Identification of tms-26 as an allele of the gcaD gene, which encodes N-acetylglucosamine 1-phosphate uridyltransferase in Bacillus subtilis.

The temperature-sensitive Bacillus subtilis tms-26 mutant strain was characterized biochemically and shown to be defective in N-acetylglucosamine 1-phosphate uridyltransferase activity. At the permissive temperature (34 degrees C), the mutant strain contained about 15% of the wild-type activity of this enzyme, whereas at the nonpermissive temperature (48 degrees C), the mutant enzyme was barely detectable. Furthermore, the N-acetylglucosamine 1-phosphate uridyltransferase activity of the tms-26 mutant strain was much more heat labile in vitro than that of the wild-type strain. The level of N-acetylglucosamine 1-phosphate, the substrate of the uridyltransferase activity, was elevated more than 40-fold in the mutant strain at the permissive temperature compared with the level in the wild-type strain. During a temperature shift, the level of UDP-N-acetylglucosamine, the product of the uridyltransferase activity, decreased much more in the mutant strain than in the wild-type strain. An Escherichia coli strain harboring the wild-type version of the tms-26 allele on a plasmid contained increased N-acetylglucosamine 1-phosphate uridyltransferase activity compared with that in the haploid strain. It is suggested that the gene for N-acetylglucosamine 1-phosphate uridyltransferase in B. subtilis be designated gcaD.     

Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistance

Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth. The CBSH activity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wild-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutants without CBSH activity. In resting cell experiments, the strain showed reduced activity. Differences between growth parameters determined for wild-type and mutant strains were not detected. Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant. Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant. The specific growth rate of the mutant strain was affected more by bile salts than the wild-type strain. Nevertheless, bile was more toxic for the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner. No difference was apparent between the two strains. In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant. The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (ZDeltapH) was involved in Lactobacillus bile salt resistance.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N(2) Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO(3)-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a "conventional" Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Deoxyribonucleic Acid Repair in a Highly Radiation-Resistant Strain of Salmonella typhimurium

Deoxyribonucleic acid repair was studied in gamma-irradiated wild-type Salmonella typhimurium and in a radiation-resistant derivative 20 times more resistant than wild type. After exposure to 20 or 50 krad, the wild-type strain (DB21) degraded 30 to 50% of its prelabeled DNA into acid-soluble fragments, whereas the radioresistant strain degraded less than 15% after 4 h of incubation. Post-irradiation synthesis of DNA in the wild-type strain DB21 was reduced after a dose of 20 krad and totally inhibited after exposure to 200 krad. With radiation-resistant strain, D21R6008, on the other hand, DNA synthesis was delayed after a dose of 200 krad but not inhibited. Doses of 20 and 200 krad produced a similar number of single-strand breaks in the DNA of both strains as determined by zone sedimentation analysis in alkaline sucrose gradients. The radiation-resistant strain D21R6008, on the other hand, DNA synthesis was strand breaks in its DNA and repairs these damages more rapidly than wild-type Salmonella.     

Penicillin production by glucose-derepressed mutants ofPenicillium chrysogenum

Summary Wild-type strains ofPenicillium chrysogenum produce lower penicillin V titers in media containing excess glucose. Two mutant strains were isolated and shown to produce normal penicillin V titers in the presence of excess glucose. These strains, designated as glucose-repression insensitive (GRI) mutants, produced higher penicillin V titers than the wild-type strain in media containing lactose as the main carbohydrate source. In lactose-based media, the production of penicillin V was depressed to a much lesser extent by in-cycle additions of glucose with the GRI mutants when compared to the wild-type strain. In short-term biosynthesis experiments using washed cells in a medium containing glucose as the sole carbon source, the GRI mutants produced penicillin V at a faster rate than the wild-type strain. In fed-batch fermentations in 14-liter fermentors, where glucose was fed continuously and pH controlled, both GRI mutants produced more than 10% higher penicillin V titers than the wild-type strain. These results suggest that isolation of GRI mutants is an effective way to select for higher producing strains and that the synthesis of penicillin synthesizing enzymes in GRI mutants may be less repressed by glucose than in wild-type strains.     

Competitiveness of Rhizobium sp. [Leucaena leucocephala (Lam.) Dewit] strains and their genetically marked derivatives

D.M. SWELIM, L.D. KUYKENDALL, F.M. HASHEM, S.M. ABDEL-WAHAB AND N.I. HEGAZI. 1996. The competitiveness of wild-type strains of Rhizobium sp. ( Leucaena ) and their genetically marked double mutants was examined in mixed infection experiments in the greenhouse. Antibiotic resistance markers were selected for use in strain identification, but these genetic markers apparently lowered both competitiveness and effectiveness, except in the case of strain DS 144/2 where the genetically marked derivative was evidently superior to the wild-type parent strain in effectiveness. Four wild-type strains and their genetically marked derivatives were carefully evaluated using double reciprocal pairs, the results of which nevertheless allowed the formulation of some conclusions. Strains DS 65 and DS 78 were more competitive than strain DS 144/2; only strain DS 78 was more competitive than DS 158; and strains DS 158 and DS 65 were equally competitive. There was no correlation between nodule number and competitiveness. Shoot dry weight and nitrogen mass, as well as nitrogenase activity, decreased with some strain mixtures indicating that relatively ineffective symbioses had formed, as compared with single-strain inoculations using symbiotically competent strains.     

Essential role of small, acid-soluble spore proteins in resistance of Bacillus subtilis spores to UV light.

Bacillus subtilis strains containing deletions in the genes coding for one or two of the major small, acid-soluble spore proteins (SASP; termed SASP-alpha and SASP-beta) were constructed. These mutants sporulated normally, but the spores lacked either SASP-alpha, SASP-beta, or both proteins. The level of minor SASP did not increase in these mutants, but the level of SASP-alpha increased about twofold in the SASP-beta- mutant, and the level of SASP-beta increased about twofold in the SASP-alpha- mutant. The growth rates of the deletion strains were identical to that of the wild-type strain in rich or poor growth media, as was the initiation of spore germination. However, outgrowth of spores of the SASP-alpha(-)-beta- strain was significantly slower than that of wild-type spores in all media tested. The heat resistance of SASP-beta- spores was identical to that of wild-type spores but slightly greater than that of SASP-alpha- and SASP-alpha(-)-beta- spores. However, the SASP-alpha- and SASP-alpha(-)-beta- spores were much more heat resistant than vegetative cells. The UV light resistances of SASP-beta- and wild-type spores were also identical. However, SASP-alpha(-)-beta- spores were slightly more sensitive to UV light than were log-phase cells of the wild-type or SASP-alpha(-)-beta- strain (the latter have identical UV light resistances); SASP-alpha- spores were slightly more UV light resistant than SASP-alpha(-)-beta- spores. These data strongly implicate SASP, in particular SASP-alpha, in the UV light resistance of B. subtilis spores.     

Physiological and enzymatic properties of a thymidine-requiring Pediococcus cerevisiae mutant.

We describe the isolation and characterization of a Pediococcus cerevisiae thymidine-requiring mutant and its thymidine-independent revertant. The mutant strain lacked thymidylate synthetase activity and had an absolute requirement for low concentrations (2 micrograms/ml) of thymidine in addition to a requirement for N-5-formyl tetrahydrofolic acid (folinate). Even at high concentrations (up to 500 micrograms/ml), thymine could not replace thymidine. In contrast to its wild-type parent, which grows only on folinate, the thymidine-requiring mutant (Thy- Fol+) was able to take up and grow on picogram quantities of unreduced folic acid. When both strains were grown on folinate, the Thy- Fol+ strain was at least 10(3)-fold more resistant to the folic acid analogs aminopterin and methotrexate than the wild-type strain. On the other hand, when grown on folic acid, the Thy- Fol+ strain was as sensitive to the folic acid analogs as the Thy+ Fol+ strain and was 10(2)-fold more sensitive than the wild-type strain grown on folinate. The thymidine-independent revertant (Thy+ Fol+) regained the wild-type level of thymidylate synthetase activity, but maintained the ability to take up and grow on unreduced folic acid like its Thy- Fol+ parent.     

Isolation and Characterization of Clostridium butyricum DSM 5431 Mutants with Increased Resistance to 1,3-Propanediol and Altered Production of Acids

Clostridium butyricum mutants were isolated from the parent strain DSM 5431 after mutagenesis with N-methyl-N(prm1)-nitro-N-nitrosoguanidine and two selection procedures: osmotic pressure and the proton suicide method. Isolated mutants were more resistant to glycerol and to 1,3-propanediol (1,3-PD) than was the wild type, and they produced more biomass. In batch culture on 62 g of glycerol per liter, the wild type produced more acetic acid than butyrate, with an acetate/butyrate ratio of 5.0, whereas the mutants produced almost the same quantities of both acids or more butyrate than acetate with acetate/butyrate ratios from 0.6 to 1.1. The total acid formation was higher in the wild-type strain. Results of analysis of key metabolic enzymatic activities were in accordance with the pattern of fermentation product formation: either the butyrate kinase activity increased or the acetate kinase activity decreased in cell extracts of the mutants. A decreased level of the hydrogenase and NADH-ferredoxin activities concomitant with an increase in ferredoxin-NAD(sup+) reductase activities supports the conclusion that the maximum percentage of NADH available and used for the formation of 1,3-PD was higher for the mutants (97 to 100%) than for the wild type (70%). In fed-batch culture, at the end of the fermentation (72 h for the wild-type strain and 80 to 85 h for the mutants), 44% more glycerol was consumed and 50% more 1,3-PD was produced by the mutants than by the wild-type strain.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N2 Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO₃-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a “conventional” Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Siliceous scales in the centrohelid heliozoan Raphidocystis contractilis facilitate settlement to the substratum

The centrohelid heliozoan Raphidocystis contractilis has hundreds of small scales on the surface of the cell body. To understand the biological functions of the scales, comparative examinations were conducted between wild-type and scale-deficient strains that has naturally lost scales after long-term cultivation. The scale-deficient strain exhibited decreased adhesion to the substratum and had a lower sedimentation rate in water than the wild-type strain, suggesting that the scale may have the ability to attach quickly and strongly to the substratum. Percoll density gradient centrifugation showed that the scale-deficient strain had a lower density than that of the wild-type strain. In the wild-type strain, more scaled cells were observed in the higher specific gravity fractions. During the long-term culture of cells, only the cells suspended in the upper area of the flask were transferred to fresh medium. By repeating this procedure, we may have selected only cells that did not possess normal scales. In the natural environment, centrohelid heliozoans are easily flushed away if they cannot adhere strongly to the bottom. These results suggest that they use scales to ensure effective adhesion to the substratum.     

Enhancing Transport of Hydrogenophagaflava ENV735 for Bioaugmentation of Aquifers Contaminated with Methyl tert-Butyl Ether

The gasoline oxygenate methyl tert-butyl ether (MTBE) has become a widespread contaminant in groundwater throughout the United States. Bioaugmentation of aquifers with MTBE-degrading cultures may be necessary to enhance degradation of the oxygenate in some locations. However, poor cell transport has sometimes limited bioaugmentation efforts in the past. The objective of this study was to evaluate the transport characteristics of Hydrogenophaga flava ENV735, a pure culture capable of growth on MTBE, and to improve movement of the strain through aquifer solids. The wild-type culture moved only a few centimeters in columns of aquifer sediment. An adhesion-deficient variant (H. flava ENV735:24) of the wild-type strain that moved more readily through sediments was obtained by sequential passage of cells through columns of sterile sediment. Hydrophobic and electrostatic interaction chromatography revealed that the wild-type strain is much more hydrophobic than the adhesion-deficient variant. Electrophoretic mobility assays and transmission electron microscopy showed that the wild-type bacterium contains two distinct subpopulations, whereas the adhesion-deficient strain has only a single, homogeneous population. Both the wild-type strain and adhesion-deficient variant degraded MTBE, and both were identified by 16S rRNA analysis as pure cultures of H. flava. The effectiveness of surfactants for enhancing transport of the wild-type strain was also evaluated. Many of the surfactants tested were toxic to ENV735; however, one nonionic surfactant, Tween 20, enhanced cell transport in sand columns. Improving microbial transport may lead to a more effective bioaugmentation strategy for MTBE-contaminated sites where indigenous oxygenate degraders are absent.     

Enhancing transport of hydrogenophaga flava ENV735 for bioaugmentation of aquifers contaminated with methyl tert-butyl ether

The gasoline oxygenate methyl tert-butyl ether (MTBE) has become a widespread contaminant in groundwater throughout the United States. Bioaugmentation of aquifers with MTBE-degrading cultures may be necessary to enhance degradation of the oxygenate in some locations. However, poor cell transport has sometimes limited bioaugmentation efforts in the past. The objective of this study was to evaluate the transport characteristics of Hydrogenophaga flava ENV735, a pure culture capable of growth on MTBE, and to improve movement of the strain through aquifer solids. The wild-type culture moved only a few centimeters in columns of aquifer sediment. An adhesion-deficient variant (H. flava ENV735:24) of the wild-type strain that moved more readily through sediments was obtained by sequential passage of cells through columns of sterile sediment. Hydrophobic and electrostatic interaction chromatography revealed that the wild-type strain is much more hydrophobic than the adhesion-deficient variant. Electrophoretic mobility assays and transmission electron microscopy showed that the wild-type bacterium contains two distinct subpopulations, whereas the adhesion-deficient strain has only a single, homogeneous population. Both the wild-type strain and adhesion-deficient variant degraded MTBE, and both were identified by 16S rRNA analysis as pure cultures of H. flava. The effectiveness of surfactants for enhancing transport of the wild-type strain was also evaluated. Many of the surfactants tested were toxic to ENV735; however, one nonionic surfactant, Tween 20, enhanced cell transport in sand columns. Improving microbial transport may lead to a more effective bioaugmentation strategy for MTBE-contaminated sites where indigenous oxygenate degraders are absent.     

Viral hemorrhagic septicemia of rainbow trout: selection of a thermoresistant virus variant and comparison of polypeptide synthesis with the wild-type virus strain.

Serial passage of viral hemorrhagic septicemia virus at gradually increasing temperature selected for a variant virus that replicates at 25 degrees C and has a low pathogenicity for rainbow trout. Viral hemorrhagic septicemia virus-specific polypeptide synthesis was examined in epithelioma papulosum cyprini cells infected with either a wild-type strain or a thermoresistant variant. The wild-type N and M1 proteins were synthesized throughout the course of infection, whereas L, G, and M2 were more actively translated later in the replication cycle. The wild-type strain was more cytotoxic at 25 than at 14 degrees C despite the fact that no translation could be evidenced when the temperature was raised. When epithelioma papulosum cyprini cells were infected with the variant virus, the kinetic study was obstructed since protein synthesis was difficult to observe by the pulse method at a low multiplicity of infection and aborted when the multiplicity of infection was raised. The variant was less cytotoxic at 25 degrees C than wild-type virus.     

NHEJ protects mycobacteria in stationary phase against the harmful effects of desiccation

The physiological role of the non-homologous end-joining (NHEJ) pathway in the repair of DNA double-strand breaks (DSBs) was examined in Mycobacterium smegmatis using DNA repair mutants (DeltarecA, Deltaku, DeltaligD, Deltaku/ligD, DeltarecA/ku/ligD). Wild-type and mutant strains were exposed to a range of doses of ionizing radiation at specific points in their life-cycle. NHEJ-mutant strains (Deltaku, DeltaligD, Deltaku/ligD) were significantly more sensitive to ionizing radiation (IR) during stationary phase than wild-type M. smegmatis. However, there was little difference in IR sensitivity between NHEJ-mutant and wild-type strains in logarithmic phase. Similarly, NHEJ-mutant strains were more sensitive to prolonged desiccation than wild-type M. smegmatis. A DeltarecA mutant strain was more sensitive to desiccation and IR during both stationary and especially in logarithmic phase, compared to wild-type strain, but it was significantly less sensitive to IR than the DeltarecA/ku/ligD triple mutant during stationary phase. These data suggest that NHEJ and homologous recombination are the preferred DSB repair pathways employed by M. smegmatis during stationary and logarithmic phases, respectively.     

The type of mutations induced by carbon-ion-beam irradiation of the filamentous fungus Neurospora crassa

Heavy-ion beams are known to cause great damage to cellular components and are particularly renowned for their ability to generate DNA double-strand breaks (DSBs). To gain insight into the mutagenic effect of carbon-ion beams and how such damage is repaired by the cell, Neurospora crassa mutants deficient in one of three components involved in the repair of DSBs, nonhomologous end-joining (NHEJ), homologous recombination repair (HR), and the Mre11-Rad50-Xrs2 (MRX) complex, were irradiated with a carbon-ion beam and killing effect, mutation frequencies, and the type of mutation incurred by survivors were analysed. The sensitivity of the NHEJ-deficient strain (mus-52) was higher than that of the wild-type and the HR-deficient (mei-3) strains at low doses of radiation, but was little changed as the level increased. As a result both the wild-type and HR-deficient strains were more sensitive than the NHEJ-deficient strain at high radiation levels. In addition, the frequency of forward mutation at the adenine-3 (ad-3) loci of the NHEJ-deficient mutant was lower than that of the wild-type strain at all levels, while the mutation frequency of the HR-deficient strain was consistently ∼3-fold higher than the wild-type. From the comparison of mutation type of each strain, deletions were frequently observed in wild-type strain, whilst base substitution and deletion in the mus-52 and mei-3 strains. These mutations resulting from carbon-ion-beam irradiation depend on the mechanism invoked to cope with DSBs. Furthermore, in wild-type cells, these mechanisms likely compete to repair DSBs.     

Isolation and characterization of a mutant of Escherichia coli K12 synthesizing DNA polymerase I and endonuclease I constitutively

A mutant of Escherichia coli K12, highly resistant to ultraviolet radiation, has been isolated. Preliminary tests show that this mutant is also resistant to mitomycin C, nalidixic acid, fluorouracil and thymineless death. The mutant strain apparently repairs its damaged DNA more efficiently than wild-type E. coli K12 strains and, to do so, constitutively produces 35 times more DNA polymerase I and 12 times more endonuclease I than the wild-type strain.     

Ectopic expression of sweet potato MuS1 increases acquired stress tolerance and fermentation yield in Saccharomyces cerevisiae

The MuS1 gene is highly homologous to many stress-related proteins in plants. Here, we characterized whether a new candidate gene, MuS1, is related to multiple stress tolerance in yeast as it is in plants. Transgenic yeast strain expressing MuS1 were more resistant to hydrogen peroxide, menadione, high salinity, metals (i.e., cadmium, copper, iron, and zinc), ethanol, and lactic acid than wild-type strain transformed with a vector alone. In addition, the alcohol yield of the transgenic yeast strain was higher than that of the wild-type strain during the batch fermentation process. These results show that MuS1-expressing transgenic yeast strain exhibits enhanced alcohol yield as well as tolerance to abiotic stresses, especially metal stress.     

Disruption of the structural gene for farnesyl diphosphate synthase in Escherichia coli

The chromosomal ispA gene encoding farnesyl diphosphate synthase of Escherichia coli was disrupted by inserting a neo gene cassette. The null ispA mutants were viable. The growth yield of the mutants was 70% to 80% of that of the wild-type strain under aerobic conditions, and was almost the same as the wild-type under anaerobic conditions. The levels of ubiquinone-8 and menaquinone-8 were both significantly lower (less than 13% and 18% of normal, respectively) in the mutants than in the wild-type. The undecaprenyl phosphate level in the mutants was modestly lower (40% to 70% of normal) than in the wild-type strain. Thus the synthesis of all-E-octaprenyl diphosphate, the precursor of ubiquinone-8 and menaquinone-8, was decreased more severely than that of Z,E-mixed undecaprenyl diphosphate, the precursor of undecaprenyl monophosphates, under the conditions where the synthesis of farnesyl diphosphate was decreased. The condensation of isopentenyl diphosphate with dimethylallyl diphosphate was detected in the cell-free extracts of the mutants, although it was 5% of that in the wild-type strain. A low level of farnesyl diphosphate seems to be synthesized in the mutants by other prenyltransferases such as octaprenyl diphosphate synthase or undecaprenyl diphosphate synthase.