Mutations in the gene for a tRNA that functions as a regulator of a transcriptional attenuator in Bacillus subtilis.

It has been proposed that uncharged tRNA molecules may act as positive regulatory factors to control the expression of a number of operons in Bacillus subtilis and related bacteria by interacting with leader sequences to cause antitermination. In this study we report the isolation and characterization of regulatory mutations that modify one of the tRNA molecules predicted to have such a regulatory role. Three different alleles of the B. subtilis leucine tRNA gene leuG were found that resulted in higher expression of the ilv-leu biosynthetic operon. Each resulted in a base change in the D-loop of the leucine tRNA molecule with the anticodon 5'-GAG-3' (leucine tRNAGAG). Experiments with strains that are diploid for mutant and wild-type alleles suggested that both charged and uncharged tRNA molecules may interact with leader sequences to control expression of the operon.

Transcriptional termination. Charging it cancels the order.

In a newly discovered mechanism for regulating transcription termination, a cognate uncharged tRNA acts directly to promote transcription readthrough.

Regions of the Bacillus subtilis ilv-leu operon involved in regulation by leucine.

The ilv-leu operon of Bacillus subtilis is regulated in part by transcription attenuation. The cis-acting elements required for regulation by leucine lie within a 683-bp fragment of DNA from the region upstream of ilvB, the first gene of the operon. This fragment contains the ilv-leu promoter and 482 bp of the ilv-leu leader region. Spontaneous mutations that lead to increased expression of the operon were shown to lie in an imperfect inverted repeat encoding the terminator stem within the leader region. Mutations within the inverted repeat of the terminator destroyed most of the leucine-mediated repression. The remaining leucine-mediated repression probably resulted from a decrease in transcription initiation. A systematic analysis of other deletions within the ilv-leu leader region identified a 40-bp region required for the derepression that occurred during leucine limitation. This region lies within a potential RNA stem-and-loop structure that is probably required for leucine-dependent control. Deletion analysis also suggested that alternate secondary structures proximal to the terminator are involved in allowing transcription to proceed beyond the terminator. Additional experiments suggested that attenuation of the ilv-leu operon is not dependent on coupling translation to transcription of the leader region. Our data support a model proposed by Grundy and Henkin (F. J. Grundy and T. M. Henkin, Cell 74:475-482, 1993) in which uncharged tRNA acts as a positive regulatory factor to increase gene expression during amino acid limitation.

The Bacillus subtilis ochre suppressor sup-3 is located in an operon of seven tRNA genes.

Most Bacillus subtilis tRNA genes have been isolated from lambda libraries by use of probes that hybridize to tRNA or rRNA sequences. None of those genes map to the region of the sup-3 mutation. By cloning of the sup-3 allele, a cluster of seven tRNA genes (the trnS operon) that had not been isolated by other methods was identified. In principle, this approach could be used to isolate at least one more predicted tRNA-containing operon in this bacterium. The trnS operon was shown to contain tRNA genes for Asn (GUU), Ser (GCU), Glu (UUC), Gln (UUG), Lys (UUU), Leu (UAG), and Leu (GAG). The sup-3 mutation was found to be a T-to-A transversion that changes the anticodon of the lysine tRNA from 5'-UUU-3' to 5'-UUA-3'. This result agrees with previous work that determined that the sup-3 mutation causes lysine to be inserted at ochre nonsense mutations.

Regulation of the Bacillus subtilis alsS, alsD, and alsR genes involved in post-exponential-phase production of acetoin.

Acetoin is a major extracellular product of Bacillus subtilis grown on glucose and other fermentable carbon sources. The enzymes responsible for the formation of acetoin, acetolactate synthase, and acetolactate decarboxylase are synthesized in detectable amounts only in cells that have reached stationary phase. We have cloned and sequenced the genes encoding these enzymes, alsS and alsD, as well as a gene, alsR, that regulates their expression. alsS and alsD appear to compose a single operon, while alsR is transcribed divergently from the alsSD operon. AlsR shows significant homology to the LysR family of bacterial activator proteins, and when alsR is disrupted the alsSD operon is not expressed. Transcriptional fusions to alsS and alsR revealed that AlsR is required for the transcription of the alsSD operon, which increases during stationary phase. Two mutations that cause increased expression of the alsSD operon have been isolated, cloned, and sequenced. They each change an amino acid in the AlsR protein.

Cloning and nucleotide sequence of the leucyl-tRNA synthetase gene of Bacillus subtilis.

The leucyl-tRNA synthetase gene (leuS) of Bacillus subtilis was cloned and sequenced. A mutation in the gene, leuS1, increases the transcription and expression of the ilv-leu operion, permitting monitoring of leuS alleles. The leuS1 mutation was mapped to 270 degrees on the chromosome. Sequence analysis showed that the mutation is a single-base substitution, possibly in a monocistronic operon. The leader mRNA predicted by the sequence would contain a number of possible secondary structures and a T box, a sequence observed upstream of leader mRNA terminators of Bacillus tRNA synthetases and the B. subtilis ilv-leu operon. The DNA of the B. subtilis leuS open reading frame is 48% identical to the leuS gene of Escherichia coli and is predicted to encode a polypeptide with 46% identity to the leucyl-tRNA synthetase of E. coli.

Transcriptional regulation of the ilv-leu operon of Bacillus subtilis.

We used primer extension and mutational analysis to identify a promoter upstream of ilvB, the first gene in the ilv-leu operon of Bacillus subtilis. Between the promoter and ilvB, there is a 482-bp leader region which contains a sequence that resembles a factor-independent transcription terminator. In in vitro transcription experiments, 90% of transcripts initiated at the ilvB promoter ended at a site near this terminator. Primer extension analysis of RNA synthesized in vivo showed that the steady-state level of mRNA upstream of the terminator was twofold higher from cells limited for leucine than it was from cells grown with excess leucine. mRNA downstream of the terminator was 14-fold higher in cells limited for leucine than in cells grown with excess leucine. Measurement of mRNA degradation rates showed that the half-life of ilv-leu mRNA was the same when the cells were grown with or without leucine. These data demonstrate that the ilv-leu operon is regulated by transcription attenuation.

M.H2I, a multispecific 5C-DNA methyltransferase encoded by Bacillus amyloliquefaciens phage H2.

Bacillus amyloliquefaciens phage H2 codes for a multispecific cytosine-5-DNA- methyltransferase (MTase), M.H2I, which methylates GGCC, GCNGC and [sequence: see text] target sequences. The gene coding for M.H2I was cloned in Escherichia coli and its nucleotide (nt) sequence was determined. It consists of 1509 bp, corresponding to a protein of 503 amino acids (aa) with a calculated Mr of 57,166. A comparison of the aa sequence of M.H2I with those of the multispecific MTases encoded by Bacillus subtilis phages SPR, phi 3T and rho 11S, revealed that M.H2I is closely related to these enzymes. A very high degree of homology was observed between M.H2I and M.rho 11SI, with 96.2% aa identity and 97.8% nt identity of the corresponding genes.

The conjugative transposon Tn925: enhancement of conjugal transfer by tetracycline in Enterococcus faecalis and mobilization of chromosomal genes in Bacillus subtilis and E. faecalis.

The effects of tetracycline on transfer of the conjugative, tetracycline-resistance transposon. Tn925, as well as the ability of the transposon to promote the transfer of chromosomal genes was examined in Enterococcus faecalis and Bacillus subtilis. To test for chromosomal transfer, multiply-marked strains of each organism, each carrying a single chromosomal copy of Tn925, were mated on filters with suitable recipient strains, under conditions where transformation and transduction were precluded. In both cases, transfer of a variety of chromosomal genes, at frequencies comparable to the frequency of Tn925 transfer, was detected readily. The presence of Tn925 in one of the members of the mating pair was absolutely required for chromosomal transfer, but transfer of Tn925 did not accompany every chromosomal transfer event. The results were consistent with a mating event resembling a type of cell fusion, allowing for extensive recombination between the genomes of the mating partners. Growth of Tn925-containing donor cells in the presence of tetracycline increased the transfer frequency of Tn925 by about tenfold in E. faecalis, but not in B. subtilis.

Genome homology and host range of some SP beta-related bacteriophages of Bacillus subtilis and Bacillus amyloliquefaciens.

Temperate Bacillus subtilis bacteriophages SB beta, phi 3T and SPR and B. amyloliquefaciens phage H2 were compared with respect to DNA-DNA homology by Southern blot analysis to each other and to members of the genus Bacillus. The results show that H2 is a distantly related member of the group III B. subtilis phages. Detectable homology to group III phages could be found in the DNA of several other Bacillus species, including B. natto and B. amyloliquefaciens, demonstrating the widespread occurrence of this group of phages. The host ranges for the phages SP beta, SPR and H2 were determined by adsorption efficiency and by the ability of erythromycin resistance- and chloramphenicol resistance-transducing phages to convert susceptible host strains. Of the three phages examined, only H2 was capable of infecting B. amyloliquefaciens. Based on these results we propose that group III phages should be divided into three subgroups: SP beta, phi 3T, Rho11, IG1, IG3 and Z (subgroup 1), SPR (subgroup 2) and H2 (subgroup 3).

H2, a temperate bacteriophage isolated from Bacillus amyloliquefaciens strain H.

Bacillus amyloliquefaciens strain H is lysogenic for a large temperate phage we call H2. H2 has a polyhedral head 85 nm in diameter and a tail of about 17 x 434 nm. H2 lysogenizes Bacillus subtilis between the tyrA and metB genes, and gives specialized transduction of metB and, at lower frequencies, of ilvD and ilvA. The phage carries a thymidylate synthase gene and converts thymine auxotrophs of B. subtilis to prototrophy. The H2 genome is a linear DNA molecule about 129 kb in length. DNA extracted from phage particles grown in B. subtilis is not cut by the restriction endonucleases HaeIII, Fnu4HI, Bsp1286I, and BamHI; the latter enzyme is produced by B. amyloliquefaciens strain H. The prophage in lysogenic B. subtilis cells can be cut by these enzymes. We have isolated H2 mutants that carry the transposon Tn917, or a mutation resulting in clear-plaque morphology, or both.

Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis.

The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcus faecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J. Christie and G. M. Dunny, Plasmid 15:230-241, 1986). When S. faecalis cells were mixed with Bacillus subtilis in broth, tetracycline resistance was transferred from S. faecalis. The tetracycline-resistant B. subtilis cells contained a 16-kb region of pCF10 (distinct from TRA) that carried the tetracycline resistance determinant (Tetr). This Tetr element was found to transfer between S. faecalis and B. subtilis strains in the absence of plasmids. Genetic and molecular techniques were used to establish locations of the element at several different sites on the B. subtilis chromosome. The Tetr element could be transferred in filter matings from B. subtilis to S. faecalis strains and between recombination-proficient and -deficient S. faecalis strains in the absence of any plasmid DNA. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The Tetr element was shown to transpose from the S. faecalis chromosome to various locations within the hemolysin plasmid pAD1. Together, the data indicate that the Tetr element, termed transposon Tn925, is very similar to the conjugative transposon Tn916 in both structure and function. A derivative of Tn925, containing transposon Tn917 inserted into a site approximately 3 kb from one end, exhibited elevated transfer frequencies and may provide a useful means for delivering Tn917 by conjugation into various gram-positive species.

Chromosomal insertions of Tn917 in Bacillus subtilis.

We describe 46 insertions of the Streptococcus faecalis transposon Tn917 into the chromosome of Bacillus subtilis. These insertion mutations were mapped genetically. Some caused auxotrophic requirements, and others were cryptic. These insertions were scattered around the B. subtilis chromosome. The mutant strains were useful in several ways for mapping and cloning B. subtilis genes and were added to the Bacillus Genetic Stock Center collection. Among the auxotrophic markers were a new serine auxotrophy and deletion-insertions that caused auxotrophy in one case for homoserine and threonine, in another case for uracil and either cysteine or methionine, and in a third case for leucine, isoleucine, and valine.

Interaction of duramycin with artificial and natural membranes.

Duramycin is a polypeptide antibiotic (molecular weight 2012) obtained from culture filtrates of Streptomyces cinnamomeus forma azacoluta. In this work, we show that low concentrations of duramycin induced aggregation of lipid vesicles containing unsaturated phosphatidylethanolamine and unsaturated monogalactosyl diglyceride, and of sarcoplasmic reticulum vesicles from rabbit skeletal muscle. Furthermore, duramycin inhibited the ATP-dependent Ca2+ uptake in sarcoplasmic reticulum vesicles without affecting the hydrolysis of ATP or the permeability of Ca2+. Also, duramycin only inhibited the bacteriorhodopsin proton pump reconstituted into phospholipid vesicles containing phosphatidylethanolamine. We have isolated a duramycin-resistant strain of Bacillus subtilis and have mapped the location of duramycin resistance. In this strain, the secretion of protons and influx of calcium were resistant to duramycin, and its lipid composition was profoundly different from that of the parent strain. No phosphatidylethanolamine was detected in the resistant strain. Our findings are consistent with the idea that duramycin recognizes a particular membrane conformation determined by the presence of phosphatidylethanolamine or monogalactosyl diglyceride.

Genetic and physical analysis of the ilvBC-leu region in Bacillus subtilis.

We describe the cloning of a 6.0-kb PstI fragment of the Bacillus subtilis genome which contains much of the ilvBC-leu gene cluster. This plasmid clone and two others that had been previously isolated were characterized physically and genetically to permit the construction of a physical map of this region that is correlated to the genetic map.

Restriction fragment maps of the genome of Bacillus subtilis bacteriophage SP beta.

Six different restriction endonucleases were used to generate restriction fragment maps of the genome of the temperate Bacillus subtilis phage SP beta. AvaI and SalI each had six target sites in the phage DNA, AvaII had three, BamHI had seven, PstI had twenty, and SacI had sixteen. Restriction analysis and heteroduplex analysis were used to locate a 10-kb region of DNA that is deleted in the clear-plaque mutant, SP beta cl. The deletion lay approx. 50 kb from the left end of the 126-kb phage genome.

Insertion of bacteriophage SP beta into the citF gene of Bacillus subtilis and specialized transduction of the ilvBC-leu genes.

We isolated a strain of Bacillus subtilis in which the SP beta c2 prophage is inserted into the citF (succinate dehydrogenase) gene. Defective specialized transducing particles for the ilvBC-leu genes were isolated from phage-induced lysates of this lysogen. We isolated a group of phages that differ in the amount of genetic material they carry from this region. Also, we incorporated mutant ilv and leu alleles into the genomes of several transducing phages. Our phage collection enables us to identify the cistron of new ilv and leu mutations by complementation analysis. In this process we discovered a fourth leu cistron, leuD. Characterization of the phages confirmed the published gene order: ilvB-ilvC-leuA-leuC-leuB; leuD lies to the right of leuB.

Specialized transduction of the ilvD-thyB-ilvA region mediated by Bacillus subtilis bacteriophage SP beta.

Specialized transducing SP beta particles were found that carried the Bacillus subtilis genes lying to the left of the prophage attachment site. Three classes of transducing particles were differentiated, depending upon whether they carried ilvA only, thyB and ilvA, or ilvD, thyB, and ilvA. Lysates prepared by the induction of strains that carried both a transducing phage and a plaque-forming phage contained the two particles in a ratio of about 1:3,000. When the transducing particles were used to transduce a phage-sensitive auxotrophic strain to prototrophy, some of the transductants carried only the transducing phage genomes which, by themselves, were defective. One putative nondefective transducing phage (for ilvA only) is also described. SP beta can mediate specialized transduction even in the absence of the major (recE) bacterial recombination system.

Defective specialized SP beta transducing bacteriophages of Bacillus subtilis that carry the sup-3 or sup-44 gene.

We isolated defective specialized transducing phages of SP beta that carry one of the extracistronic suppressors, sup-3 or sup-44. Lysates containing these phages can be used in a simple spot test to determine whether an auxotrophic mutation can be suppressed. The sup-3 and sup-44 mutations are distinct, in that their suppression patterns differ for the markers hisA1, metC3, and thr-5; and they are not alleles.

Bacillus subtilis bacteriophages SP beta c1 is a deletion mutant of SP beta.

The restriction fragment patterns of two mutants forms of the temperate Bacillus subtilis bacteriophage SP beta have been examined. The DNA of a heat-inducible mutant, SP beta c2, which has a molecular size of 128 kilobases (kb), yields the same restriction pattern as the wild type SP beta c+ DNA. The DNA of a clear-plaque mutant, SP beta c1, has a molecular size of 117 kb, and is deleted for an 11 kb region of phage DNA. Neither SP beta c1 nor SP beta c2 DNA is cleaved by the endonuclease HaeIII.