Comparison of Lysyl-Transfer Ribonucleic Acid Species from Vegetative Cells and Spores of Bacillus subtilis by Methylated Albumin-Kieselguhr and Reversed-Phase Chromatography

Lysyl-transfer ribonucleic acid (tRNA) species from a spore-forming strain of Bacillus subtilis (168 trp2(-)) and an early blocked asporogenous mutant (spoA 12) were compared on reversed-phase and methylated albumin-kieselguhr columns. Lysyl-tRNA species from spores and the asporogenous mutant in stationary phase both exhibited altered chromatographic profiles compared to that of log-phase cells. The major peak in spore lysyl-tRNA species eluted later than that characteristic of vegetative cells, whereas the major peak of the lysyl-tRNA species from the asporogenous mutant in stationary phase eluted earlier. Although the early eluting lysyl-tRNA species was observable on methylated albumin columns, the late eluting peak was not detectable by that column technique. By using a shallower gradient on an RPC-2 column, the resolution of all lysyl-tRNA species increased. Several subspecies were revealed. The chromatographic comparisons clearly show that both the spore-forming strain and the asporogenous mutant undergo relative increases in different lysyl-tRNA species when grown to late stationary phase. No new species seem to be involved but rather altered amounts of minor species existing in log-phase cells. The experiments also demonstrate the usefulness of reversed-phase columns for such comparisons.     

Two active forms of valyl-tRNA synthetase from Bacillus subtilis: alteration related to the early stages of sporulation

Two enzymatically active forms, E-I and E-II, of valyl-tRNA synthetase [EC 6.1.1.9] from cells at various stages in the life cycle of Bacillus subtilis 168 LTT, germinated cells, vegetative cells (t-0.5), sporulating cells (t0, t1, t3, and t4), forespores and mature spores, were analyzed by hydroxyapatite column chromatography. The E-II activity was detected in the main fraction of valyl-tRNA synthetase during the life cycle of B. subtilis 168 LTT. The high activity of E-II at t0 decreased rapidly in the stationary and sporulating phases. On the other hand, the E-I activity increased in the early sporulating stage and was about twofold higher at t3 than at t0. After t3, this activity also decreased rapidly and was not detected in forespores and mature spores. The relative amount of E-I at t0 was 3.4% of the total valyl-tRNA synthetase activity eluted from the hydroxyapatite column, 12.9% at t1 and 29.2% at t3, but it was less than 10% at t4 and in germinated cells. The alteration in E-I and E-II activities was also observed in cells of B. subtilis NIG 1121 (spo+), W23 and 168W, but not in any asporogenous mutant strain studied. These results show that the alteration in the valyl-tRNA synthetase activity appears only during the early stages of sporulation and is closely related to the sporulation of B. subtilis.     

A new chemically-defined medium for RAC-certified and other strains of Bacillus subtilis

Summary Conventional chemically-defined media were found to be inadequate for extensive batch culture growth of an asporogenous mutant of Bacillus subtilis certified as an HV-1 host for recombinant DNA. We developed a chemically-defined medium for rapid and extensive growth of one such strain, BGSC-1S53. The medium contains no components requiring separate sterilization. It is also useful for the prototrophic 168 strain (from which 1S53 was derived), a triple auxotroph of 168, and a highly debilitated (HV-2) 168 strain (BGSC 1S76) upon addition of the specific growth factors required by these strains. Excellent growth was also obtained with W-23, a Bacillus subtilis strain unrelated to the 168 series.     

The origins of 168, W23, and other Bacillus subtilis legacy strains

Bacillus subtilis is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of B. subtilis genetics. For three--NCIB 3610T, PY79, and SMY--we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more "W23 islands" in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its trp and sac loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these B. subtilis legacy strains.     

Temperature-Sensitive Induction of Bacteriophage in Bacillus subtilis 168

In a temperature-sensitive mutant of Bacillus subtilis 168, induction of the defective phage PBSX occurred at 48 C. Cell lysis began after 90 min of growth at 48 C, and cell viability began to decrease after 10 to 30 min. The loss in viability at the nonpermissive temperature was prevented by azide or cyanide. Deoxyribonucleic acid (DNA), ribonucleic acid, and protein synthesis were not inhibited at 48 C. Temperature induction of the temperate phage SPO2 also occurred in this mutant. The temperature-sensitive mutation, designated tsi-23, was linked by transduction to purB6 and pig, the order being purB6 pig tsi-23. Mutation tsi-23 was transformable to wild type by B. subtilis 168 DNA but not by DNA from the closely related strains W23 or S31. DNA from the latter two strains transformed auxotrophic markers of strain 168 at frequencies close to those found with 168 donor DNA. Upon temperature induction, cellular DNA was broken to a size of 22S, characteristic of DNA in PBSX particles. The DNA isolated from temperature-induced PBSX did not give an increased Ade(+)/Met(+) transformant ratio relative to cellular DNA nor contain preferential break points as determined by transformation of four closely linked markers.     

Unrelatedness of Bacillus amyloliquefaciens and Bacillus subtilis

Eight strains of highly amylolytic, sporeforming bacilli (hereafter referred to as Bacillus amyloliquefaciens) were compared with respect to their taxonomic relationship to B. subtilis. The physiological-biochemical properties of these two groups of organisms showed that B. amyloliquefaciens differed from B. subtilis by their ability to grow in 10% NaCl, characteristic growth on potato plugs, increased production of alpha-amylase, and their ability to ferment lactose with the production of acid. The base compositions of the deoxyribonucleic acid (DNA) of the B. subtilis strains consistently fell in the range of 41.5 to 43.5% guanine + cytosine (G + C), whereas that of the B. amyloliquefaciens strains was in the 43.5 to 44.9% G + C range. Hybrid formation between B. subtilis W23 and B. amyloliquefaciens F DNA revealed only a 14.7 to 15.4% DNA homology between the two species. Transducing phage, SP-10, was able to propagate on B. subtilis W23 and B. amyloliquefaciens N, and would transduce B. subtilis 168 (indole(-)) and B. amyloliquefaciens N-10 (arginine(-)) to prototrophy with a frequency of 3.9 x 10(-4) and 2.4 x 10(-5) transductants per plaque-forming unit, respectively. Attempts to transduce between the two species were unsuccessful. These data show that Bacillus amyloliquefaciens is a valid species and should not be classified as a strain or variety of B. subtilis.     

Comparison of the α-Amylase of Bacillus subtilis and Bacillus amyloliquefaciens

The alpha-amylase (alpha-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) of Bacillus subtilis strain W23 is less negatively-charged than the alpha-amylase of B. amyloliquefaciens strain F, as determined by electrophoretic mobility in polyacrylamide gel at pH 8.6. The alpha-amylase of strain W23 is immunologically unrelated to the alpha-amylase of strain F, as judged by lack of cross-reaction in Ouchterlony immunodiffusion studies. The pH range of maximal activity for the enzyme of strain W23 was 5.7 to 6.7, with a maximum at 6.3. The pH range of activity for the alpha-amylase of strain F was 5.5 to 6.5, with a maximum at 5.9. No significant difference was found in the effect of temperature on the activity of the alpha-amylase of strain W23 and strain F. alpha-Amylase production by strain W23 occurs throughout the 7-hr growth period, whereas enzyme production by strain F does not begin until the culture enters the stationary phase of growth. The total amounts of enzyme produced by strains W23 and F after 7 hr of growth were 0.3 and 25.5 units/ml, respectively.     

DNA and protein sequence conservation at the replication terminus in Bacillus subtilis 168 and W23.

Cloned DNA from the replication terminus region of Bacillus subtilis 168 was used to identify and construct a restriction map of the homologous region in B. subtilis W23. With this information, DNA from the terminus region of W23 was cloned and the sequence was determined for a 1,499-base-pair segment spanning the expected terC site. The position of the site was then located more precisely. Use of the cloned DNA from strain W23 as a probe for digests of DNA from exponentially growing cells of the same strain established the presence of the slowly migrating replication termination intermediate (forked DNA). The orientation and dimensions of the forked molecule were consistent with arrest of the clockwise fork at the terC site in W23, as has been shown to occur in strain 168. Thus, despite significant differences between the two strains, the same termination mechanism appears to be used. The DNA sequences spanning the terC site in strains 168 and W23 showed a high level of homology (90.2%) close to the site but very little at a distance of approximately 250 base pairs from the site in one particular direction. The overall sequence comparison emphasised the importance of the open reading frame for a 122-amino-acid protein adjacent to terC. Although there were 22 base differences in the open reading frames between the strains, the amino acid sequence of the encoded protein was completely conserved. It is suggested that the amino acid sequence conservation reflects a role for the protein in the clockwise fork arrest mechanism as proposed earlier (M.T. Smith and R.G. Wake, J. Bacteriol. 170:4083-4090, 1988).     

Pseudo-allelic relationship between non-homologous genes concerned with biosynthesis of polyglycerol phosphate and polyglycerol phosphate teichoic acids in Bacillus subtilis strains 168 and W23

A 60 kbp region of the Bacillus subtilis chromosome encompassing the genes concerned with teichoic acid biosynthesis has been subjected to physical analysis. No homology was detected by Southern hybridization between DNA segments encoding the tag genes of strain 168, concerned with polyglycerol phosphate (poly(groP)) biosynthesis, and the tar genes of strain W23, concerned with polyribitol phosphate (poly-(rboP)) biosynthesis. Analysis of 168/W23 interstrain hybrids that incorporate poly(rboP) instead of poly-(groP) into their cell walls revealed that, in every case, integral substitution of the W23 tar genes for the 168 tag genes had occurred. Interstrain hybrids of the 'W23-like' type have inherited larger segments of W23 DNA than interstrain hybrids of the 'mixed' type. The tag and tar genes are located at equivalent positions on the chromosomes of strains 168 and W23, behaving, in genetic crosses, like an allelic pair. They provide the first example of a pseudo-allelic relationship between non-homologous genes in B. subtilis.     

Completed Chromosomes in Thymine-Requiring Bacillus subtilis Spores

Origin:terminus genetic marker ratios (both purA: metB and purA:ilvA) were measured in extracts of spores of Bacillus subtilis strains W23 thy his and 168 thy. For strain W23 thy his, normalized to W23 spore deoxyribonucleic acid, both ratios were equal to unity and were consistent with the presence of only completed chromosomes in the spores. The same ratios in extracts of spores of 168 thy, normalized to strain 168 or the prototroph SB19, were abnormal, i.e., 2.26 +/- 0.10 and 0.71 +/- 0.06 for purA:metB and purA:ilvA, respectively. These values were unaffected by the extent of extraction of the spore deoxyribonucleic acid, the richness of the medium on which they are formed, and the thymine phenotype. The high ratio for purA:metB is in agreement with the results of earlier workers but, because of the low purA:ilvA ratio, cannot be explained simply by the presence of partially replicated chromosomes in spores of strain 168 thy. Furthermore, purA:leuA in such extracts is 1.01 +/- 0.06, consistent with the presence of only completed chromosomes. It is concluded that the abnormal origin:terminus marker ratios are only apparent and result from non-isogenicity between strains 168 thy and 168 in the metB thyB ilvA chromosome region introduced during construction of 168 thy by transformation of strain 168 with W23 thy deoxyribonucleic acid. It is concluded further that the chromosomes of strain 168 thy spores are in a completed form.     

Identification of Bacillus subtilis NRRL B-3275 as a Strain of Bacillus pumilus

The physiological and biochemical properties of a species of Bacillus previously identified as B. subtilis NRRL B-3275 (B-3275) were compared with those of seven strains of B. pumilus and five strains of B. subtilis. The biotin requirement of B-3275, its inability to hydrolyze starch, and its failure to reduce nitrate indicate that the organism is more closely related to the B. pumilus strains than to those of B. subtilis. Hybridization of deoxyribonucleic acid (DNA) from B-3275 with that of the strains of B. pumilus showed a binding efficiency (compared with the homologous reaction) of 58 to 99%, depending on the strain. Hybridization with the DNA from any of the strains of B. subtilis did not exceed 24%. DNA from B-3275 was unable to transform two amino acid auxotrophic markers to prototrophy in a highly competent strain of B. subtilis 168. We conclude that B-3275 is a strain of B. pumilus which we designate as B. pumilus NRRL B-3275.     

Analysis of Isoaccepting Transfer Ribonucleic Acid Species of Bacillus subtilis: Changes in Chromatography of Transfer Ribonucleic Acids Associated with Stage of Development

Changes in chromatographic profiles of tyrosyl-, leucyl-, tryptophanyl-, and lysyl-transfer ribonucleic acids (tRNAs) are presented as a function of the growth stage in Bacillus subtilis. All of the tRNA groups investigated expressed different temporal patterns of change in isoaccepting species. Tyrosyl-tRNAs were the earliest to change and were followed by changes in leucyl- and then tryptophanyl-tRNAs. Lysyl-tRNAs were unique in having two times of change: one early and one very late. As an aid in understanding the temporal aspect of tRNA alterations during sporulation, the chromatographic profiles of aminoacyl tRNAs from an early blocked asporogenous mutant were studied. The asporogenous mutant used was blocked at the axial filament stage, stage 0 of sporulation. Nevertheless, those tRNAs which showed differences between the spore and cells in exponential growth exhibited similar changes in the asporogenous mutant after 24 h of growth. The data suggest that several tRNA changes occur during development in B. subtilis but that the events leading to these changes are either independent of, or occur before, stage 0 of sporulation, except in the case of lysyl-tRNA.     

Identification of poly-gamma-glutamyl chain lengths in folates of Bacillus subtilis.

Bacillus subtilis strains 168 met ile leu and 23 thy contain folates which differ from one another in the number of glutamyl residues. The folate species were identified by reductive cleavage to the corresponding p-aminobenzoylglutamyl poly-gamma-glutamates and chromatography on diethylaminoethyl-cellulose. Pteroyltriglutamate is the predominant folate type, accounting for 86 to 88% of the total. Pteroyltetraglutamate is the only other type present in appreciable quantities, accounting for 5 to 6% of the total folates. Pteroyldiglutamate and pteroylpentaglutamate are present in small amounts, accounting for 1 to 3% and 1% of the total folates, respectively. Strain 168 met ile leu contains a very small amount of pteroylmonoglutamate (less than 0.5% of the total folates), but the other strain contains none.     

多效调控基因degU32(Hy),degQa和degR对枯草芽孢杆菌Ki-2-132生长和蛋白酶发酵的影响

通过向枯草芽孢杆菌Ki-2-132染色体和／或细胞质导入来自枯草杆菌168菌株的degU32（Hy）和degR基因,以及来自芽孢杆菌解淀粉菌株（Bacillus amyloliquefaciens）的degQa基因,对上述基因对枯草芽孢杆菌Ki-2-132细胞的生长、孢子发生、蛋白酶发酵的影响进行了研究。尽管上述多效调控基因来自不同的芽孢杆菌种和菌株,它们在枯草芽孢杆菌Ki-2-132中依然表现多效性。枯草杆菌Ki-2-132degU32（Hy）表现出增高了的蛋白酶产量;当和质粒或染色体上的degQa基因协作,可以进一步依赖葡萄糖的水平和degQa的基因剂量影响细胞生长,增加蛋白酶产量,以及影响孢子的形成。与此不同,degR在degU32（Hy）突变体中并不显著影响其蛋白酶的产量,这一发现支持DegR蛋白通常稳定磷酸化的DegU,而其在degU32（Hy）菌株中不再进一步放大该突变体内已被磷酸化的DegU的调控作用。     

Adenosine 5′-Triphosphate Release and Membrane Collapse in Glycerol-Requiring Mutants of Bacillus subtilis

Glycerol-requiring mutants of Bacillus subtilis could not sporulate in nutrient sporulation medium even when additional glycerol was added from the beginning of growth. Sporulation could be partially restored either by the frequent addition of small amounts of glycerol during the developmental period or by the single addition of both 10 mM glycerol and 10 mM malate. But sporulation could be completely restored by the addition of 50 mM glycerol-phosphate from the beginning. At the end of growth of the glycerol mutants in nutrient sporulation medium, the cell membrane collapsed and separated from the cell wall, and much of the cellular adenosine 5'-triphosphate was released into the medium. These observations were made in two glycerol mutants, one derived from strain 168 containing glycerol-teichoic acid in the cell wall and the other derived from strain W23 containing ribitol-teichoic acid.     

Transposition of the Arsenate Resistance Locus of BACILLUS SUBTILIS Strains 23 and 168

Wild-type Bacillus subtilis strains 23 and 168 are resistant to high concentrations of sodium arsenate. The genetic configurations of the arsenate resistance loci of these two related strains of B. subtilis have been characterized. The transformable 168 strain has a single resistance locus which maps between phe and aroD in the terminal third of the genome. In contrast, strain 23 is shown to have its single arsenate resistance locus between purB and thr in the first third of the bacterial chromosome. Moreover, in strain 23 the chromosomal segment equivalent to the phe-linked asa region of 168 strains is missing. DNA isolated from 23 strains is able to transform 168 arsenate-sensitive strains to resistance and the heterologous 23 DNA is found to preferentially establish a new purB linked asa locus in such transformed cells. Thus, the majority of phenotypically arsenate-resistant cells recovered after exposure of competent 168 sensitive mutants to 23 DNA are "heterozygous" and still retain their phe-linked mutated asa locus. The tolerance of several of these heterologously transformed hybrid strains to arsenate suggests that the 168 and 23 asa gene products are similar, and a transposition model for the evolution of arsenate resistance in B. subtilis is proposed.     

D-Tyrosine as a metabolic inhibitor of Bacillus subtilis

The d-isomer of tyrosine is a potent inhibitor of growth in transformable strain 168 of Bacillus subtilis. A d-tyrosine-resistant mutant of the inhibited strain was isolated which excreted l-tyrosine, had a diminished growth rate, and required l-phenylalanine to attain the growth rate of the wild-type parent. Mapping by deoxyribonucleate transformation located this resistance in the gene coding for prephenate dehydrogenase. This enzyme in the d-tyrosine-resistant mutant was insensitive to the usual feedback inhibition exerted by l-tyrosine in extracts of strain 168. In contrast, the growth of poorly transformable strain 23 of B. subtilis, as well as that of several other Bacillus species, was not affected by the analogue. Transformation mapping demonstrated no linkage of this latter "natural resistance" to several different aromatic markers. Prephenate dehydrogenase in extracts from strain 23 was as sensitive as that from strain 168 to feedback inhibition by l-tyrosine in vitro. The relationships of the latter results to the regulation of tyrosine biosynthesis and the possible nature of strain differences in d-tyrosine sensitivity are discussed.     

Physiological and genetic strategies for enhanced subtilisin production by Bacillus subtilis.

Defined minimal media conditions were used to assess and subsequently enhance the production of subtilisin by genetically characterized Bacillus subtilis strains. Subtilisin production was initiated by the exhaustion or limitation of ammonium in batch and fed-batch cultures. Expression of the subtilisin gene (aprE) was monitored with a chromosomal aprE::lacZ gene fusion. The beta-galactosidase production driven by this fusion reflected subtilisin accumulation in the culture medium. Subtilisin gene expression was temporally extended in sporulation-deficient strains (spoIIG), relative to co-genic sporogenous strains, resulting in enhanced subtilisin production. Ammonium exhaustion not only triggered subtilisin production in asporogenous spoIIG mutants but also shifted carbon metabolism from acetate production to acetate uptake and resulted in the formation of multiple septa in a significant fraction of the cell population. Fed-batch culture techniques, employing the spoIIG strain, were investigated as a means to further extend subtilisin production. The constant provision of ammonium resulted in linear growth, with doubling times of 11 and 36 h in each of two independent experiments. At the lower growth rate, the responses elicited (subtilisin production, glucose metabolism, and morphological changes) during the feeding regime closely approximated the ammonium starvation response, while at the higher growth rate a partial starvation response was observed.     

黑水虻幼虫肠道和体表产酶细菌的分离和鉴定

从野外收集和室内饲养的黑水虻Hermetia illucens L.幼虫体表和肠道分离出同时具蛋白质和有机磷分解能力的细菌10株,其中编号为T2、T4、T6、T7和T8的菌株分离自体表,编号为S14、S15、S16、S19和S20的菌株分离自肠道。克隆细菌的16S rDNA和DNA促旋酶B亚基编码基因gyrB对10株细菌进行了鉴定。通过16S rDNA序列确定10株菌为芽孢杆菌属,根据gyrB基因以及BLAST结果构建系统发育树,将10株细菌鉴定为枯草芽孢杆菌。然后将10株菌的gyrB基因以B.subtilis subsp.subtilisstr.168和模式菌株B.subtilis subsp.subtilis BCRC10255相应基因序列为参照构建系统发育树,分析10株菌在种的水平上的亲缘关系,发现10株菌存在亲缘关系差异,没有重复菌株。