Physiological Modifications in the Production and Repair of Methyl Methane Sulfonate-Induced Breaks in the Deoxyribonucleic Acid of Escherichia coli K-12

The medium in which Rec(+) strains of Escherichia coli K-12 are grown affected their sensitivity to treatment with methyl methane sulfonate (MMS). Rec(+) cells grown to the stationary phase in glucose-enriched nutrient broth (GNB) were more resistant to MMS than cells grown in nutrient broth (NB). The repair of MMS-induced breaks (or alkali-labile bonds) in the deoxyribonucleic acid (DNA) from E. coli K-12 strains AB1157, AB1886 uvrA6, and SR111 recA13 recB21 grown in GNB and NB media was examined by means of alkaline sucrose gradient centrifugation. It appeared that essentially all of the repair of breaks that occurred, as evidenced by an increase in "molecular weight," took place within 10 min after treatment with MMS under our conditions. Cell survival was highest in cells for which the size of the DNA after the post-treatment incubation was the largest. The largest DNA after post-treatment incubation was found in Rec(+) cells grown in GNB medium. The results suggest that these cells may have an enhanced capacity for repairing breaks in DNA.     

Comparison of the effects of ultraviolet light and ethylmethanesulphonate upon the frequency of mitotic recombination in yeast

Summary Host-cell reactivation of gamma-irradiated phage T1 in strains of E. coli K-12 has been compared with HCR of UV-irradiated phage in these same strains and with the radiation sensitivities of these strains (Fig. 1–4). The pattern of the HCR of gammairradiated phage in these strains is like that of the HCR of UV-irradiated phage. HCR in strains whose genotype is uvr ⁺rec^- is like that of the wild type; whereas, HCR is minimal in strains which are uvr ^-. It is suggested that some type of gamma-ray-induced base damage in phage DNA is repaired in uvr ⁺ strains.This work was supported by the United States Atomic Energy Commission Contract No. AT(11-1)-1686. — This is report No. COO-1686-6.Supported in part by the United States Public Health Service Training Grant No. 5T1 RH-80-02(67).     

Effects of respiratory activity on starvation survival of marine vibrios

The marine bacterium Vibrio fluvialis NCTC11328 responded to nutrient depletion by a reduction in cell volume, and this was prevented by conditions that eliminated respiration as a source of energy. Addition of the protonophore, CCCP, removal of oxygen and introduction of mutations leading to defects of the respiratory chain prevented size reduction during periods of nutrient limitation. Further, survival of the wild-type strain during starvation was reduced under anaerobic conditions and survival of respiratory mutants under aerobic conditions was reduced compared with that of the parent strain. Removal by mutation of the respiratory Na⁺ pump from Vibrio alginolyticus did not inhibit size reduction or lead to reduced viability in starved cultures.Abbreviations ANa medium A containing 0.4 M sodium chloride - ANaS ANa containing 50 mM sodium succinate - CCCP carbonyl cyanide-m-chlorophenylhydrazone - CFU colony forming units - FMNH flavine mononucleotide - MMS modified Morita's salts solution - MMSGC MMS containing 20 mM glucose and 0.1% (w/v) casamino acids - MMST MMS buffered to pH 8.5 with 50 mM tricine - NM nutrient Morita's broth - Ox oxidase - DH dehydrogenase - NTG N-methyl-N'-nitro-N-nitrosoguanidine     

Behavior of Some Episomal Elements in a Recombination-Deficient Mutant of Escherichia coli

Conjugal transfer and autonomous replication of some episomes occurred normally in a recombination-deficient (Rec^–) mutant of Escherichia coli K-12. Transduction with phage Plbt of an R factor also occurred normally in this Rec^– mutant, but complete or abortive transduction with Plbt of chromosomal genes did not occur. In contrast, transduction of galactose genes by phage λ_dg occurred in the Rec^– bacteria as frequently as in the Rec⁺ strain. It was shown that phage Plbt does not grow at all on the Rec–bacteria. Recombination between two different R factors, two mutants of phage λ and two mutants of phage T4 occurred normally in the Rec^– bacteria, but did not give a Rec⁺ phenotype to the host bacteria. Colicinogenic factor I made the Rec^– host bacteria more resistant to ultraviolet light but the colicinogenic strain was still infertile in the crosses with the Hfr srains of E. coli K-12.     

Isolation and characterisation of Hfr strains from a recombination-deficient strain of Escherichia coli

Summary A T6^s RecA^- strain carrying a lac proB deletion and F_ts lac ⁺ was challenged with phage T6 and survivors which were both T6^r and Lac⁺ at 42° were tested for fertility. Among these were a number of Hfr strains which had their points of origin at or near the tsx locus and which still carried the recA allele. These arose in comparable frequencies in the RecA^- strain and in a Rec⁺ analogue. We conclude that such integration does not require the RecA function. The rate of chromosome transfer was similar in one such RecA^- Hfr and its Rec⁺ derivative; the yield of recombinants from the RecA^- strain was slightly lower than from its Rec⁺ derivative.     

The effect of the source of inorganic nitrogen on growth and enzymes of nitrogen assimilation in soybean and wheat cells in suspension cultures

Summary Soybean (Glycine max L. cv. Mandarin) and wheat (Triticum monococcum L.) cells were grown in media with NO₃ ^- plus NH₄ ⁺ (B₅) and NO₃ ^- without NH₄ ⁺ (B₅-NH₄) as nitrogen sources. Changes in pH, [NO₃ ^-] and [NH₄ ⁺] in media, and dry weight, protein content, nitrate reductase (NR) and glutamate dehydrogenase (GDH) in the cells were followed for about 170 h. With both NH₄ ⁺ and NO₃ ^- in the medium, NH₄ ⁺ was utilized very quickly. Soybean cells grew poorly in the absence of NH₄ ⁺ while wheat cells grew equally well on media with or without NH₄ ⁺. When soybean cells were grown in medium with NO₃ ^- plus NH₄ ⁺, dry weight and NR activity remained relatively low for several hours after which both increased rapidly. This coincided with the time NH₄ ⁺ was depleted from the medium. In the absence of NH₄ ⁺, soybean cell growth and NR activity remained low. NR activity in wheat cells, and GDH activity in soybean and wheat cells, did not vary significantly in the presence or absence of NH₄ ⁺.This work was supported by a grant in aid of research from the National Research Council of Canada to one of us (J. K.). NRCC No. 12521.     

A correlation between sensitization to gamma-rays by purine starvation and the rec genes in Escherichia coli K-12

Summary After purine staration the recombination proficient (Rec⁺) strains of E. coli K-12 tested were sensitized to inactivation by gamma-rays (Figs. 1–4). No such change was noted in the Rec^– strains tested (Fig. 5). These results suggest that purine starvation may sensitize cells to ionizing radiation by inhibiting repair controlled by the recA, recB and recC genes.This paper was reported in part at the Fourteenth Annual meeting of the Biophysical Society in Baltimore, Md., 25–27 February 1970.     

Effects of Al on nitrogen (NH 4 + and NO 3 − ) uptake,nitrate reductase activity and proton release in two sorghum cultivars differing in Al tolerance

After growth for 17 to 36 days on nutrient solutions with NH₄NO₃ as nitrogen source (pH 4.2) dry matter of sorghum genotype SC0283 was much less affected by Al (1.5 and 3.0 ppm) than that of genotype NB9040. In the absence of Al both cultivars released protons into the nutrient solution as a result of an excess of cationic nutrients taken up. When Al was present, this proton efflux per unit dry weight increased drastically, especially with the sensitive genotype NB9040. Chemical analysis of plant material and continuous analyses of NO ₃ ⁻ and NH ₄ ⁺ in the nutrient solution indicated, that the Al-induced shift in H⁺-balance of both genotypes could almost completely be attributed to a decreased NO ₃ ⁻ /NH ₄ ⁺ uptake ratio. In vivo nitrate reductase activity (NRA) was reduced in the shoot of NB9040 and to a lesser degree in SC0283. Al-induced decrease in NRA was accompanied by similar percentual decreases in NO ₃ ⁻ tissue concentrations. Therefore this decrease is interpreted as being indirect,i.e., the consequence of the reduced NO ₃ ⁻ uptake of the plants. A direct repression of NRA by Al seems also unlikely because nitrate reductase activity of the roots (where cellular Al-concentrations should be higher than in shoots) was not affected in Al-treated plants of either genotype.     

Non-receptivity for kappa phage of kappa-lysogenic Serratia and reactions to superinfection of receptive cells with a mutant prophage

Summary l_I-lysogenic cells of Serratia marcescens as opposed to l_I ⁺-lysogenic cells, are receptive to further infection. After superinfection with wild type or several clear plaque mutants killing and lytic response were observed to a varying extent. From some of the surviving cells doubly lysogenic colonies with a l_I ⁺ and a l_I prophage could originate, l_I ⁺ being dominant and the cells therefore non-receptive. By this property, combined with a special color reaction, the colonies could be easily screened. Evidence is presented that the l_I ⁺ gene product probably does not interfere with the formation of new phage receptors but that under its influence receptors are masked.Two mutants resembling int^- mutants are described which only rarely give double lysogenization and prophage substitution following superinfection. The phage-coded function normally achieving these reactions is likely to work constitutively after superinfection. Transduction experiments performed with one of the two mutants pointed at integration of prophage into the host DNA near to the trp gene.Abbreviations moi multiplicity of infection - OD optical density - NG N-methyl-N-nitro-N-nitrosoguanidine - NB nutrient broth - BS buffered saline - EMB eosmemethylene blue - leu leucine - met methionine - pro proline - trp tryptophan     

Photoproduction of molecular hydrogen by Rhodospirillum rubrum immobilized in composite agar layer/microporous membrane structures

Summary Viable cells of Rhodospirillum rubrum were immobilized by entrapment in a planar agar matrix bounded by a microporous membrane filter and were tested for H₂ photoproduction in synthetic waste water provided with malate and glutamate. Optimum H₂ production was obtained at 15 klx for a C/N ratio of 7–8. Production rates as high as 565 mm³ H₂ · h^-1 per cubic centimetre of agar were recorded. The composite structures, however, suffered from high diffusion limitations which increased with the population of immobilized bacteria. The H₂-evolving activity could be maintained over several months by periodically incubating the biocatalytic structures in a rich nutrient broth. No contamination of the nutrient broth due to leakage of photosynthetic organisms from the gel appeared during incubation of the structures.     

Relationship of K+-Uptaking System with H+-Translocating ATPase in Enterococcus hirae, Grown at a High or Low Alkaline pH

Potassium ion pool was studied in glycolyzing Enterococcus hirae, grown at high or low alkaline pH (pH 9.5 and 8.0, respectively). Energy-dependent increase of K⁺ pool was lower for the wild-type cells, grown at pH 9.5, than that for the cells grown at pH 8.0. It was inhibited by N,N′-dicyclohexylcarbodiimide (DCCD). The stoichiometry of DCCD-inhibited K⁺ influx to DCCD-inhibited H⁺ efflux for the wild-type cells, grown at pH 9.5 or 8.0, was fixed for different K⁺ external activity. DCCD-inhibited ATPase activity of membrane vesicles was significantly stimulated by K⁺ for the wild-type cells grown at pH 9.5, and required K⁺ for the wild-type cells grown at pH 8.0, while the levels of α and β subunits of the F₁ and b subunit of the F₀ were lower for the cells grown at pH 9.5 than that for the cells grown at pH 8.0. Such an ATPase activity was residual in membrane vesicles from the atpD mutant with a nonfunctional F₀F₁. ATPase activity of membrane vesicles from the mutant with defect in Na⁺-ATPase was higher for the cells grown at pH 9.5 than that for the cells grown at pH 8.0, and was inhibited by DCCD. An energy-dependent increase of K⁺ pool in this bacterium, grown at a high or low alkaline pH, is assumed to occur through a K⁺ uptaking system, most probably the Trk. The latter functions in a closed relationship with the H⁺-translocating ATPase F₀F₁. Received: 30 June 1997 / Accepted: 4 August 1997     

UNEXPECTED RESPONSE TO VITAMIN B12 OF DOMINANT CENTRIC DIATOMS FROM THE SPRING BLOOM IN THE GULF OF MAINE (NORTHEAST ATLANTIC OCEAN)1,2

Twelve clones (seven species) representative of centric diatoms dominant in the spring phytoplankton bloom in the Gulf of Maine were isolated and rendered axenic. Genera included were Thalassiosira, Porosira and Chaetoceros. Unlike most centric diatoms studied previously, none of these has an absolute requirement for vitamin B_12. However, B₁₂ (5 ng.l^-1) stimulated growth of most clones by eliminating or reducing the lag phase and increasing the growth rate. Bloom population densities developed 4–54 days earlier with B₁₂ present. Several clones grown with B₁₂ removed more than 80% of the vitamin from the medium. When grown in vitamin-free medium the cells put 0.01–0.7 ng.l^-1 B₁₂ into the medium. We conclude that vitamin B₁₂ is of ecological significance even though the requirement for it is not absolute.     

Nitrogen metabolism inRhodopseudomonas globiformis

Rhodopseudomonas globiformis strain 7950 grew with a variety of amino acids, urea, or N₂ as sole nitrogen sources. Cultures grown on N₂ reduced acetylene to ethylene; this activity was absent from cells grown on nonlimiting NH ₄ ⁺ . Glutamate dehydrogenase could not be detected in extracts of cells of strain 7950, although low levels of an alanine dehydrogenase were present. Growth ofR. globiformis on NH ₄ ⁺ was severely inhibited by the glutamate analogue and glutamine synthetase inhibitor, methionine sulfoximine. High levels of glutamine synthetase (as measured in the -glutamyl transferase assay) were observed in cell extracts of strain 7950 regardless of the nitrogen source, although N₂ and amino acid grown cells contained somewhat higher glutamine synthetase contents than cells grown on excess NH ₄ ⁺ . Levels of glutamate synthase inR. globiformis were consistent with that reported from other phototrophic bacteria. Both glutamate synthase and alanine dehydrogenase were linked to NADH as coenzyme. We conclude thatR. globiformis is capable of fixing N₂, and assimilates NH ₄ ⁺ primarily via the glutamine synthetase/glutamate synthase pathway.Abbreviations GS glutamine synthetase - GOGAT Glutamineoxoglutarate aminotransferase - GDH Glutamate dehydrogenase - ADH Alanine dehydrogenase - MSO Methionine sulfoximine     

Potassium transport in Chlamydomonas reinhardtii: isolation and characterization of transport-deficient mutant strains

Putative potassium-transport-deficient mutant strains of Chlamydomonas reinhardtii Dang. were induced by ultra-violet mutagenesis and were identified by their dependence on abnormally high concentrations of potassium for growth. Potassium transport studies employing ⁸⁶Rb as a tracer were carried out with wild-type cells and with three independently isolated KDP (potassium-dependent phenotype) clones. Wildtype cells exhibit two transport activities. Transport activity A was expressed when cells were grown in medium supplemented with 10 mM KCl. The transporter with type-A activity does not discriminate between either Rb⁺ or K⁺ as a substrate and has a K_m for Rb⁺ equal to 1 mM and a V_max equal to 31 nmol Rb⁺ h^-1 10^-6 cells. Transport activity B was expressed when cells were starved of potassium for 24 h. The transporter with type-B activity prefers K⁺ to Rb⁺ as a substrate; it has a K_m for Rb⁺ equal to 2.5 mM and a V_max equal to 210 nmol Rb⁺ h^-1 10^-6 cells. All three mutant clones exhibit transport activity comparable to type-A when grown in 10 mM KCl. When starved of potassium for 24 h, two KDP clones demonstrate no transport activity and the third clone continues to exhibit only type-A activity.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DES diethylstilbesterol - KDP potassium-dependent phenotype     

Utilization of n-Paraffins and Vitamin B6 Production by Pichia guilliermondii Wickerham

The accumulation of vitamin B₆ by Pichia guilliermondii Wickerham NK–2 strain grown on hydrocarbon was investigated. Ammonium acetate was more effective than other nitrogen sources tested. Satisfactory utilization by the yeast strain was observed in n-alkanes of C₁₀–C₁₈, and n-pentadecane was the best for vitamin B₆ production. Vitamin B₆ was excreted in the cultural broth mainly in the form of pyridoxal, The maximal vitamin B₆ production was approximately 25 mg per liter of the culture broth.     

Physiological Streptomycin Resistance in a Multiauxotroph of Escherichia coli Strain 15 T

Escherichia coli strain WWU was found to be moderately resistant to streptomycin when grown in a minimal medium, although the strain was sensitive if grown in nutrient broth. Transfer experiments showed that cells grown in minimal medium retain the resistant state for a period of time after dilution into nutrient broth; and conversely, sensitive cells grown in nutrient broth were sensitive after dilution into minimal medium for a period of time. The kinetics of transition from resistant to sensitive and from sensitive to resistant were observed, and kinetics of ³H-dihydrostreptomycin accumulation by resistant and sensitive cells were compared. The data suggested that cells grown in minimal medium were physiologically resistant because they accumulated streptomycin poorly. Inactivation per incorporated antibiotic molecule was the same in resistant and sensitive cells.     

Cell surface structure enhancing uptake of polyvinyl alcohol (PVA) is induced by PVA in the PVA-utilizing <Emphasis Type="Italic">Sphingopyxis</Emphasis> sp. strain 113P3

Polyvinyl alcohol (PVA)-utilizing Sphingopyxis sp. 113P3 (reidentified from Sphingomonas sp. 113P3) removed almost 0.5% PVA from culture supernatants in 4 days. Faster degradation of 0.5% PVA was performed by the periplasmic fraction. The average molecular size of PVA in the culture supernatant or cell-bound PVA was gradually shifted higher, suggesting that lower molecular size molecules are degraded faster. Depolymerized products were found in neither the culture supernatant nor the cell-bound fraction; however they were recovered from the periplasmic fraction. As extracellular or cell-associated PVA oxidase activity was almost undetectable in strain 113P3, degradation of PVA must be performed by periplasmic PVA dehydrogenase after uptake into the periplasm. Following the consumption of PVA, a dent appeared on the cell surface on day 2 and increased in size and depth for 4 days and was maintained for 8 days. Ultrastructural change on the cell surface was only observed in PVA medium, but not in nutrient broth (NB), suggesting that the change is induced by PVA. Fluorescein-4-isothiocyanate-labeled PVA was bound more to cells grown in PVA than to cells grown in NB. No binding was found with PVA-grown cells treated with formaldehyde. Thus, a dent on the cell surface seems to be related to the uptake of PVA.     

Soybean response to nodulation by rhizobitoxine-producing bradyrhizobia as influenced by nitrate application

Foliar chlorosis of soybean (Glycine max [L.] Merr.) resulting from nodulation by rhizobitoxine-producing (RT⁺) strains of Bradyrhizobium japonicum is commonly less severe in the field than under greenhouse conditions. Differences in nutritional conditions between the field and greenhouse may contribute to this phenomenon. In particular, field-grown plants obtain some N from soil sources, whereas in the greenhouse soybean is often grown in low-N rooting media to emphasize symbiotic responses. Therefore, we examined the effect of NO₃ ^- on the expression of RT-induced symptoms. Soybean plants inoculated with RT⁺ bradyrhizobia were grown for 42 days in horticultural vermiculite receiving nutrient solution amended with 0.0, 2.5, or 7.5 mM KNO₃. Foliar chlorosis decreased with increasing NO₃ ^- application whereas nodule mass per plant was generally increased by NO₃ ^- application. Total amounts of nodular RT remained constant or increased with NO₃ ^- application, but nodular concentrations of RT decreased. Chlorosis severity was negatively correlated with shoot dry weight, chlorophyll concentration, and total shoot N content. It was concluded that application of NO₃ ^- can reduce the negative effects of RT production on the host plant. This suggests that any NO₃ ^- present in field soils may serve to limit chlorosis development in soybeans.Abbreviations RT rhizobitoxine - RT⁺ rhizobitoxine-producing - Lb leghemoglobin Published as Miscellaneous Paper No. 1429 of the Delaware Agricultural Experiment Station.     

Multiple effects of salinity on photosynthesis of the protist Euglena gracilis

The effect of NaCl in the culture medium on growth, photosynthesis and cell content of chlorophyll, K⁺, Na⁺, Ca²⁺ and Mg²⁺ in Euglena gracilis was studied. O₂ production, quantum yield of photosystem II (PSII), the non-photochemical quenching of chlorophyll fluorescence (q_N) and the chlorophyll alb ratio all diminished by 0.2 M NaCl. Respiration and chlorophyll a and b increased, whereas the photochemical quenching (q_p) of chlorophyll fluorescence was not affected by 0.2 M NaCl. Salt stress also induced an increase in cell volume and in K⁺ and Na⁺ concentrations, but decreased the concentrations of Ca²⁺ and Mg²⁺. Except for a protective effect on O₂ production, additional Ca²⁺ in the culture medium did not attenuate the salt effect on the parameters measured. The addition of HCO₃^? restored the PSII quantum yield of O₂ production in cells grown in high salt. Salt stress promoted a decrease in the apparent rate of quinone A (Q_A) reduction and an apparent obstruction of Q_B reduction, which were not prevented by excess HCO₃^?; the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not increase chlorophyll fluorescence in salt-grown cells. These results indicate that photosynthesis in Euglena grown under salt stress exhibits: (1) diminution of the HCO₃^? dependent water-splitting activity of PSII; (2) inhibition of the electron transfer at the quinone pool level; (3) probable increase in thylakoid stacking (as indicated by the effect on the chlorophyll alb ratio); and (4) dissipation of the H⁺ gradient across the thylakoid membranes (as indicated by the decrease of q_N).     

Catabolism of 1,3-dinitrobenzene by Rhodococcus sp. QT-1

The 1,3-dinitrobenzene-degrading Rhodococcus strain QT-1 was isolated under nitrogen limiting conditions from contaminated soil samples. Experimental data indicate that 1,3-dinitrobenzene is metabolized via 4-nitrocatechol. Both compounds were oxidized by resting cells and nitro groups were completely eliminated as nitrite. Strain QT-1 utilizes both 1,3-dinitrobenzene and 4-nitrocatechol as source of nitrogen in the absence as well as in the presence of high amounts of ammonia. Growth on 4-nitrocatechol does not induce the enzyme(s) for the initial oxidation of 1,3-dinitrobenzene.Abbreviations TNT 2,4,6-trinitrotoluene - 1,3DNB 1,3-dinitrobenzene - 4NC 4-nitrocatechol - 3NA 3-nitroaniline - NB nutrient broth; t_d doubling time - OD₅₄₆ optical density at 546 nm