Effects of surrounding sequence on the suppression of nonsense codons.

Using a lacI-Z fusion system, we have determined the efficiency of suppression of nonsense codons in the I gene of Escherichia coli by assaying beta-galactosidase activity. We examined the efficiency of four amber suppressors acting on 42 different amber (UAG) codons at known positions in the I gene, and the efficiency of a UAG suppressor at 14 different UGA codons. The largest effects were found with the amber suppressor supE (Su2), which displayed efficiencies that varied over a 35-fold range, and with the UGA suppressor, which displayed a 170-fold variation in efficiency. Certain UGA sites were so poorly suppressed (less than 0.2%) by the UGA suppressor that they were not originally detected as nonsense mutations. Suppression efficiency can be correlated with the sequence on the 3' side of the codon being suppressed, and in many cases with the first base on the 3' side. In general, codons followed by A or G are well suppressed, and codons followed by U or C are poorly suppressed. There are exceptions, however, since codons followed by CUG or CUC are well suppressed. Models explaining the effect of the surrounding sequence on suppression efficiency are considered in the Discussion and in the accompanying paper.

In vivo generation of hybrids between two Bacillus thuringiensis insect-toxin-encoding genes.

The parasporal crystal of Bacillus thuringiensis is composed of polypeptides highly toxic to a number of insect larvae. The structural genes (cryIA) encoding the Lepidoptera-specific toxin from different bacterial strains diverge primarily in a single hypervariable region, whereas the N-terminal and C-terminal parts of the proteins are highly conserved. In this report, we describe the generation of hybrid genes between two cryIA genes. Two truncated cryIA genes were cloned in a plasmid vector in such way as to have only the hypervariable region in common. The two truncated cryIA genes were separated by the tetracycline-resistance determinant (or part of it). In vivo recombination between the hypervariable regions of the cryIA genes reconstituted an entire hybrid cryIA gene. Direct sequence analysis of 17 recombinant plasmids identified eleven different crossover regions which did not alter the reading frame and allowed the production of eight different hybrid proteins. The recombination events were independent from the RecA function of Escherichia coli. Some of the hybrid gene products were more specific in their insecticidal action and one had acquired a new biological activity.

A Bacillus subtilis large ORF coding for a polypeptide highly similar to polyketide synthases.

The nucleotide (nt) sequence of 13.6 kb of the outG locus of Bacillus subtilis, which maps at approximately 155 degrees between the genetic markers nrdA and polC, was determined. One putative coding sequence was identified corresponding to a large polypeptide of 4427 amino acids (aa). Structural organization at the nt and aa sequence level and extensive similarities of the deduced product, especially to EryA, suggest that the locus is potentially responsible for the synthesis of a polyketide molecule. The locus has been renamed pksX. Comparison of the deduced product with known fatty acid and polyketide synthases (PKS) suggested the presence of beta-ketosynthase, dehydratase, beta-ketoreductase and acyl-carrier protein domains. Preliminary data obtained with deletion mutants indicate that pksX is not an essential gene.

The Bacillus subtilis outB gene is highly homologous to an Escherichia coli ntr-like gene.

The Bacillus subtilis outB gene was found to have strong similarities to an Escherichia coli gene complementing ntr-like mutations in Rhodobacter capsulatus. The deduced gene products had 52% identical amino acids (65% similar residues). The phenotype of strains affected in the OutB function indicates that this B. subtilis gene may be involved in nitrogen utilization.

Amplification of a chromosomal region in Bacillus subtilis.

We report on the amplification in Bacillus subtilis of a defined DNA sequence after exposure of the bacteria to increasing levels of antibiotic. The experimental system consisted of transformation of competent cells with a plasmid (pRHA39) unable to replicate in the host and carrying the alpha-amylase gene derived from B. subtilis. Selection of transformants resistant to 5 micrograms of chloramphenicol per ml resulted in the isolation of strains with the plasmid integrated into the chromosome at the site of homology, by a Campbell type mechanism. Starting from such a nontandem duplication, amplification was achieved by growing the bacteria in increasing concentrations of chloramphenicol. By dilution, Southern blotting, and hybridization to a radioactive probe, we estimated a copy number of about 10 for the amplified sequence of samples grown in the presence of 50 micrograms of chloramphenicol per ml. No free plasmid could be detected in the amplified strains. The extent of the amplified region was the same for all transformants, and the endpoints appeared to be the same in all isolates. As a consequence of the amplification, there was a noticeable increase in amylase production, and the amount of enzyme produced correlated with gene dosage. The amplification did not occur in a recE genetic background.

Functional analysis of the Bacillus subtilis y shD gene, a mutS paralogue.

In the course of the Bacillus subtilis genome sequencing project, an ORF called yshD was identified, and its product was classified as a mismatch repair protein. Further analysis of the YshD primary sequence showed that the protein belongs to the MutS2 protein family, sharing a high degree of identity with the Thermootoga Inaritima protein TM1278 (34%) and with the so-called MutS2 protein sl11772 of Synechocystis (32%). The COG1193 family of MutS-like proteins is made up of polypeptides that have been predicted from genomic sequencing data from various prokaryotes, but their biological role has not yet been analysed. The functional study of yshD revealed that the gene is constitutively transcribed during the life cycle of B. subtilis, and in minimal medium expression remains at appreciable levels until very late in stationary phase. Fluctuation tests with yshD knock-out mutants did not indicate any role for the protein in preventing the accumulation of spontaneous forward mutations to RifR, nor was any functional interaction with MutS or MutL suggested in fluctuation experiments with mutants lacking combinations of the three genes. Nevertheless, the mutation spectrum observed in the rpoB gene in the deltayshD strain has some characteristic features. The gene does not seem to be involved in the prevention of interspecific recombination in transformation-competent cells.

Mutant of Bacillus subtilis with a temperature-sensitive lesion in ribonucleic acid synthesis during germination.

We have isolated a mutant of Baccillus subtilis with a temperature-sensitive lesion in the process of spore germination. The temperature-sensitive mutation affects only germination and outgrowth, and the earliest defect observed is an early block of ribonucleic acid synthesis during germination at 46 C. Upon return to 35 C there is a complete repair of the impaired function, even in the absence of protein synthesis. Protein synthesis inhibition during germination of the mutant spores at 46 C has the effect of increasing the amount of ribonucleic acid made. The temperature-sensitive mutation is located near aroI.

Pattern of RNA transcription during Bacillus subtilis spore outgrowth.

During the outgrowth of Bacillus subtilis spores, there is a period of RNA and protein synthesis in the absence of DNA replication. Two mutants of B. subtilis, PB2442 (gsp-4) and PB2452 (gsp-81), were used to study the pattern of RNA synthesis during this period. The two mutants are temperature-sensitive in the outgrowth phase, and show a limited amount of incorporation of (3H)uridine at 47 degrees C. The RNAs synthesized during a 2 min pulse with (3H)uridine were hybridized to EcoRI-digested DNA, after agarose gel electrophoresis and transfer to nitrocellulose paper (Southern technique). For both mutants the transcripts synthesized at 35 degrees C at different times were different. Differences were also observed in the transcripts made at 47 degrees C. For both mutants, in the presence of chloramphenicol, the same hybridization pattern was obtained for RNAs pulse-labelled at different periods during outgrowth.

The outB gene of Bacillus subtilis regulates its own transcription.

The outB gene of Bacillus subtilis is under the control of two promoters (P1 and P2). To study the regulation of expression from the P1 promoter we have constructed a set of multicopy plasmids carrying different portions of the outB region and analyzed the transcripts present in vivo by RNase protection experiments. The data indicate that the product of gene outB regulates its own transcription from the P1 promoter. We also constructed an outB-lacZ fusion in an insertional plasmid. The plasmid was inserted into the chromosome adjacent to or distal from the outB gene. Assays of beta-galactosidase activity and RNase protection experiments are in accordance with a model implying that the product of gene outB regulates the initiation of transcription from the P1 promoter acting in the cis configuration.

Bacillus subtilis mutS mutL operon: identification, nucleotide sequence and mutagenesis.

The Bacillus subtilis mutS and mutL genes, involved in the DNA mismatch repair system, have been cloned and characterized. From sequence analysis the two genes appear to be organized in a single operon, located immediately downstream of the cotE gene (approximately 150 degrees on the genetic map). The deduced MutS protein is 49% identical to HexA and MutL is 46% identical to HexB of Streptococcus pneumoniae. Deletion of both mutS and mutL resulted in an increase in the frequency of spontaneous mutations and abolished the marker effect observed in transformation. The expression of the mut operon was studied with the use of a mutSL-lacZ transcriptional fusion. An increase in expression was observed during late exponential growth.

Synthesis of RNA and protein in a mutant of Bacillus subtilis temperature sensitive during spore germination.

Bacillus subtilis strain PB 2427 temperature sensitive in the synthesis of RNA during spore germination and outgrowth has been characterized to some extent. At non permissive temperature (46 degrees C) strain PB 2427 synthesizes stable and unstable RNA for 50 min from the beginning of germination and then stops. Most of the stable RNA is degraded to shorter molecules but can be identified as ribosomal RNA by hybridization-competition experiments. At non permissive temperature, in the presence of chloramphenicol, synthesis of RNA proceeds, though at a reduced rate, for at least 90 min. By hybridization-competition experiments it can also be shown that the RNA synthesized at 46 degrees C in the presence of chloramphenicol includes transcripts that are absent, from the RNA synthesized at 46 degrees C in the absence of drug. The RNA polymerase (holo and core) purified from vegatative cells of the mutant strain does not appear to have a greater heat-lability as compared with the enzyme purified from the parental strain. At non permissive temperature only six polypeptide chains with MW ranging from 47,000 to 78,000 daltons are synthesized by the germinating spores of the mutant.

Integration and excision of a plasmid in Bacillus subtilis.

We have studied the behaviour in Bacillus subtilis of a plasmid (pPV21) carrying the thymidylate synthetase gene of phage phi3T (thyP3). The plasmid can transform efficiently the competent cells of all the strains tested. Polyethylene glycol (PEG)-mediated protoplast transformation is efficient only for recE, recD or recF mutants. When present in recombination proficient strains, the plasmid can be integrated into the chromosome, primarily at the thyA locus. This has been shown by genetic mapping and by blot-hybridization. A second less efficient site is at (or near to) the attachment site of phage phi3T. Excision of the plasmid restores the EcoRI restriction pattern of the parental DNA, although with the loss of the defective thyA endogenotic allele and the retention of the thyP exogenotic gene.

Gene amplification in the lac region of E. coli.

We have characterized strains of E. coli in which the lac region, together with varying amounts of surrounding DNA, is amplified 40 to 200 fold. The amplification events involve regions of 7 to 37 kb and result in a tandem array of repeated units. Restriction digest patterns of DNA from over 100 independent strains reveal that the amplified units are different in each case. Mechanisms of gene duplication and amplification, and the relationship of gene amplification in bacteria to that in eucaryotic cells, are considered.

Functional analysis of the outB gene of Bacillus subtilis.

Three additional alleles of the outB gene of Bacillus subtilis, whose activity is required for spore outgrowth, were identified. The nucleotide sequence of three mutant genes was determined. Analyses of dominance-recessivity showed that the wild-type allele is dominant over the mutant ones. When the outB gene was placed under the control of the inducible spac-1 promoter, the presence of IPTG was necessary to obtain normal growth. The results suggested that the outB gene is required for growth of B. subtilis. Expression of outB from the sporulation promoter spoIID negatively affected subsequent spore outgrowth, without altering vegetative growth and sporulation.

Nucleotide sequence of the outB locus of Bacillus subtilis and regulation of its expression.

The outB gene is one of the genes involved in the process of spore outgrowth in Bacillus subtilis. The gene has been cloned in bacteriophage lambda and subcloned in plasmids. We have determined the sequence of 2,553 base pairs around the outB locus. The locus was found to code for a protein of about 30,000 daltons. Analysis of the in vivo transcripts from this region by RNase protection experiments revealed the presence of two start sites for transcription. Two potential promoters for these transcripts can be tentatively assigned from the sequence data. The amount of one transcript is highest during outgrowth and vegetative growth and absent during the stationary phase. The second transcript is present at a low level throughout the cell cycle.

Expression of a cloned Bacillus thuringiensis delta-endotoxin gene in Bacillus subtilis.

The entire coding region of the Bacillus thurigiensis HD73 crystal protein gene was subcloned from plasmid pJWK20 into the integration vector pUG2-15. This plasmid expresses chloramphenicol resistance when integrated into the Bacillus subtilis chromosome in the outH locus near the recE region. The correct molecular organization of the integrated plasmid was verified by hybridization to Southern blots of chromosomal DNA digests. Production of the toxic crystal protein was monitored at different time points during the life cycle of B. subtilis. Toxicity assays against Anagasta (Ephestia) larvae, direct electron microscopy crystal detection, and immunoblotting assays proved that the expression of the gene in B. subtilis is time regulated and restricted mainly to the sporulation stage. RNase protection experiments defined the transcription initiation start point and the transcription timing. All tests were made in a strain containing one to three copies of the integrated plasmid and in a strain subjected to an amplification regimen.

Properties of the Bacillus subtilis chemotaxis protein CheF, a homolog of the Salmonella typhimurium flagellar protein FliJ.

The nucleotide sequence of Bacillus subtilis cheF was corrected. It encodes an 18-kDa protein that is homologous to FliJ, a protein required for formation of basal bodies in Escherichia coli and Salmonella typhimurium. Methanol release is abnormal in cheF mutants, suggesting that the morphology and functioning of the motor affects methanol formation.

The flaA locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPase-like polypeptide.

We cloned and sequenced 8.3 kb of Bacillus subtilis DNA corresponding to the flaA locus involved in flagellar biosynthesis, motility, and chemotaxis. The DNA sequence revealed the presence of 10 complete and 2 incomplete open reading frames. Comparison of the deduced amino acid sequences to data banks showed similarities of nine of the deduced products to a number of proteins of Escherichia coli and Salmonella typhimurium for which a role in flagellar functioning has been directly demonstrated. In particular, the sequence data suggest that the flaA operon codes for the M-ring protein, components of the motor switch, and the distal part of the basal-body rod. The gene order is remarkably similar to that described for region III of the enterobacterial flagellar regulon. One of the open reading frames was translated into a protein with 48% amino acid identity to S. typhimurium FliI and 29% identity to the beta subunit of E. coli ATP synthase.

The Bacillus subtilis genes for ribonucleotide reductase are similar to the genes for the second class I NrdE/NrdF enzymes of Enterobacteriaceae.

We have cloned and sequenced the nrd (nucleotide reductase) locus of Bacillus subtilis. The locus seems to be organized in an operon comprising four ORFs. The first three encode polypeptides highly similar to the product of the coding sequences characterizing the nrdEF operons of Enterobacteriaceae. The sequencing of the conditional lethal mutation ts-A13, localized in the nrdE cistron, and the lethality of insertional mutations targeted in the internal region of nrdE and nrdF, demonstrated the essential role of this locus. The fourth ORF, ymaB, part of the putative operon, which is not similar to any known protein, is also essential. The regulation of expression of the operon, monitored by lacZ transcriptional fusions, is similar to the regulation of the functionally relevant nrdAB operon of Escherichia coli. The operon was induced by thymidine starvation and its expression was directly or indirectly affected by RecA function. Genetic and functional analysis strongly indicates that in B. subtilis the class I ribonucleotide reductase encoded by this nrd operon is evolutionarily distant from the homologous class I enzyme of Enterobacteria.