The Bacillus subtilis spo0J gene: evidence for involvement in catabolite repression of sporulation.

Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spo0J and to make wild-type sporulation catabolite resistant suggested that spo0J had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spo0J locus (spo0J::Tn917 omega HU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spo0J::Tn917 omega HU261 to sporulate in medium with glucose. Sequencing the spo0J locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spo0J mutations. Analysis of the deduced amino acid sequence of the spo0J locus indicated that the spo0J gene product contains an alpha-helix-turn-alpha-helix unit similar to the motif found in lambda Cro-like DNA-binding proteins.     

The isolation, cloning and identification of a vegetative catalase gene from Bacillus subtilis.

A Bacillus subtilis library of Tn917::lacZ insertions was screened for mutants that were unable to grow in the presence of normally sublethal concentrations of hydrogen peroxide. The identification and subsequent analysis of one mutant strain, YB2003, which carried the mutation designated kat-19, revealed that this strain was deficient in the expression of a vegetative catalase. Regions of the chromosome both 5' and 3' to the site of the Tn917 insertion, as well as the gene without the insertion (kat-19+) were cloned. The presence of the functional kat-19+ gene on a high-copy plasmid restored catalase activity to the kat-19::Tn917 strain as well as to strains of B. subtilis that carried the katA 1 mutation. While the katA+ locus is believed to represent the structural gene for the vegetative catalase of B. subtilis [Loewen and Switala, J. Bacteriol. 169 (1987) 5848-5851], the sequence analysis of the cloned kat-19+ DNA fragments revealed an open reading frame that showed significant homology between the deduced amino acid sequence of this gene product and that of known eukaryotic catalases.     

The protonophore resistance of Bacillus megaterium is correlated with elevated ratios of saturated to unsaturated fatty acids in membrane phospholipids

Growth of the protonophore-resistant strain of Bacillus megaterium, strain C8, in the presence of oleic acid markedly reduced its resistance to low concentrations of carbonylcyanide m-chlorophenylhydrazone (CCCP). Growth of the CCCP-sensitive wild-type strain in the presence of stearic acid increased the resistance of that strain to growth inhibition by protonophore. Studies of the membrane lipids indicated that in the absence of additions to the medium, membranes from C8 contained greatly reduced levels of monounsaturated fatty acids relative to the wild type; wild-type levels were restored by growth of C8 in the presence of oleic acid, concomitant with the loss of resistance. Conversely, growth of the wild type on stearic acid increased the ratio of saturated/unsaturated fatty acids in the membrane, concomitant with a modest increase in the resistance of the wild-type strain to CCCP. The exogenous oleic acid was preferentially incorporated into phosphatidylethanolamine, diphosphatidylglycerol, and 1,2-diacylglycerol, whereas stearic acid was incorporated preferentially into phosphatidylglycerol, and into the small component of free fatty acids. Depending upon the growth conditions, changes in membrane lipid-to-membrane protein ratio and in the ratios of polar lipid components were observed, but none of those changes correlated as did the changes in saturated fatty-acid-to-unsaturated fatty-acid ratio with protonophore resistance. This latter correlation was further suggested by experiments in which the protonophore resistance of wild type B. megaterium was shown to increase with increasing growth temperature without any temperature-dependent loss of protonophore efficacy. The experiments here support the hypothesis developed from work with Bacillus subtilis that changes in the fatty acid composition of the membrane phospholipids affect energy coupling, and make it clear that simple increases or decreases in the hydrolytic activity of ATPase in the uncoupler-resistant mutants of bacilli are not correlated with resistance in some direct way.     

Isolation and characterization of a new chromosomal virulence gene of Agrobacterium tumefaciens.

A mutant (strain B119) of Agrobacterium tumefaciens with a chromosomal mutation was isolated by transposon (Tn5) mutagenesis. The mutant exhibited growth rates on L agar and minimal medium (AB) plates similar to those of the parent strain (strain A208 harboring a nopaline-type Ti plasmid). The mutant was avirulent on all host plants tested: Daucus carota, Cucumis sativus, and Kalanchoe diagremontiana. The mutant was not impaired in attachment ability to carrot cells. The mutant had one insertion of Tn5 in its chromosome. The avirulent phenotype of B119 was shown to be due to the Tn5 insertion in the chromosome by the marker exchange technique. A wild-type target chromosomal segment (3.0 kb) which included the site of mutation was cloned and sequenced. Two open reading frames, ORF-1 (468 bp) and ORF-2 (995 bp), were identified in the 3.0-kb DNA segment. Tn5 was inserted in the middle of ORF-2 (acvB gene). Introduction of the acvB gene into the mutant B119 strain complemented the avirulent phenotype of the strain. Homology search found no genes homologous to acvB, although it had some similarity to the open reading frame downstream of the virA gene on the Ti plasmid. Thus, the acvB gene identified in this study seems to be a new chromosomal virulence gene of A. tumefaciens.     

A cold-sensitive Listeria monocytogenes mutant has a transposon insertion in a gene encoding a putative membrane protein and shows altered (p)ppGpp levels

A cold-sensitive Listeria monocytogenes mutant designated cld-14 was obtained by transposon Tn917 mutagenesis. The gene interrupted by Tn917 in cld-14 was the L. monocytogenes LMOf2365_1485 homolog, which exhibits 45.7% homology to the Bacillus subtilis yqfF locus. LMOf2365_1485, here designated pgpH, encodes a putative integral membrane protein with a predicted molecular mass of 81 kDa. PgpH is predicted to contain a conserved N-terminal signal peptide sequence, seven transmembrane helices, and a hydrophilic C terminus, which likely extends into the cytosol. The Tn917 insertion in pgpH is predicted to result in production of a premature polypeptide truncated at the fifth transmembrane domain. The C terminus of PgpH, which is probably absent in cld-14, contains a highly conserved HD domain that belongs to a metal-dependent phosphohydrolase family. Strain cld-14 accumulated higher levels of (p)ppGpp than the wild type accumulated, indicating that the function of PgpH may be to adjust cellular (p)ppGpp levels during low-temperature growth. The cld-14pgpH(+) complemented strain was able to grow at a low temperature, like the parent strain, providing direct evidence that the activity of PgpH is important in low-temperature adaptation. Because of its predicted membrane location, PgpH may play a critical role in sensing the environmental temperature and altering cellular (p)ppGpp levels to allow the organism to adapt to low temperatures.     

Tn917 transposition in Lactobacillus plantarum using the highly temperature-sensitive plasmid pTV1Ts as a vector

pTV1Ts, a temperature-sensitive plasmid coding for chloramphenicol (Cm) resistance and carrying the macrolide-lincosamide-steptogramin B (MLS) resistance transposon Tn917, was introduced into strains of Lactobacillus plantarum by electroporation. After two passages in broth medium selecting for MLS resistance at 40 degrees C and subsequent plating on solid medium, two strains, L. plantarum NC4Ts1 and L. plantarum NC7Ts5, lost chloramphenicol resistance but retained MLS resistance, indicative of Tn917 transposition into host DNA. Analysis of DNA from MLSrCms isolates from both strains revealed Tn917 insertions into resident plasmids. Restriction analysis of plasmid DNA from four MLSrCms isolates from NC7Ts5 indicated four different insertion sites.     

Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements.

New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli (pT21delta2TetM). In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S. mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn9l7, in JH1005 at the permissive temperature (30 degrees C) versus that at the nonpermissive temperature (45 degrees C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10(-5) to 10(-4)) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45 degrees C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7%. Recovery of transposon-interrupted genes was achieved by two methods: (i) marker rescue in E. coli with the recovery vector pTV21delta2TetM, a tetracycline-resistant and ampicillin-sensitive Tn9l7-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19. Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp.fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylxM-ffh (58% identity), icd (citC [69% identity]), and argD (61% identity). Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements. This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization. This represents the first published report of successful Tn9l7 mutagenesis in the genus Streptococcus.     

Bacillus subtilis DEAD protein YdbR possesses ATPase, RNA binding, and RNA unwinding activities

The product of an open reading frame (ORF) (called YdbR) identified while analyzing the Bacillus subtilis genome has been classified as an Asp-Glu-Ala-Asp (DEAD) protein, but the biological function and enzymology of YdbR have not been characterized in detail. Here we show that recombinant YdbR-His(6) purified from Escherichia coli is an ATP-independent RNA binding protein. It also possesses RNA-dependent ATPase activity stimulated not only by total RNA from B. subtilis but also by an RNA that is irrelevant to that of B. subtilis. Functional analysis indicated that the growth rate of a DeltaydbR mutant strain of B. subtilis was reduced as compared with that of the wild type not only at 37 degrees C, but more severely at 22 degrees C.     

Identification of genes and gene products whose expression is activated during nitrogen-limited growth in Bacillus subtilis.

The levels of urease and asparaginase were elevated 25- and 20-fold, respectively, in extracts of Bacillus subtilis cells grown in medium containing nitrogen sources that are poor sources of ammonium (NH4+) compared with the levels seen in extracts of cells grown in medium containing nitrogen sources that are good sources of NH4+. To determine whether a collection of genes whose expression responds to nitrogen availability could be isolated, a library of Tn917-lacZ insertions was screened for nitrogen-regulated beta-galactosidase expression. Two fusion strains were identified. beta-Galactosidase expression was 26- and 4,000-fold higher, respectively, in the nrg-21::Tn917-lacZ and the nrg-29::Tn917-lacZ insertion strains during NH4(+)-restricted growth than during growth on nitrogen sources that are good sources of NH4+. PBS1 transduction analysis showed that the nrg-21::Tn917-lacZ insertion mapped between gutB and purB and that the nrg-29::Tn917-lacZ insertion mapped between degSU and spoIID. The repression of expression of these four gene products during growth on good sources of NH4+ required the wild-type glutamine synthetase protein but not the glutamine synthetase regulatory protein, GlnR.     

Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80.

Transposon Tn917-LTV1 was used to produce a collection of Lactococcus lactis strains with fusion of a promoterless lacZ gene to chromosomal loci. Screening 2,500 Tn917-LTV1 integrants revealed 222 that express beta-galactosidase on plates at 30 degrees C. Pulsed-field gel electrophoresis revealed Tn917-LTV1 insertions in at least 13 loci in 15 strains analyzed. Integrants in which beta-galactosidase expression was regulated by temperature or pH and/or arginine concentration were isolated. In most cases, the regulation observed on plates was reproducible in liquid medium. One integrant, PA170, produces beta-galactosidase at pH 5.2 but not at pH 7.0, produces more beta-galactosidase at 15 degrees C than at 30 degrees C, and has increased beta-galactosidase activity in the stationary phase. DNA fragments potentially carrying promoters from selected Lactococcus lactis integrants were cloned in Escherichia coli. A new promoter probe vector, pAK80, containing promoterless beta-galactosidase genes from Leuconostoc mesenteroides subsp. cremoris and the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid replication region was constructed, and the lactococcal fragments were inserted. Plasmid pAK80 was capable of detecting and discriminating even weak promoters in Lactococcus lactis. When inserted in pAK80, the promoter cloned from PA170 displayed a regulated expression of beta-galactosidase analogous to the regulation observed in PA170.     

Isolation and initial characterization of a Bacillus subtilis mutant with novel protease secretion capability

A bank of pTV32 (Tn 917 lacZ) - generated Bacillus subtilis mutants were examined on milk agar for the ability to produce proteases at 48 degrees C. A single mutant, BUL786, was isolated, which could hydrolyze casein after overnight incubation at 48 degrees C. This mutant secreted protease 10 fold more at 48 degrees C when compared to 37 degrees C, and part of the activity appears to be 48 degrees C-specific. At high temperatures, other strains of B. subtilis, including hyperprotease secretors, were unable to secrete protease to any significant degree. The BUL786 strain is missing the 97K major heat shock protein. Since a number of other proteins also appear to be secreted at 48 degrees C, this mutant may be a hypersecretor of exported proteins at temperatures greater than 45 degrees C.     

Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome.

Delivery vectors for mini-Tn10 transposons function in Bacillus subtilis (M. A. Petit, C. Bruand, L. Janniére, and S. D. Ehrlich, J. Bacteriol. 172:6736-6740, 1990). Using this system, we identified a new gene (sytA) whose inactivation affected regulation of genes of sucrose metabolism. For cloning the sytA::Tn10 insertion in Escherichia coli, we developed a methodology similar to that commonly used for B. subtilis Tn917 insertions. We constructed a plasmid which can be used to insert (by in vivo recombination) a ColE1 origin linked to a spectinomycin resistance gene (ori-spc element) into mini-Tn10 transposons inserted into the B. subtilis chromosome. DNA extracted from a sytA::Tn10::ori-spc transformant was cut with restriction enzymes that do not cut into the Tn10::ori-spc sequence; plasmids containing the sytA::Tn10 insertion were cloned by self-ligation, followed by transformation of E. coli. To obtain the wild-type sytA region, one of these plasmids was ligated with an E. coli-B. subtilis shuttle vector conferring erythromycin resistance, and the hybrid was used to transform the wild-type B. subtilis strain. Erythromycin-resistant transformants, detected as spectinomycin sensitive, resulted from conversion of the insertion mutation by the resident wild-type locus. The shuttle plasmid containing the wild-type locus could then be recovered in E. coli.