Complete nucleotide sequence of the fumarase gene (citG) of Bacillus subtilis 168.

The nucleotide sequence of a 2.14 kb fragment of Bacillus subtilis DNA containing the citG gene encoding fumarase was determined using the dideoxy chain termination method. The citG coding region of 1392 base pairs (464 codons) was identified, and the deduced Mr (50425) is in good agreement with that of the protein identified from expression in Escherichia coli maxicells. There is no sequence homology between the B. subtilis and E. coli fumarases. Overlapping potential promoter sequences have been identified for sigma 28, sigma 37 and sigma 55 RNA polymerase holoenzymes. The DNA fragment also contains the proximal part of the gerA locus, responsible for L-alanine-sensitive spore germination.     

苏云金芽孢杆菌gerA操纵子在芽孢萌发中的作用

芽孢萌发的营养诱导剂通过与特异的萌发受体结合激活下游的萌发过程,从而使芽孢经过一系列的遗传变化及生化反应恢复营养生长.从苏云金芽孢杆菌(Bacillus thuringiensis)中克隆到一个与枯草芽孢杆菌(Bacillus subtilis)gerA操纵子和蜡状芽孢杆菌(Bacillus cereus)gerR操纵子同源的gerA操纵子.苏云金芽孢杆菌gerA操纵子含有3个开放读码框:gerAA、gerAC和gerAB,该操纵子在产孢起始3个小时后开始转录.gerA的破坏阻断了L-丙氨酸诱导的芽孢萌发并且延迟了肌苷诱导的萌发.在L-丙氨酸诱导芽孢萌发的过程中D-环丝氨酸能够提高芽孢的萌发率.     

Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168.

The gene coding for an extracellular lipase of Bacillus subtilis 168 was cloned and found to be expressed in Escherichia coli. Enzyme activity measurements showed no fatty acid chain length preference. A set of Tn5 insertions which inactivate the gene were localized and used to initiate its sequencing. The nucleotide sequence was determined on two independent clones expressed in E. coli. In one of these clones, the sequence revealed a frameshift, due to the presence of an additional adenine in the N-terminal region, which caused the interruption of the open reading frame, probably allowing translation to initiate at a second ATG codon. The sequence of the wild-type lip gene from B. subtilis was confirmed on the chromosomal fragment amplified by polymerase chain reaction (PCR). When compared to other lipases sequenced to date, the enzyme described here lacks the conserved pentapeptide Gly-X-Ser-X-Gly supposed to be essential for catalysis. However, alignments of several microbial lipase sequences suggest that the pentapeptide Ala-X-Ser-X-Gly present in the lipase B. subtilis may function as the catalytic site. Homologies were found in the N-terminal protein region with lipases from different Pseudomonas species. The predicted M(r) and isoelectric point for the mature protein are 19,348 and 9.7 respectively.     

The nucleotide sequence of Bacillus subtilis tRNA genes

Clones carring Bacillus subtilis tRNA genes were isolated from a lambda 816 library. A recombinant phage lambda 816-BS83 which was hybridized effectively with unfractionated tRNA probes contained a 3-kb fragment. By a Southern's blot analysis, it was found that tRNA genes were located in Eco RI-Hinc II region of this fragment. Sequence determination revealed the presence of a cluster of four tRNA genes in this region. The gene organization was as follows: tDNALys-9bp-tDNAGlu-81bp-tDNAAsp-30bp-tDNAPhe. The RNA sequences expected from tDNALys and tDNAPhe were identical with the reported RNA sequences. Two tRNA genes, tDNALys and tDNAAsp encoded the CCA sequence of 3'-terminal region, but the other two, tDNAGlu and tDNAPhe did not. A promoter-like sequence which corresponds to the sigma 55-recognition site was found in a region about 100bp upstream from tDNALys.     

RNA synthesis during spore germination in Bacillus subtilis.

Summary We have analyzed the RNA synthesized during spore germination in Bacillus subtilis. Early in germination there is little incorporation of [³H]uridine into RNA. A large increase in incorporation into RNA was found at 45–60 min into germination which was in part due to increases in the specific activity of the UTP pool. When corrected for specific activity changes, the instantaneous rate of RNA synthesis showed a seven to tenfold increase between 30 and 45 min of germination. Polyacrylamide gel electrophoresis studies showed that the RNA synthesized during germination appeared very similar to the RNA made during vegetative growth. DNA-RNA hybridization studies indicated that mRNA and rRNA were synthesized throughout germination. Their relative proportions remained constant and were very similar to the composition of RNA synthesized during vegetative growth.In partial fulfillment of the requirements for the doctoral degree by A.S. in the Department of Microbiology at the New York University School of Medicine     

Biochemical analysis of the Bacillus subtilis 1604 spore germination response

Germination at 37 degrees C of spores of Bacillus subtilis 1604 in the L-alanine and potassium phosphate (ALA) and the glucose, fructose, L-asparagine, potassium chloride (GFAK) germinant systems was triggered following heat activation at 70 degrees C for 1 h. In these conditions, 50% of the spore population became committed to germinate after exposure for 10 min and 14 min to ALA and GFAK, respectively, at which time 38% and 30% losses of OD600 had taken place. Dipicolinic acid (DPA) release, loss of heat resistance and release of soluble hexosamine-containing fragments occurred after commitment and were closely associated with loss of refractility in both the ALA and GFAK pathways. Net ATP synthesis could not be detected until 3-4 min after initiation of germination in both ALA and GFAK, by which time greater than 20% of the spore population was committed to germinate. The ALA and GFAK germination pathways were greater than 99% inhibited by 3 and 1 mM-HgCl2, respectively, as measured by OD600 loss. Reversible post-commitment HgCl2-sensitive sites were present in the ALA and GFAK pathways which were 50% inhibited by 0.125 mM and 0.05 mM-HgCl2, respectively. A pre-commitment HgCl2-sensitive site was identified in the ALA pathway which was 55% inhibited by 6 mM-HgCl2. At 3 mM-HgCl2, 70% of the spore population became committed to germinate in the ALA pathway, whereas less than 5% OD600 loss occurred. In this system, loss of heat resistance was associated with commitment, whereas OD600 loss and DPA release were identified as post-commitment events. The ALA and GFAK pathways were insensitive to a variety of metabolic inhibitors. Protease inhibitors had different effects on the ALA and GFAK pathways: phenylmethanesulphonyl fluoride (PMSF) solely inhibited ALA germination at a pre-commitment site and had little effect on GFAK germination, whereas N alpha-p-tosyl-L-arginine methyl ester (TAME) inhibited both the ALA and GFAK pathways at pre- and post-commitment sites. These results are discussed in relation to a recently proposed model for the triggering of Bacillus megaterium KM spore germination.     

Genetics analysis of spore germination mutants of Bacillus subtilis 168: the correlation of phenotype with map location.

The isolation and characterization of 29 new germination (Ger) mutants of Bacillus subtilis 168 is described. These were classified, along with previously described mutants, into seven groups according to map location. The mutations in 26 GerA mutants mapped between cysB and thr; detailed mapping of two of these has located them very close to citG. These mutants were deficient in germination in alanine, but responded to the germinative combination of asparagine, glucose, fructose and KCl. One GerB mutant mapped on the origin-proximal side of hisA; it was normal in germination in alanine, but deficient in termination in a mixture of asparagine, glucose, fructose and KCl. Two GerC mutants were linked to lys, but were separable from a temperature-sensitive growth deficiency mapping between lys and trp. The GerC mutants had a similar germination phenotype to the GerA mutants. Three GerD mutants did not germinate in either of the above germinants or in Penassay Broth. They were located on the side of ery distal to cysA. The GerE mutant, which did not germinate in any of the three germinants, was located very close to citF and possessed an altered spore coat. The two GerF mutants were defective in germination in all three germinants and mapped on the origin proximal-side of hisA, but much closer to his than did the GerB mutant. A phosphoglycerate kinase-negative mutant altered in germination mapped between cysB and hisA (GerG). These mutants have established a minimum of seven locations important to germination, and will be useful in the development and appraisal of theories of spore germination.     

The nucleotide sequence of phenylalanine tRNA from Bacillus subtilis

The nucleotide sequence of tRNA(Phe) from Bacillussubtilis W 23 has been determined using (32)P labeled tRNA. This is the second B. subtilis tRNA so far reported. The nucleotide sequence was found to be pG-G-C-U-C-G-G-U-A-G-C-U-C-A-G-U-D-G-G-D-A-G-A-G-C-A-A-C-G-G-A-C-U-Gm-A-A- ms(2)i(6)A-A-psi-C-C-G-U-G-U-m(7)G-U-C-G-G-C-G-G-T-psi- C-G-A-U-U-C-C-G-U-C-C-C-G-A-G-C-C-A-C-C-A(OH).Images     

A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination.

The Bacillus subtilis sleB gene, which codes for the enzyme homologous to the germination-specific amidase from Bacillus cereus, was cloned and its nucleotide sequence was determined. Sequence analysis showed that it had an open reading frame of 918 bp, coding for a polypeptide of 305 amino acids with a putative signal sequence of 29 residues. Enzyme activity was not found in germination exudate of B. subtilis spores, which differs from the case of B. cereus enzyme. A B. subtilis mutant with an insertionally inactivated sleB gene revealed normal behavior in growth and sporulation. However, the sleB mutant was unable to complete germination mediated by L-alanine.