Chromosomal tetA(L) gene of Bacillus subtilis: regulation of expression and physiology of a tetA(L) deletion strain.

Deletion of the tetA(L) chromosomal region of Bacillus subtilis in a strain designated JC112 increased the strain's sensitivity to low tetracycline concentrations. It also resulted in phenotypic changes that correlate with the previously found role of TetA(L) in mediating electrogenic NA+/H+ antiport. Growth of JC112 was impaired relative to that of the wild type at both pH 7.0 and 8.3; Na(+)- and K(+)-dependent pH homeostases were impaired at alkaline pH. The phenotype of JC112 was complemented by plasmid-borne tetA(L) and related tet(K) genes; the antiport activity conferred by the tet(K) gene had an apparently higher preference for K+ over Na+ than that conferred by tetA(L). The data were consistent with TetA(L) being the major Na+(K+)/H+ antiporter involved in pH homeostasis in B. subtilis as well as a significant Na+ extrusion system. The phenotype of JC112 was much more pronounced than that of an earlier transposition mutant, JC111, with a disruption in the putative tetA(L) promoter region. Northern (RNA) blot analysis of tetA(L) RNA from wild-type and JC111 strains revealed the same patterns. That JC111 nevertheless exhibited some Na+ and alkali sensitivity may be accounted for by disruption of regulatory features that, in the wild type, allow increased tetA(L) expression under specific conditions of pH and monovalent cation concentration. Evidence for several different regulatory effects emerged from studies of lacZ expression from the transposon of JC111 and from a tetA(L)-lacZ translational fusion introduced into the amyE locus of wild-type and JC112 strains.     

Characterization of Polynucleotide Phosphorylase Mutants of Escherichia coli

Three polynucleotide phosphorylase mutations, isolated in heavily mutagenized Escherichia coli strains Q7, Q13, and Q27, were characterized after their transfer by P1 transduction to nearly isogenic strains which lack ribonuclease I. Each strain has a different altered form of polynucleotide phosphorylase. One enzyme exhibited sharply reduced activity under all conditions tested. A second had reduced activity which was stimulated by Mn(++). The third enzyme was thermolabile and could be >95% inactivated in vivo at 44 C and pH 6 if the cells were prevented from growing; during growth under these and other conditions, the full enzyme level was maintained. The strains showed no differences from the wild type in their growth rates, their adjustments to changes in media and temperature, or their recoveries from starvation.     

Isolation and Mapping of Polynucleotide Phosphorylase Mutants of Escherichia coli

Three strains of Escherichia coli with altered polynucleotide phosphorylase, Q7, Q13, and Q27, were isolated by screening clones from heavily mutagenized cultures for low levels of the enzyme. The three mutations were found to cotransduce with argG and asp, and the pnp locus which they define was mapped with respect to these loci. An explanation for the nonreciprocal cotransduction frequencies observed with asp is provided by the demonstration of an unlinked asp-suppressing locus.     

Comparison Between the Escherichia coli and Bacillus subtilis Genomes Suggests That a Major Function of Polynucleotide Phosphorylase is to Synthesize CDP

Genome comparison permits identification of chromosome regionsconserved during evolution. Bacillus subtilis and Escherichiacoli are so distant that there exists veryfew conserved landmarksin their genome organisation. Analysis of the conserved cmkrpsA cluster pinpointed the importance of cytosine nucleotidemetabolism. In these bacteria, mRNA turnover provides an efficientmeans to fulfil the need for CDP as a precursor of DNA synthesis.The cmk rpsA operon is responsible for CDP synthesis. This functionis self-explained in the case of the cmk gene (which codes forcytidylate kinase). The case of rpsA, that codes for ribosomalprotein S1, is more subtle. It is suggested here that S1 isa RNA-binding protein helping polynucleotide phosphorylase (PNPase,known to be phylogenetically related to S1) to degrade mRNA,or helper molecule involved in other RNase activities. Thisprovides an explanation for the elusive function of PNPase,which generates nucleoside diphosphates (not monophosphates)when degrading RNA. This also accounts for the discoverythatthe B. subtilis comR gene product is PNPase. This article brieflydiscussesthe availabilityof cytosine nucleotides in eukaryotes, and suggeststhat they are derived from phospholipids turnover. Finally,the GC content of genomes is discussed in this new light.     

Hyperprotease-Producing Mutants of Bacillus subtilis

A number of mutants of Bacillus subtilis producing high levels of extracellular protease have been isolated. Analysis of culture supernatants of these mutants has shown that the total amount of proteolytic activity is elevated from 16- to 37-fold over the wild strain. The elevated activity was due to a simultaneous increase in both the neutral and alkaline protease. All of the mutants genetically analyzed were found linked to the argC4 marker by PBS-1 transduction analysis.     

胞苷生产菌的选育

目的：筛选得到胞苷发酵单位较高的菌株,并对发酵过程作初步研究。方法：以胞苷脱氨酶缺失枯草芽孢杆菌DOS7为出发菌株,对其进行紫外诱变、5-氟胞苷（5FCR＋）和2-杂氮尿嘧啶抗性（2AU＋）抗性筛选。结果：通过紫外诱变和抗性筛选得到突变株DOS7-2-1000-15,抗5-氟胞苷和2-杂氮尿嘧啶的临界浓度分别为800mg/L和1 000mg/L。同时检测了抗5-氟胞苷突变株中CTP合成酶的活性,比原始菌株提高了12.4%,突变株DOS7-2-1000-15发酵过程结果为：36℃发酵72h能积累胞苷最高为3.5g/L。结论：筛选得到的突变株DOS7-2-1000-15的遗传稳定性较好,可稳定发酵。     

酿酒酵母HOR2基因缺失突变株的构建

目的:构建酿酒酵母HOR2基因缺失的突变株并研究其对甘油和乙醇产量的影响。方法:以PCR为基础,通过同源重组的方式使目的基因缺失。结果:通过设计含有与HOR2(GPP2)基因两侧序列同源的长引物,以质粒PUG6为模板进行PCR构建含有Cre/loxP系统的酿酒酵母HOR2基因敲除组件,转化酿酒酵母(Saccharomyces cerevisiae)YS2,获得为loxP-kan-loxP序列组件所替换而产生kanr的阳性克隆子。然后再将质粒PSH65转入阳性克隆子诱导表达Cre酶切除筛选标记,在原ORF基因处保留一个loxP位点,丢失质粒后获得HOR2单倍体缺陷型菌株。重复转化敲除组件实现另一条等位基因的敲除。发酵实验表明,突变株甘油产量降低3.34%,乙醇产量提高1.96%。结论:成功获得了酿酒酵母HOR2基因缺失的突变株,并命名为YS2-HOR2。     

Mutagen Stability of Alkylation-Sensitive Mutants of Bacillus subtilis

The phosphotransacetylase of Veillonella alcalescens catalyzes a reversible reaction with Michaelis-Menten kinetics for all substrates. The rate of the reverse reaction (the synthesis of acetyl coenzyme A from acetyl phosphate) was 6.5 times greater than the rate of the forward reaction (the synthesis of acetyl phosphate from acetyl coenzyme A). The apparent K(m) values determined for the forward reaction were 8.6 x 10(-6)m for acetyl coenzyme A and 9.3 x 10(-3)m for phosphate. In the reverse reaction, the K(m) values were 3.3 x 10(-4)m for coenzyme A and 5.9 x 10(-4)m for acetyl phosphate. The results of an analysis of the inhibition by end products in the forward and reverse directions were compatible with a random bi- bi- mechanism. The enzyme was inhibited by adenosine triphosphate and adenosine diphosphate but was not affected by reduced nicotinamide adenine dinucleotide or pyruvate. The inhibition by adenosine triphosphate was noncompetitive with respect to acetyl phosphate and competitive with respect to coenzyme A. MgCl(2) reversed the inhibition by adenosine triphosphate or adenosine diphosphate. The role of Mg(2+) and adenylates in the regulation of phosphotranscetylase activity is discussed.     

Double Mutants of Bacillus subtilis Growing as Helices

Double mutants of Bacillus subtilis, deficient in autolysin and rod in genotype, grow as helical filaments of unseparated cells when changed from the cocci to rods.     

Mutants of Bacillus subtilis defective in protein export

We have isolated a set of strains with mutations (designated prs) that decrease secretion of alpha-amylase and have a pleiotropic effect on secretion of other exoproteins. The seven mutants were selected in a strain of Bacillus subtilis which overproduces alpha-amylase due to the presence of an alpha-amylase gene on a multicopy plasmid. The mutations were mapped to four different chromosomal loci. The phenotype of the mutants, especially their pleiotropic effects and the accumulation of alpha-amylase precursor, indicated that they have defects in the mechanism of protein export. Double mutants with certain pairwise combinations of mutations in different loci had additive effects on secretion, suggesting that these prs genes encode different components of the secretion pathway.     

Two types of Bacillus subtilis tetA(L) deletion strains reveal the physiological importance of TetA(L) in K(+) acquisition as well as in Na(+), alkali, and tetracycline resistance

The chromosomally encoded TetA(L) protein of Bacillus subtilis is a multifunctional tetracycline-metal/H(+) antiporter that also exhibits monovalent cation/H(+) antiport activity and a net K(+) uptake mode. In this study, B. subtilis mutant strains JC112 and JC112C were found to be representative of two phenotypic types of tetA(L) deletion strains that are generated in the same selection. Both strains exhibited increased sensitivity to low tetracycline concentrations as expected. The mutants also had significantly reduced ability to grow in media containing low concentrations of K(+), indicating that the net K(+) uptake mode is of physiological consequence; the deficit in JC112 was greater than in JC112C. JC112 also exhibited (i) greater impairment of Na(+)- or K(+)-dependent growth at pH 8.3 than JC112C and (ii) a greater degree of Co(+2) as well as Na(+) sensitivity. Studies were initiated to explore the possibility of two different patterns of compensatory changes in other ion-translocating transporters in these mutants. Increased expression of two loci has thus far been shown. Increased expression of czcD-trkA, a locus with a proposed involvement in K(+) uptake, occurred in both mutants. The increase was highest in the presence of Co(2+) and was higher in JC112 than in JC112C. Deletion of czcD-trkA resulted in diminished growth of the wild-type and both mutant strains at low [K(+)], supporting a significant role for this locus in K(+) uptake. Expression of yheL, which is a homologue of the Na(+)/H(+) antiporter-encoding nhaC gene from Bacillus firmus OF4, was also increased in both tetA(L) deletion strains, again with higher up-regulation in JC112. The phenotypes resulting from deletion of yheL were consistent with a modest role for YheL in Na(+)-dependent pH homeostasis in the wild type. No major role for YheL was indicated in the mutants in spite of the overexpression. The studies underscore the multiple physiological functions of TetA(L), including tetracycline, Na(+), and alkali resistance and K(+) acquisition. The studies also reveal and begin to detail the complexity of the response to mutational loss of these functions.     

Biotechnological Potential of the Bacillus subtilis 20 Strain

Molecular Biology - Bacillus subtilis bacteria play an important role in veterinary medicine, medicine, and biotechnology, and the permanently growing demand for biotechnological products fuels the...     

Accumulation of Xanthosine by Auxotrophic Mutants of Bacillus subtilis

Several guanineless mutants derived from Bacillus subtilis IAM 1145 were found to accumulate xanthosine in the culture broth. Further mutation of the guanineless mutants to adenine dependence led to remarkable increase in the accumulation of xanthosine. One of the guanine-adenine doubleless mutants, strain Gu-Ad-3-35, accumulated 8.9g of xanthosine per liter. Xanthosine was isolated in a crystalline form from the culture broth by a procedure involving charcoal treatment and ion-exchange chromatography.     

Sporulation of Tricarboxylic Acid Cycle Mutants of Bacillus subtilis

A mutant of Bacillus subtilis 168 lacking aconitase (EC 4.2.1.3) was found to be blocked at stage 0 or I of sporulation. Although adenosine triphosphate levels, which normally decrease in tricarboxylic acid cycle mutants at the completion of exponential growth, could be maintained at higher levels by feeding metabolizable carbon sources, this did not permit the cells to progress further into the sporulation sequence. When post-exponential-phase cells of mutants blocked in the first half of the tricarboxylic acid cycle were resuspended with an energy source in culture fluid from post-exponential-phase wild-type B. subtilis or Escherichia coli, good sporulation occurred. The spores produced retained the mutant genotype and were heat stable but lost refractility and heat stability several hours after their production.     

Transformation and Transduction in Recombination-defective Mutants of Bacillus subtilis

The effects on transformation and transduction of an ultraviolet sensitivity (uvr(-)) and two ultraviolet sensitivity-recombination deficiency (rec-1(-) and rec-2(-)) mutations in isogenic strains of Bacillus subtilis were investigated. Transformation frequency in the rec-1(-) and rec-2(-) strains was reduced to approximately 5 and 25%, respectively, of the parental strains. Normal kinetics of deoxyribonucleic acid dose response in transformation were found for the rec-1(+) and rec-2(-) strains. Biphasic curves were obtained with the rec-1(-) strains. Transduction frequency with bacteriophage SP-10 decreased parallel to transformation frequency in the rec-1(-) and rec-2(-) strains. This result suggests that transformation and SP-10 transduction share a common mechanism for genetic recombination. It also indicates that the reduction in transformation frequency of these strains was not due to altered competence. Transduction frequency with bacteriophage PBS-1 or 3NT, on the contrary, was not diminished in rec-1(-) strains. This frequency was reduced in rec-2(-) strains but not as severely as that of transformation or SP-10 transduction. Several hypotheses to interpret these differences are presented. Recombination frequency between linked markers was reduced more than 50% in transformation by the presence of the rec-1(-) mutation. Linkage was unaffected in the rec-2(-) strains. Neither the rec-1(-) nor the rec-2(-) mutation had an effect on linkage in PBS-1 or 3NT transduction. The uvr(-) strains were transformed at a frequency equal to or greater than that of the parental strains. These strains were transduced by all bacteriophage systems at frequencies about twofold higher than those of parental strains.