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.

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.

The prophage of SP beta c2dcitK1, A defective specialized transducing phage of Bacillus subtilis.

The defective specialized transducing phage SP beta c2dcitK1 carries two known bacterial genes, kauA and citK, as well as SP beta hage markers including the heat-sensitive repressor allele, c2. Some phage genes (including essential ones) are missing. When SP beta c2dcitK1 transduces SP beta-sensitive cells of Bacillus subtilis, the defective prophage is inserted into sites in the homologous bacterial DNA of the attSP beta-kauA-citK region of the recipient chromosome. During the growth of these transductants, occasional excisions occur that result in the loss of the phage genes and of the heterogenotic state. These excisions increase greatly in frequency during growth at repressor-inactivating temperatures. The kinds of insertions and excisions seen suggest that a Campbell-type (CAMPBELL 1962) circular phage genome may occur transiently. If the transductants are superinfected by SP beta c2 or by the clear-plaque mutant SP beta c1, the resulting double lysogen can be heat induced to release high-frequency-of-transduction (HFT) lysates for kauA and citK.

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.

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.

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.

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.

Nutrition of Myxococcus xanthus FBa and some of its auxotrophic mutants.

A defined medium containing 15 amino acids plus salts was used to study the nutrition of Myxococcus xanthus FBa. The amino acids phenylalanine, leucine, isoleucine, valine, and methionine were essential for growth, whereas glycine, proline, asparagine, alanine, lysine, and threonine stimulated growth. An unusual pattern of requirement was found in the aromatic amino acids. Phenylalanine was essential and served as the precursor of tyrosine. Growth in the absence of tryptophan was adaptive, with cells reaching a growth rate equal to that of controls after a lag of about a week. (14)C-labeled ribose and glucose were not appreciably metabolized. Auxotrophs requiring purines and pyrimidines were isolated and were used to study the fate of externally supplied nucleic acid derivatives. Appropriate mutants could satisfy their requirements with free bases, nucleosides, and nucleotides, and could hydrolyze nucleic acids and use the products. However, studies using (14)C-ribose-labeled uridine (isolated from a Salmonella typhimurium pyrimidine auxotroph) showed that externally supplied nucleic acid derivatives were incorporated almost solely into the nucleic acids of the myxobacters, with little used either for energy-yielding oxidations or other cell anabolism.

Nutritional induction and suppression of fruiting in Myxococcus xanthus FBa.

A defined agar medium (A agar) containing 15 amino acids in concentrations between 0.5 and 2 mm was developed for studying the fruiting cycle of Myxococcus xanthus FBa. Cells grew only vegetatively in this medium unless the initial concentration of one of nine required or stimulatory amino acids was lowered about 50-fold. In the latter circumstance, fruiting bodies developed after several days of vegetative growth. The conclusion was that fruiting occurred when any amino acid required for normal growth became limiting in the environment. High concentrations (10 mm) of phenylalanine, tryptophan, or methionine prevented fruiting without affecting growth. Mutants requiring arginine, thymidine, or adenine could not be induced to fruit by limiting their unique requirement although they responded to the same deprivations which brought about fruiting of the wild type. A histidine auxotroph formed fruiting bodies when histidine was lowered to growth-limiting concentrations, provided that the medium was supplemented with purines. A uracil auxotroph was isolated that, perhaps secondarily, had lost some of the mechanisms which control the formation of fruiting bodies; if uracil was present, it formed fruits even when no amino acid was limiting. No concentration of uracil was sufficient to prevent fruiting. Fruiting bodies were formed when mixtures of the uracil auxotroph and wild-type cells were inoculated on A agar plus uracil, even when 75% of the cells were wild type. Microcysts of both strains were present in the fruiting bodies.

Genetic studies of leucine biosynthesis in Bacillus subtilis.

The mutations in a series of leucine auxotrophs isolated after treatment with nitrosoguanidine, ultraviolet light, and ICR-191 have been mapped between ilvC and pheA on the Bacillus subtilis chromosome. A fine structure map of the region was constructed by transformation. Analysis of several strains by assaying levels of their leucine bioysnthetic enzymes has shown that the region encodes three enzymes. The order of the genes with respect to the biosynthetic steps catalyzed by the gene products is 1-3-2.

Regulation of leucine biosynthesis in Bacillus subtilis.

The biosynthesis of alpha-isopropylmalate (alphaIPM) synthetase, IPM isomerase, and betaIPM dehydrogenase in Bacillus subtilis can be derepressed in leucine auxotrophs by limiting them for leucine. The derepression of the three enzymes is apparently coordinate. A class of mutants resistant to 4-azaleucine excretes leucine and has derepressed levels of all three enzymes. The azaleucine-resistance mutations may lie in a gene (azlA) encoding a repressor. Efforts to find mutations characteristic of a constitutive operator have been unsuccessful. No polar mutations have been found among nine leucine auxotrophs that have characteristics of frameshift mutations. The enzyme catalyzing the first step in leucine biosynthesis, alphaIPM synthetase, is sensitive to feedback inhibition by leucine. We conclude that leucine biosynthesis is controlled by the inhibition of the activity of the first biosynthetic enzyme by leucine, and by the repression of the synthesis of the first three biosynthetic enzymes by leucine. The repression of the three enzymes may be under the control of a single repressor and a single operator, or of a single repressor and a separate operator for each structural gene.

Lytic enzyme produced by Myxococcus xanthus.

Hart, Beth A. (Cornell University, Ithaca, N.Y.), and Stanley A. Zahler. Lytic enzyme produced by Myxococcus xanthus. J. Bacteriol. 92:1632-1637. 1966.-Strain FBa of Myxococcus xanthus releases into its culture medium an enzyme capable of lysing Micrococcus lysodeikticus cells and of releasing N-acetyl amino sugars from their cell walls. The lysin is stable at pH values near neutrality and at temperatures below 50 C. It lyses a number of bacterial species sensitive to egg-white lysozyme, and fails to lyse lysozyme-resistant species. Sensitivity of M. lysodeikticus cell walls to FBa lysin and to lysozyme is changed in a similar manner by addition and removal of O-acetyl groups. We describe methods for obtaining 190-fold purification of the lysin from culture filtrates, using acetone and ammonium sulfate precipitations and filtration through diethylaminoethyl cellulose. We also describe a gel filtration method for separating the lysin from a protease which is also produced by strain FBa.

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.

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.

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).

Uptake of branched-chain alpha-keto acids in Bacillus subtilis.

Bacillus subtilis has a constitutive system for the uptake of alpha-keto-beta-methylvalerate, alpha-ketoisovalerate, and (probably) alpha-ketoisocaproate. A mutation, kauA1, which blocks the uptake of alpha-keto-beta-methylvalerate and alpha-ketoisovalerate, is located between metB and citK on the B. subtilis chromosome.

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.

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.

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.