to streptomycin, sulfonamides, and colicin E1, but colicin E1 itself was not synthesized. In contrast, colicin E1 was synthesized by cells containing simultaneously both colicin E1 factor and RSF 1010 as separate entities. In the presence of chloramphenicol, the composite plasmid continued to replicate for 6 h. whereas replication of RSF 1010 and chromosomal DNA stopped within 2 h. Continued replication in the presence of chloramphenicol suggests that the replicator of the colicin E1 factor is functional in the composite plasmid. Images

Outer membrane of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants.

Abstract: The outer membrane layer of the cell wall was isolated from wild-type Salmonella typhimurium LT2 as well as from its mutants producing lipopolysaccharides with shorter saccharide chains. Chemical analysis of these preparations indicated the following. (i) The number of lipopolysaccharide molecules per unit area was constant, regardless of the length of the saccharide side chain in lipopolysaccharide. (ii) In contrast, in "deep rough" (Rd or Re) mutants producing the lipopolysaccharides with very short saccharide chains, the amount of outer membrane protein per unit surface area decreased to about 60% of the value in the wild type. (iii) In the wild type, the amount of phospholipids is slightly less than what is needed to cover one side of the membrane as a monolayer. In comparison with the wild type, the outer membrane of Rd and Re mutants contains about 70% more phospholipids, which therefore must be distributed in both the outer and inner leaflets of the membrane. Freeze-fracture studies showed that the outer membrane of Re mutants were easily fractured, but fracture became increasingly difficult in strains producing lipopolysaccharides with longer side chains. The convex fracture face was always nearly smooth, but the concave fracture face or the outer half of the membrane was densely covered with particles 8 to 10 nm in diameter. The density of particles was decreased in Re mutants to the same extent as the reduction in proteins, suggesting the largely proteinaceous nature of particles. A model for the supramolecular structure of the outer membrane is presented on the basis of these and other results.

Genetics of resistance to colicins in Escherichia coli K-12: cross-resistance among colicins of group A.

Abstract: By using each of the available colicins, we have isolated a large number of colicin-resistant mutants. They included both receptor and tolerant mutants and each was screened for cross-resistance to all other colicins. On the basis of the cross-resistance of these mutants it was possible to place known colicins into two mutually exclusive groups, group A and group B. Mutants selected as resistant to colicins of group A may or may not be cross-resistant to other colicins of group A, BUT Are never resistant to colicins of group B. The reverse also applies. The mutants isolated as resistant to colicins of group A (A, E1, E2, E3, K, L, N, S4, and X) have been divided into 21 phenotypic classes on the basis of their colicin resistance patterns. These include most of the tolerant and receptor mutants previously isolated, some of which were previously shown to also have an increased sensitivity to certain antibiotics and detergents. Type strains from each of the phenotypic classes were therefore tested for sensitivity to a range of antibiotics, detergents, and surfactants that included all those previously used. With these new data, it has been possible to speculate informatively on the mode of action of the different colicins. We have confirmed the position of previously isolated mutations on the Escherichia coli K-12 genetic map, and located approximately the loci conferring colicin resistance in some of the newly isolated mutants.

Maltose transport in Escherichia coli K-12: involvement of the bacteriophage lambda receptor.

Abstract: Mutants affected in lamB, the structural gene for phage lambda receptor, are unable to utilize maltose when it is present at low concentrations (less than or equal 10 muM). During growth in a chemostat at limiting maltose concentrations, the lamB mutants tested were selected against in the presence of the wild-type strain. Transport studies demonstrate that most lamB mutants have deficient maltose transport capacities at low maltose concentrations. When antibodies against purified phage lambda receptor are added to a wild-type strain, transport of maltose at low concentrations is significantly reduced. These results strongly suggest that the phage lambda receptor molecule is involved in maltose transport.

Novel type of murein transglycosylase in Escherichia coli.

Abstract: The purification and properties of a novel type of murein transglycosylase from Escherichia coli are described. The purified enzyme appears as a single band on sodium dodecyl sulfate-polyacrylamide gels and has an apparent molecular weight of approximately 65,000 as estimated by gel filtration and gel electrophoresis. It degrades pure murein sacculi from E. coli almost completely into low-molecular-weight products. The two prominent muropeptide fragments in the digest are the disaccharide-tripeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid and the corresponding disaccharide-tetrapeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid-D-alanine. The unique feature of these compounds is that the disaccharide has no reducing end group and that the muramic acid residue possesses an internal 1 leads to 6 anhydro linkage. The new lytic enzyme is designated as a murein: murein transglycosylase. Its possible role in the rearrangement of murein during cell growth and division is discussed.

Phosphorylation of D-glucose in Escherichia coli mutants defective in glucosephosphotransferase, mannosephosphotransferase, and glucokinase.

Abstract: Genetic studies show that Escherichia coli has three enzymes capable of phosphorylating glucose: soluble adenosine 5'-triphosphate-dependent glucokinase, which plays only a minor role in glucose metabolism; an enzyme II, called glucosephosphotransferase, with high specificity for the D-glucose configuration; and another enzyme II, called mannosephosphotransferase, with broader specificity. The former enzyme II is active on glucose and methyl-alpha-glucopyranoside, whereas the latter is active on D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, and D-mannosamine. Mutations leading to loss of glucosephosphotransferase activity and designated by the symbol gpt are between the purB and pyrC markers in a locus previously called cat. The locus of mutations to loss of mannosephosphotransferase, mpt, is between the eda and fadD genes. Mutations to loss of glucokinase, glk, are between the ptsI and dsd genes.

Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans.

Abstract: The enzyme 4-hydroxyphenylacetate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating) (EC 11413 ; 4-hydroxyphenylacetate 1-monooxygenase; referred to here as 4-HPA 1-hydroxylase) was induced in Pseudomonas acidovorans when 4-hydroxyphenylacetate (4-PHA) was utilized as carbon source for growth; homogentisate and maleylacetoacetate were intermediates in the degradation of 4-HPA A preparation of the hydroxylase that was free from homogentisate dioxygenase and could be stored at 4 C in the presence of dithioerythritol with little loss of activity was obtained by ultracentrifuging cell extracts; but when purified 18-fold by affinity chromatography the enzyme became unstable Flavin adenine dinucleotide and Mg2+ ions were required for full activity 4-HPA 1-hydrocylase was inhibited by KCl, which was uncompetitive with 4-HPA Values of Ki determined for inhibitors competitive with 4-HPA were 17 muM dl-4-hydroxymandelic acid, 43 muM 3,4-dihydroxyphenylacetic acid, 87 muM 4-hydroxy-3-methylphenylacetic acid, and 440 muM 4-hydroxyphenylpropionic acid Apparent Km values for substrates of 4-HPA 1-hydroxylase were 31 muM 4-HPA, 67 muM oxygen, 95 muM reduced nicotinamide adenine dinucleotide (NADH); AND 250 muM reduced nicotinamide adenine dinucleotide phosphate (NADPH) The same maximum velocity was given by NADH and NADPH A chemical synthesis is described for 2-deutero-4-hydroxyphenylacetic acid This compound was enzymatically hydroxylated with retention of half the deuterium in the homogentisic acid formed Activity as substrate or inhibitor of 4-HPA 1-hydroxylase was shown only by those analogues of 4-HPA that possessed a hydroxyl group substituent at C-4 of the benze nucleus A mechanism is suggested that accounts for this structural requirement and also for the observation that when 4-hydroxyphenoxyacetic acid was attacked by the enzyme, hydroquinone was formed by release of the side chain, probably as glycolic acid Only one enantiometer of racemic 4-hydroxyhydratropic acid was attacked by 4-HPA 1-hydroxylase; the product, alpha-methylhomogentisic acid (2-(2,5-dihydroxyphenyl)-propionic acid), exhibited optical activity This observation suggests that, during its shift from C-1 to C-2 of the nucleus, the side chain of the substrate remains bound to a site on the enzyme while a conformational change of the protein permits the necessary movement of the benzene ring

Effect of growth rate on the amounts of ribosomal and transfer ribonucleic acids in yeast.

Abstract: The steady-state growth rate of Saccharomyces cerevisiae was varied by growing the cells in different media. The total amount of ribonucleic acid (RNA) per cell was found to decrease as a nonlinear function of decreasing growh rate. The RNA from cells growing in different media was analyzed by polyacrylamide gel electrophoresis. Although the amounts of both ribosomal RNA and transfer RNA decreased with decreasing growth rate, the ratio of ribosomal to transfer RNA was not constant. As the growth rate was reduced the ribosomal RNA fraction decreased slightly, whereas the transfer RNA fraction increased slightly. Thus the levels of ribosomal and transfer RNA were regulated to similar yet different extents. The levels of the different ribosomal RNA species were more closely coordinated. At all growth rates the ribosomal RNAs (including 5S RNA) were present in equimolar amounts. The rate of protein synthesis in yeast cells also decreased with decreasing growth rate. The low rates of protein synthesis did not appear to be due to limiting numbers of ribosomes or transfer RNA molecules.

Origin of the TEM-beta-lactamase gene found on plasmids.

Abstract: A sequence of deoxyribonucleic acid of 2.7 times 10-6 to 3.3 times 10-6 daltons which includes the TEM beta-lactamase gene is present on the small plasmid RSF 1030 (R-Amp). This same sequence is present on plasmid derivatives that have received a translocation of deoxyribonucleic acid specifying the TEM beta-lactamase and is also present on naturally occurring plasmids of the F1, F11, N, X, O, I, C, and W incompatibility groups that do not specify ampicillin resistance or specify O-type beta-lactamases.

Regulation of lactate dehydrogenase and change of fermentation products in streptococci.

Abstract: Streptococcus mutans JC 2 produced mainly lactate as a fermentation product when grown in nitrogen-limited continuous culture in the presence of an excess of glucose and produced formate, acetate, and ethanol, but no lactate, under glucose-limited conditions. The levels of lactate dehydrogenase (LDH) in these cultures were of the same order of magnitude, and the activity of LDH was completely dependent on fructose-1,6-diphosphate (FDP). The intracellular level of FDP was high and the level of phosphoenolpyruvate (PEP) was low under the glucose-excess conditions. In the glucose-limited cultures, all glycolytic intermediates studied, except PEP, were low. S. mutans FIL, which had an FDP-independent LDH and similar levels of glycolytic intermediates as S. mutans JC2, produced mainly lactate under glucose-excess or under glucose-limited conditions. LDH of Streptococcus bovis ATCC 9809 was dependent on FDP for activity at a low concentration of pyruvate but had a significant activity without FDP at a high concentration of pyruvate. This strain also produced mainly lactate both under glucose-excess and glucose-limited conditions. The levels and characteristics of these LDHs were not changed by the culture conditions. These results indicate that changes in the intracellular level of FDP regulate LDH activity, which in turn influences the type of fermentation products produced by streptococci. PEP, adenosine 5'-monophosphate, adenosine 5'-diphosphate, and inorganic phosphate significantly inhibited LDH activity from S. mutans JC 2 and may also participate in the regulation of LDH activity in other streptococci.

Regulation of nitrogen metabolism in Escherichia coli and Klebsiella aerogenes: studies with the continuous-culture technique.

Abstract: Ammonia-nitrogen-limited continuous cultures of Escherichia coli and Klebsiella aerogenes contain induced levels of glutamine synthetase that is deadenylyated (i.e., fully active). In the presence of excess ammonia or glutamate in glucose-limited cultures of E. coli, glutamine synthetase is repressed and adenylylated (inactive). The average state of adenylylation (n) is a linear function of the specific growth rate. At low specific growth rates, glutamine synthetase is adenylylated; as the specific growth rate increases, n decreases, approaching 0 to 2 at rapid growth rates. The average state of adenylylation correlates well with the intracellular concentrations and ratios of alpha-ketoglutarate and glutamine, which are key effectors in the adenylylation-deadenylylation systems. E. coli and K. aerogenes differ markedly in their growth yields, growth rates, and enzymatic composition during nitrogen limitation. The data suggest that, unlike K. aerogenes, E. coli W uses glutamate dehydrogenase to incorporate ammonia during nitrogen limitation. In E. coli, glutamate dehydrogenase is progressively induced during nitrogen limitation when mu (growth rate) approaches mumax. In contrast, in K. aerogenes glutamate dehydrogenase is repressed during nitrogen limitation, whereas glutamate synthase, an alternative supplier of glutamate to the cell, is induced. Data are presented that support the regulatory schemes proposed for the control of glutamine synthetase activity by induction-repression phenomena and adenylylation-deadenylylation reaction. We propose that the intracellular ratio of alpha-ketoglutarate to glutamine may be the most important physiological parameter in determining the activity of glutamine synthetase.

Interactions among substrates and inhibitors of nitrogenase.

Abstract: Examination of interactions among various substrates and inhibitors reacting with a partially purified nitrogenase from Azotobacter vinelandii has shown that: nitrous oxide is competitive with N2; carbon monixide and acetylene are noncompetitive with N2; carbon monoxide, cyanide, and nitrous oxide are noncompetitive with acetylene, whereas N2 is competitive with acetylene; carbon monoxide is noncompetitive with cyanide, whereas azide is competitive with cyanide; acetylene and nitrous oxide increase the rate of reduction of cyanide. The results are understandable if nitrogenase serves as an electron sink and substrates and inhibitors bind at multiple modified sites on reduced nitrogenase. It is suggested that substrates such as acetylene may be reduced by a less completely reduced electron sink than is required for the six-electron transfer necessary to reduce N2.

Facultative anoxygenic photosynthesis in the cyanobacterium Oscillatoria limnetica.

Abstract: An isolate from H2S-rich layers of the Solar Lake, the cyanobacterium Oscillatoria limnetica, exhibits both oxygenic and anoxygenic photosynthesis. It can use Na2S as an electron donor for CO2 photoassimilation (photosystem I supplies the energy) in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or 700-nm light. A stoichiometric ratio of approximately 2 is observed between the Na2S consumed and the photoassimilated CO2. The anoxygenic phototrophic capability of this cyanobacterium explains its growth in nature in high sulfide concentrations and indicates a selective advantage.

Transmissible toxin (hemolysin) plasmid in Streptococcus faecalis and its mobilization of a noninfectious drug resistance plasmid.

Abstract: Streptococcus faecalis strain DS-5 has been shown previously to contain three plasmids, pAMalpha1, pAMbeta1, and pAMgamma1. Mixed incubation of DS-5 with strain JH2-2, a plasmid-free S. faecalis recipient, results in the transfer of pAMalpha1 (which determines resistance to tetracycline) and/or pAMgamma1. Analyses of recipients carrying various combinations of these plasmids have revealed the pAMgamma1 codes for toxin (hemolysin) production and two bacteriocin activities. JH2-2 strains carrying both pAMalpha1 and pAMgamma1, or pAMgamma1 only, can donate their plasmids to other recipients, whereas strains carrying only pAMalpha1 cannot serve as donors. This indicates that pAMgamma1 actually mobilizes the otherwise nontransferable pAMalpha1.

Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells.

Abstract: Lysogenic strains of Bacillus subtilis 168 were reduced in their level of transformation as compared to non-lysogenic strains. The level of transformation decreased even further if the competent lysogenic cells were allowed to incubate in growth media prior to selection on minimal agar. This reduction in the frequency of transformation was attributable to the selective elimination of transformed lysogenic cells from the competent population. Concurrent with the decrease in the number of transformants from a lysogenic competent population was the release of bacteriophage by these cells. The lysogenic bacteria demonstrated this dramatic release of bacteriophage only if the cells were grown to competence. Both the selective elimination of transformed lysogens and the induction of prophage was prevented by the inhibition of protein synthesis. Additionally, competent lysogenic cells released significantly higher amounts of exogenous donor transforming deoxyribonucleic acid than did competent non-lysogenic cells or competent lysogenic cells incubated with erythromycin. These data establish that the induction of the prophage from the competent lysogenic cells was responsible for the selective elmination of the lysogenic transformants. A model is presented that accounts for the induction of the prophage from competent lysogenic bacteria via the induction of a repair system. It is postulated that a repair system is induced or derepressed by the accumulation of gaps in the chromosomes of competent bacteria. This hypothetical enzyme(s) is ultimately responsible for the induction of the prophage and the selective elimination of transformants.

Evidence for common binding sites for ferrichrome compounds and bacteriophage phi 80 in the cell envelope of Escherichia coli.

Abstract: Mutants ton A and ton B of Escherichia coli K12, known to be resistant to bacteriophage phi80, were found to be insensitive as well to albomycin, an analogue of the specific siderochrome ferrichrome. Ferrichrome at micromolar concentrations strongly inhibited plaque production by phi80. Preincubation with ferrichrome did not inactivate the phage. At a concentration at which ferrichrome allowed 90% inhibition of plaque formation, the chromium analogue of ferrichrome showed no detectable activity. Similarly, ethylenediaminetetraacetic acid, ferrichrome A, and certain siderochromes structurally distinct from ferrichrome, such as ferrioxamine B, schizokinen, citrate, and enterobactin, did not show detectable inhibitory activity. However, rhodotorulic acid showed moderate activity. A host range mutant of phi80, phi80h, was also inhibited by ferrichrome, as was a hybrid of phage lambda possessing the host range of phi80. However, phage lambdacI- and a hybrid of phi80 possessing the host range of lambda were not affected by ferrichrome. Finally, ferrichrome and chromic deferriferrichrome were shown to inhibit adsorption of phi80 to sensitive cells, ferrichrome giving 50% inhibition of adsorption at a minimal concentration of 8 nM. It is suggested that a component of the ferrichrome uptake system may reside in the outer membrane of E. coli K12 and may also function as a component of the receptor site for bacteriophage phi80, and that ferrichrome inhibition of the phage represents a competition for this common site.

Maltose chemoreceptor of Escherichia coli.

Abstract: Strains carrying mutations in the maltose system of Escherichia coli were assayed for maltose taxis, maltose uptake at 1 and 10 muM maltose, and maltose-binding activity released by osmotic shock. An earlier conclusion that the metabolism of maltose is not necessary for chemoreception is extended to include the functioning of maltodextrin phosphorylase, the product of malP, and the genetic control of the maltose receptor by the product of malT is confirmed. Mutants in malF and malK are defective in maltose transport at low concentrations as well as high concentrations, as previously shown, but are essentially normal in maltose taxis. The product of malE has been previously shown to be the maltose-binding protein and was implicated in maltose transport. Most malE mutants are defective in maltose taxis, and all those tested are defective in maltose transport at low concentrations. Thus, as previously suggested, the maltose-binding protein probably serves as the recognition component of the maltose receptor, as well as a component of the transport system. tsome malE mutants release maltose-binding activity and are tactic toward maltose, although defective in maltose transport, implying that the binding protein has separate sites for interaction with the chemotaxis and transport systems. Some mutations in lamB, whose product is the receptor for the bacteriophage lamba, cause defects in maltose taxis, indicating some involvement of that product in maltose reception.

Plasmid incompatibility and control of replication: copy mutants of the R-factor R1 in Escherichia coli K-12.

Abstract: Plasmid incompatibility was studied in Escherichia coli K-12. By double-antibiotic selection, clones were constructed that carried the two R-factors R1 and R100, both belonging to the compatibility group FII. After release of the selection pressure, each of the two plasmids was lost at the same rate (8% per generation). Mutants of R-factor R1 showing an increased number of copies per chromosome (copy mutants) were tested for their incompatibility towards R-factor R100. The results indicate that plasmid incompatibility is quantitative and not just a qualitative property. All copy mutants studied affected incompatibility, and there were two classes of mutants: one increasing and one decreasing the incompatiblity exerted towards the test plasmid R100. Evidence is presented that incompatibility is related to the mechanisms that control replication. The implications of such a relation on proposed models for control of replication are discussed. The data do not support the hypothesis that plasmid incompatibility is due to competition for a replicational or segregational site.

Inheritance of low-level resistance to penicillin, tetracycline, and chloramphenicol in Neisseria gonorrhoeae.

Abstract: The genetics of low-level resistance to penicillin and other antibiotics in a clinical isolate and a multistep laboratory mutant of Neisseria gonorrhoea was studied by transformation. Mutations at three loci affected sensitivity to penicillin. Mutation at penA resulted in an eightfold increase in resistance to penicillin without affecting response to other antimicrobial agents. Mutation at ery resulted in a two- to fourfold increase in resistance to penicillin and similar increases in resistance to many other antibiotics, dyes, and detergents. Mutation at penB resulted in a fourfold increase in resistance to penicillin and tetracycline, the phenotypic expression of which was dependent on the presence of mutation at ery. The cumulative effect of mutations at penA, ery, and penB was an approximate 128-fold increase in penicillin resistance, to a minimum inhibitory concentration of 1.0 mug/ml. Low-level resistance to tetracycline or chloramphenicol was due to similar additive effects between mutations at the nonspecific ery and penB loci and a locus specific for resistance to each drug (tet and chl, respectively). No evidence was found for penicillinases or other drug-inactivating enzymes.

Adenylate energy charge in Saccharomyces cerevisiae during starvation.

Abstract: Bakers' yeast cells, Saccharomyces cerevisiae, if grown aerobically on ethanol or if grown aerobically on glucose and allowed to pass into stationary phase, with utilization of accumulated ethanol, maintain a normal value (0.8 to 0.9) of the adenylate energy charge during prolonged starvation. In contrast, cells grown anaerobically on glucose and cells in the early stages of aerobic growth on glucose exhibit a rapid decrease of energy charge if transferred to medium lacking on energy source. These results suggest that functional mitochondria or enzymes of balance of adenine nucleotides during starvation. Yeast cells remain viable at energy charge values below 0.1, in marked contrast to results previously obtained with Escherichia coli. In other respects, the engery charge responses of yeast to starvation and refeeding are generally similar to those previously reported for E. coli.

Electron microscope heteroduplex studies of sequence relations among bacterial plasmids: identification and mapping of the insertion sequences IS1 and IS2 in F and R plasmids.

Abstract: Heteroduplex experiments between the plasmid R6 and one strand of the deoxyribonucleic acid (DNA) of a lambda phage carrying the insertion sequence IS1 show that IS1 occurs on R6 at the two previously mapped junctions of resistance transfer factor (RTF) DNA with R-determinant DNA. From previous heteroduplex experiments, it then follows that IS1 occurs at the same junctions in R6-5, R100-1, and R1 plasmids. Heteroduplex experiments with the DNA from a lambda phage carrying the insertion sequence IS2 show that one copy of IS2 occurs in R6, R6-5, and R100-1 (but not R1) at a point within the RTF with coordinates 67.5 TO 68.9 kilobase units (kb). In an accompanying paper, Ptashne and Cohen (1975) show that the insertion sequence IS3 occurs on R6 and R6-5. R100-25, a traC mutant, differs from its parent R100-1 only in that it contains an additional copy of IS1 inserted within the tra gene region of 82.1 kb. R100-31, atraX, TC-s mutant of R100-1, is deleted in R100-1 sequences starting at one of the IS3 termini (46.9 kb) and extending with RTF to 61.0 kb. Heteroduplex studies of F plasmids with the DNA of a lambda phage bearing insertion sequence IS2 show that the sequence of F with coordinates 16.3-17.6F is IS2. The occurrence of IS1 at the two junctions of R-determinant DNA and RTF DNA in R plasmids provides a structural basis to explain the mechanism of the previously observed formation of molecules containing one RTF unit and several tandem copies of the R-determinant unit, when R plasmids in Proteus mirabilis are grown in the presence of antibiotics, and the segregation of an R plasmid into an RTF unit and an R-determinant unit. In general, correlation of our results with previous studies shows that insertion sequences play a role in a variety of F- and R-related intra- and intermolecular recombination phenomena.

Identification of the dicyclohexylcarbodiimide-reactive protein component of the adenosine 5'-triphosphate energy-transducing system of Escherichia coli.

Abstract: Membranes of Escherichia coli contain an adenosine 59-triphosphate (ATP) energy-transducing system that is inhibited by treatment with dicyclohexylcarbodiimide (DCCD) The carbodiimide-reactive protein component of this system has been identified after treatment with [14C]DCCD This protein has an apparent molecular weight of 9,000 as judged from acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and is extracted from the membrane with chloroform-methanol (2:1) These properties are similar to the analogous protein previously identified in mitochondria (Cattell et al, 1971) A mutant strain, RF-7, has been isolated which derives energy from oxidative phosphorylation in the presence of 5 mM DCCD The ATP hydrolase activity of the membraned system in the mutant was considerably less sensitive to inhibition by DCCD than that in the wild type The carbodiimide-reactive protein, which was easily labeled by [14C]DCCD in the wild type, was labeled much less rapidly in the carbodiimide-resistant mutant It is thus concluded that the reaction of DCCD with this specific protein leads to inhibition of the ATP energy-transducing reactions The mutation causing carbodiimide resistance in strain RF-7 was mapped It is cotransduced with the uncA gene at a frequency exceeding 90% The mutationally altered protein causing the carbodiimide resistance was not conclusively identified However, reconstitution experiments indicate that the altered protein is not one of the subunits of the soluble ATP hydrolase activity, which can be removed from the membrane by washing with 1 mM tris(hydroxymethyl)aminomethane buffer lacking Mg2+ The carbodiimide-reactive protein remains with the membrane residue after removal of the soluble ATP hydrolase and is thus distinct from these subunits as well Images

Control of cell length in Bacillus subtilis.

Abstract: During inhibition of deoxyribonucleic acid synthesis in Bacillus subtilis 168 Thy-minus Tryp-minus, the rate of length extension is constant. A nutritional shift-up during thymine starvation causes an acceleration in the linear rate of length extension. During a nutritional shift-up in the presence of thymine, the rate of length extension gradually increases, reaching a new steady state at about 50 min before the new steady-state rate of cell division is reached. The steady-state rates of nuclear division and length extension are reached at approximately the same time. The ratio of average cell length to numbers of nuclei per cell in exponential cultures is constant over a fourfold range of growth rates. These observations are consistent with: (i) surface growth zones which operate at a constant rate of length extension under any one growth condition, but which operate at an absolute rate proportional to the growth rate of the culture, (ii) a doubling in number of growth zones at nuclear segregation, and (iii) a requirement for deoxyribonucleic acid replication for the doubling in a number of sites.

Regulation of alkane oxidation in Pseudomonas putida.

Abstract: We have studied the appearance of whole-cell oxidizing activity for n-alkanes and their oxidation products in strains of Pseudomonas putida carrying the OCT plasmid Our results indicate that the OCT plasmid codes for inducible alkane-hydroxylating and primary alcohol-dehydrogenating activities and that the chromosome codes for constitutive oxidizing activities for primary alcohols, aliphatic aldehydes, and fatty acids Mutant isolation confirms the presence of an alcohol dehydrogenase locus on the OCT plasmid and indicated the presence of multiple alcohol and aldehyde dehydrogenase loci on the P putida chromosome Induction tests with various compounds indicate that inducer recognition has specificity for chain length and can be affected by the degree of oxidation of the carbon chain Some inducers are neither growth nor respiration substrates Growth tests with and without a gratuitous inducer indicate that undecane is not a growth substrate because it does not induce alkane hydroxylase activity Using a growth test for determining induction of the plasmid alcohol dehydrogenase it is possible to show that heptane induces this activity in hydroxylase-negative mutants This suggests that unoxidized alkane molecules are the physiological inducers of both plasmid activities

Molecular mechanisms of accommodation in Escherichia coli to toxic levels of Cd2

Abstract: Cells of Escherichia coli strain B develop large intracellular vacuoles and exhibit an abnormally long lag phase when inoculated into a defined medium to glucose and salts containing 3 times 10-6 M Cd2+. Early in this lag, about 95% of the cells fail to form colonies when plated on nutrient broth-NaCl-agar. Prior to the initiation of proliferation, the morphology of these cells becomes normal. They regain viability in the absence of deoxyribonucleic acid replication. The rate and extent of growth are normal once proliferation begins. This reversible phenomenon of accommodation to a growth-inhibiting concentration of Cd2+ does not appear to result from a selection of mutant cells. Cells which are proliferating in the presence of Cd2+ accumulate the ion to a very high concentration. In membranes and 31% in the cytoplasm. In unaccommodated cells, the figures are 2%, 75%, and 23%, respectively. The activity of alkaline phosphatase, a zinc-metalloenzyme which is inhibited by cadmum and is located between cell wall and membrane, is not abnormally low in accommodated cells, suggesting that the cadmim is compartmentalized in these cells. Molecular sieve chromatography of cell extracts shows that the Cd2+ is associated with two classes of macromolecules. It appears that accommodation of E. coli to the presence of Cd2+ involves exclusion of the ion from the cell and reversal of damage caused by prior exposure to the ion.

Effects of varying the carbon source limiting growth on yield and maintenance characteristics of Escherichia coli in continuous culture.

Abstract: The magnitudes of Yo (grams [dry weight] formed per gram of atom O) and mo, the maintenance respiration (milligram-atoms of O per gram [dry weight] per hour), of Escherichia coli B have been determined by growing the organism in aerobic continuous culture limited by a number of different substrates. The value found were as follows: glucose--tyo = 12.5, mo = 0.9; glucose plus 2.7 mM cyclic adenosine 3',5'-monophosphate (cAMP)--Yo = 31.2, mo = 9.3; galactose--Yo = 13.2, mo = 1.8; mannitol--Yo = 20.1, mo = 6.1; L-glutamate--Yo = 25.5, mo = 17.7; glycerol--Yo = 14.9, mo = 10.0; succinate--Yo = 11.2, mo = 12.1; and acetate--Yo = 14.7, mo = 25.4. During growth in anaerobic continuous culture with limiting glucose YATP was found to be 10.3 g (dry weight)/mol of adenosine 5'-triphosphate (ATP) and m ATP was 18.9 mmol of ATP/g (dry weight) per h. The aerobic growth yields of cells growing on glucose, glucose plus cAMP, mannitol, and glutamate were consistent with the hypothesis that carbohydrates partially repress oxidative phosphorylation, but the yields of cells growing on glycerol, succinate, acetate, and galactose were all lower than expected. We conclude that, like the efficiency of oxidative phosphorylation, both the maintenance respiration and the amount of ATP necessary to serve maintenance processes are determined by the identity of the growth substrates. Yields smaller than expected may be explained by the absence of respiratory control exerted by phosphate acceptors.

Transformation analysis of three linkage groups in Staphylococcus aureus.

Abstract: While studying a set of multiply marked mutants of Staphylococcus aureus strain 8325 by transformation, several instances of apparent genetic linkage were encountered. After showing that these linked transformations were readily inactivated by shearing of the deoxyribonucleic acid (DNA) but were resistant to dilution of the DNA, and showing that mixtures of DNA failed to form double transformants, it was concluded that the linkages were legitimate rather than the result of congression. Three linkage groups were defined: thy-101-lys-115-trp-103-thr-106, pyr-141-hisGb15-nov-pur-102, and pur-110-ilv-129. The positions of the previously studied trp and his operons corresponded to the trp-103 and hisGb15 loci. The ilv-129 position adjacent to pur-110 probably corresponds to the ilv-leu gene cluster. The distance over which linkage was detected was greater by transformation than by generalized transduction.