Ribonucleic acid-permeable mutant of Escherichia coli

An RNA-permeable mutant was isolated from a tryptophan amber auxotrophic strain of Escherichia coli after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The rationale of the isolation was based on the suppression of an amber mutation. A strain was selected, which could grow in minimal medium supplemented with transfer RNA prepared from an suI-carrying strain but not from an su⁻ strain. This mutant incorporated³H-labeled bulk RNA into the cells at a rate 40 times higher than did the parent strain. The level of tryptophan requirement, susceptibility to the lytic action of lysozyme and RNase activity in the culture medium of the mutant strain did not differ from those of the parent strain. The mutant strain incorporated ³H-labeled ribosomal RNA equally as well as it incorporated ³H-labeled transfer RNA and the incorporation was competitively inhibited by any species of cold RNA. However, the fate of ³H-labeled rRNA after incorporation resulted in degradation to yield acid-soluble fragments whereas tRNA after incorporation remained intact in the cell.     

Isolation and Characterization of an Oxygen Sensitive Mutant of Azorhizobium caulinodans

An oxygen sensitive mutant of Azorhizobium caulinodans strain IRBG 46 was isolated by NTG mutagenesis. It was defective in N₂ fixation under 3% O₂ level, while under 1% O₂ it was almost as active as the parent strain IRBG 46. The mutant was also found to be a slow grower with reduced respiratory activity, low azide tolerance and no catalase activity. However, it did not differ from its parent strain with respect to nitrate respiration. Under symbiotic condition the mutant formed smaller, light green nodules as compared to bigger, dark green nodules formed by the wild type strain. The mutant was also defective in N₂ fixation under symbiotic condition. Complementation analysis showed that the mutation might be in either fixL or fixJ gene which are involved in O₂ regulation of nif/fix gene expression. A possible role of all these factors in conferring a highly O₂ tolerant nitrogen fixing system in the organism, has been discussed.     

Efficient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain

A mutant strain of Klebsiella pneumoniae, termed GEM167, was obtained by γ irradiation, in which glycerol metabolism was dramatically affected on exposure to γ rays. Levels of metabolites of the glycerol reductive pathway, 1,3-propanediol (1,3-PD) and 3-hydroxypropionic acid (3-HP), were decreased in the GEM167 strain compared to a control strain, whereas the levels of metabolites derived from the oxidative pathway, 2,3-butanediol (2,3-BD), ethanol, lactate, and succinate, were increased. Notably, ethanol production from glycerol was greatly enhanced upon fermentation by the mutant strain, to a maximum production level of 21.5 g/l, with a productivity of 0.93 g/l/h. Ethanol production level was further improved to 25.0 g/l upon overexpression of Zymomonas mobilispdc and adhII genes encoding pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase (Adh), respectively in the mutant strain GEM167.     

PanB is involved in nicotine metabolism in Pseudomonas putida

A nicotine-sensitive mutant was generated from the nicotine-degrading bacterium, Pseudomonas putida strain J5, by mini-Tn5 transposon mutagenesis. This mutant was unable to grow with nicotine as the sole carbon source but could grow with glucose. Sequence analysis showed that the Tn5 transposon inserted at the site of the ketopantoate hydroxymethyltransferase gene (panB), which had 54% identity to PanB in Escherichia coli K-12. In-frame deletion of the panB gene abolished the nicotine-degrading ability of strain J5, while complementation with panB from P. putida J5 and E. coli K-12 restored the degrading activity of the mutant to the wild-type level. These results suggest that ketopantoate hydroxymethyltransferase is a crucial enzyme in nicotine metabolism in P. putida J5.     

Fatty acid desaturase system of yeast microsomes. Involvement of cytochrome b5-containing electron-transport chain.

The oxidative desaturation of palmitoyl CoA by microsomes from anaerobically grown Saccharomyces cerevisiae has been studied by using NADH as electron donor. The desaturation product was identified as palmitoleic acid by periodate oxidation. The desaturase activity was sensitive to relatively high concentrations of cyanide; the concentration of cyanide causing half-maximal inhibition was determined to be 7.1 mm. The rate of reoxidation of cytochrome b₅ in NADH-reduced microsomes was stimulated by the addition of palmitoyl CoA, and the amount of cytochrome b₅ reoxidized by the palmitoyl CoA added could be closely correlated to the amount of palmitoleate formed. No stimulation of the reoxidation of cytochrome b₅ was induced by palmitoyl CoA in microsomes prepared from the desaturase-repressed cells and from a desaturase-deficient mutant, strain KD-20. It is concluded that the fatty acyl CoA desaturase system of yeast microsomes involves cytochrome b₅ as an electron carrier and that the terminal desaturase is sensitive to relatively high concentrations of cyanide.     

ATP production by an AMP deaminase-impaired mutant of a methanol yeast,Candida boidinii

A mutant strain, which was impaired with respect to its ability to grow on AMP as the sole nitrogen source, was derived from Candida boidinii (Kloeckera sp.) no. 2201. AMP deaminase of the mutant strain exhibited the same allosteric patterns as those of the wild type strain. The specific activity in the cell-free extract of the mutant strain, however, was lower than that of the wild type strain. Sorbitol-treated cells of the mutant strain produced ATP from AMP at a high conversion rate (95 mol%).     

Polycaprolactone depolymerase produced by the bacterium Alcaligenes faecalis

Several microorganisms were isolated as bacteria degrading polycaprolactone (PCL), and one of them, a strain B273 identified as Alcaligenes faecalis, was selected. Because this strain produced only slight PCL depolymerase activity, the hyper-producing mutant, TS22, was isolated after UV irradiation. Synthesis of PCL depolymerase was derepressed, probably based on the altered regulation of metabolic pathways in strain TS22. The partially purified enzyme hydrolyzed p-nitrophenyl fatty acids and triglycerides other than PCL, but not poly(3-hydroxybutyrate), indicating that PCL depolymerase may be a kind of lipase.     

Cloning of rel from Listeria monocytogenes as an Osmotolerance Involvement Gene

Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.     

Fermentation characterization of an L-tryptophan producing Escherichia coli strain with inactivated phosphotransacetylase

Acetate is a primary inhibitory metabolite in Escherichia coli cultivation which is detrimental to bacterial growth and the formation of desired products. It can be derived from acetyl coenzyme A by the phosphotransacetylase (Pta)–acetate kinase (AckA) pathway. In this study, the fermentation characteristics of Pta mutant strain E. coli TRTHΔpta were compared with those of the control strain E. coli TRTH in a 30-L fermentor. The effects of glucose concentration and dissolved oxygen (DO) level were investigated, and the results suggest that DO and glucose concentration are vital influencing parameters for the production of L-tryptophan. Based on our experimental results, we then tested a DO-stat fed-batch fermentation strategy. When DO was controlled at about 20 % during L-tryptophan fermentation in the DO-stat fed-batch system, the pta mutant was able to maintain a higher growth rate at the exponential phase, and the final biomass and L-tryptophan production were increased to 55.3 g/L and 35.2 g/L, respectively. Concomitantly, as the concentration of acetate decreased to 0.7 g/L, the accumulation of pyruvate and lactate increased in the mutant strain as compared with the control strain. This characterization of the recombinant mutant strain provides useful information for the rational modification of metabolic fluxes to improve tryptophan production.     

Microbial production of squalene by a nicotinic acid-resistant mutant derived from Fusarium sp. no. 5-128B

A nicotinic acid-resistant mutant, designated NA201, was obtained from Fusarium sp. no. 5-128B by treatment with ultraviolet light. This mutant strain could grow in the presence of up to 500 mM nicotinic acid in the culture medium, although the parent strain could not grow at concentrations of nicotinic acid above 200 mM. The NA201 strain exhibited morphological mutations, neither forming aerial hyphae nor secreting a red-brown pigment. However, it retained the resistance to kabicidin at 25 mg l^−t of the parent strain. The mutant NA201 cells contained high levels of squalene and low levels of ergosterol, about 53 times higher and five to six times lower, respectively, than those of the parent strain under standard culture conditions. The volumetric oxygen transfer coefficient (Kd) affected the level of squalene in the mutant cells. The Kd for the maximum production of squalene by the mutant was 24 mmol O₂I⁻¹h⁻¹atm⁻¹ and the level of squalene in the mutant cells was 26 mg (g cell)⁻¹ on a dry weight basis. The greatest accumulation of squalene by the NA201 strain, corresponding to 323 mg per liter of culture medium and 35 mg (g cell)⁻¹ on a dry weight basis, was achieved in a culture in which the Kd was changed from a high to a low value on the third day, with the simultaneous addition of 3% glucose (w/v).     

Nitrogen Fixation and Carbon Dioxide Assimilation in Rhizobium japonicum

In free-living Rhizobium japonicum cultures, the stimulatory effect of CO₂ on nitrogenase (acetylene reduction) activity was mediated through ribulose bisphosphate carboxylase activity. Two mutant strains (CJ5 and CJ6) of R. japonicum defective in CO₂ fixation were isolated by mitomycin C treatment. No ribulose bisphosphate carboxylase activity could be detected in strain CJ6, but a low level of enzyme activity was present in strain CJ5. Mutant strain CJ5 also exhibited pleiotropic effects on carbon metabolism. The mutant strains possessed reduced levels of hydrogen uptake, formate dehydrogenase, and phosphoribulokinase activities, which indicated a regulatory relationship between these enzymes. The CO₂-dependent stimulation of nitrogenase activity was not observed in the mutant strains. Both mutant strains nodulated soybean plants and fixed nitrogen at rates comparable to that of the wild-type strain.     

Mirin-Making from Koji Prepared with a Mutant Aspergillus oryzae Producing High Activity of Carboxypeptidase

It has been desired to improve the quality of mirin without decreasing the level of productivity. Accordingly, by ultraviolet irradiation to cause mutation, we screened for mutants with high acid carboxypeptidase (ACPase) activity from Aspergillus oryzae IFO 4079 (parental strain). We obtained a mutant with ACPase with the activity of 20,000 units · g⁻¹ of koji, about three times that of the parental strain. In mirin-making, koji prepared with this mutant had the same productivity, but the concentration of amino nitrogen in the mirin obtained with this mutant was 1.2–1.4 times that of the parental strain. Results of sensory tests showed that the mirin prepared with koji of the mutant had a good taste on the whole. The degree of clouding In mirin made with glutinous rice steamed at atmospheric pressure was much less with koji of this mutant than that of the parental strain.     

Ethanol Tolerance in the Yeast Saccharomyces cerevisiae Is Dependent on Cellular Oleic Acid Content

In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Δ⁹Z-C_16:1) and oleic acid (Δ⁹Z-C_18:1), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C_16:0) and stearic acid (C_18:0), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Δ⁹ and Δ¹¹) and substrate chain-length preferences (i.e., C_16:0 and C_18:0); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Δ¹¹Z-C_18:1), whereas neither Δ¹¹Z-C_16:1 nor palmitoleic acid (Δ⁹Z-C_16:1) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.     

Role of Alkyl Hydroperoxide Reductase (AhpC) in the Biofilm Formation of Campylobacter jejuni

Biofilm formation of Campylobacter jejuni, a major cause of human gastroenteritis, contributes to the survival of this pathogenic bacterium in different environmental niches; however, molecular mechanisms for its biofilm formation have not been fully understood yet. In this study, the role of oxidative stress resistance in biofilm formation was investigated using mutants defective in catalase (KatA), superoxide dismutase (SodB), and alkyl hydroperoxide reductase (AhpC). Biofilm formation was substantially increased in an ahpC mutant compared to the wild type, and katA and sodB mutants. In contrast to the augmented biofilm formation of the ahpC mutant, a strain overexpressing ahpC exhibited reduced biofilm formation. A perR mutant and a CosR-overexpression strain, both of which upregulate ahpC, also displayed decreased biofilms. However, the introduction of the ahpC mutation to the perR mutant and the CosR-overexpression strain substantially enhanced biofilm formation. The ahpC mutant accumulated more total reactive oxygen species and lipid hydroperoxides than the wild type, and the treatment of the ahpC mutant with antioxidants reduced biofilm formation to the wild-type level. Confocal microscopy analysis showed more microcolonies were developed in the ahpC mutant than the wild type. These results successfully demonstrate that AhpC plays an important role in the biofilm formation of C. jejuni.     

Regulation of isoleucine and valine biosynthesis in Salmonella typhimurium: the effect of hisU on repression control.

The control of isoleucine and valine biosynthesis was examined in a hisU mutant of Salmonella typhimurium. It was found that the levels of expression of the ilvEDA operon and the ilvC gene were significantly reduced relative to an isogenic normal strain when grown in unsupplemented medium. In contrast, this hisU mutant exhibited only a slight reduction in total acetohydroxy acid synthase activity relative to that of the wild type. The hisU and hisU⁺ strains were examined to determine their derepressibility upon either leucine, valine or isoleucine limitation. Only during leucine limitation did the hisU strain exhibit impaired derepressibility relative to the hisU⁺ strain. In addition, repression control of threonine deaminase (the ilvA product of the ilvEDA operon) in this hisU mutant was refractory to exogenous supplementation with either leucine or valine. This response is in distinct contrast to that of the normal strain, in which the single addition of leucine or valine results in a significant reduction in the level of threonine deaminase.     

Correlation Between the Expression of an Escherichia coli Cell Surface Protein and the Ability of the Protein to Bind to Lipopolysaccharide

The ompA gene of Escherichia coli codes for a major protein of the outer membrane. When this gene was moved between various unrelated strains (E. coli K-12 and two clinical isolates of E. coli) by transduction, the gene was expressed very poorly. Recombinants carrying “foreign” genes produced no OmpA protein which could be detected on polyacrylamide gels and became resistant to bacteriophage K3, which uses this protein as receptor. The recombinants were sensitive to host-range mutants of K3, indicating a very low level of OmpA protein was produced. When an E. coli K-12 recombinant carrying an unexpressed foreign ompA allele was subjected to two cycles of selection for an OmpA⁺ phenotype, a mutant strain was obtained which was sensitive to K3 and which expressed nearly normal levels of OmpA protein in the outer membrane. This strain carried mutations in the foreign ompA gene, as indicated both by genetic mapping and the alteration of a peptide in the mutant OmpA protein. The ability of the OmpA protein to bind to lipopolysaccharide (LPS) showed similar strain specificity, and the mutant OmpA protein which was expressed in an unrelated host showed enhanced ability to bind LPS from its new host. Thus, cell surface expression of the ompA gene appears to depend upon the ability of the gene product to bind LPS, suggesting that an interaction between the protein and LPS plays an essential role in biosynthesis of this outer membrane protein.     

Nucleotide Sequence and Characterization of cdrA, a Cell Division-Related Gene of Helicobacter pylori

We identified cell division-related gene cdrA in Helicobacter pylori HPK5. The putative gene product, CdrA, is a 367-amino-acid polypeptide that exhibited a high level of homology to conserved hypothetical ATP-binding protein HP0066 of H. pylori 26695, except in the N-terminal region, and showed some similarity to the FtsK/SpoIIIE family proteins. We isolated a cdrA-disrupted mutant by allelic exchange mutagenesis. Because of the low transformation frequency, the possibility that a suppressing mutation would be found in the obtained cdrA mutant was discussed. A repressive role for CdrA on cell division was suggested by the observations that the wild-type strain formed filamentous cells in a high-salt level medium at early stationary phase, while a cdrA-disrupted mutant did not show such an abnormality. In addition, the wild-type strain adopted coccoid forms in the stationary phase, whereas the cdrA-disrupted mutant remained mostly as short rods. Furthermore, the cdrA-disrupted mutant regained the filamentation phenotype when the intact cdrA gene was introduced by allelic exchange. Taken together, these observations show that the cdrA gene plays an important role in the cell growth of H. pylori.