New integrative method to generate Bacillus subtilis recombinant strains free of selection markers

The novel method described in this paper combines the use of blaI, which encodes a repressor involved in Bacillus licheniformis BlaP beta-lactamase regulation, an antibiotic resistance gene, and a B. subtilis strain (BS1541) that is conditionally auxotrophic for lysine. We constructed a BlaI cassette containing blaI and the spectinomycin resistance genes and two short direct repeat DNA sequences, one at each extremity of the cassette. The BS1541 strain was obtained by replacing the B. subtilis P(lysA) promoter with that of the P(blaP) beta-lactamase promoter. In the resulting strain, the cloning of the blaI repressor gene confers lysine auxotrophy to BS1541. After integration of the BlaI cassette into the chromosome of a conditionally lys-auxotrophic (BS1541) strain by homologous recombination and positive selection for spectinomycin resistance, the eviction of the BlaI cassette was achieved by single crossover between the two short direct repeat sequences. This strategy was successfully used to inactivate a single gene and to introduce a gene of interest in the Bacillus chromosome. In both cases the resulting strains are free of selection marker. This allows the use of the BlaI cassette to repeatedly further modify the Bacillus chromosome.     

Generation of auxotrophic mutants of Enterococcus faecalis.

A 22-kb segment of chromosomal DNA from Enterococcus faecalis OG1RF containing the pyrimidine biosynthesis genes pyrC and pyrD was previously detected as complementing Escherichia coli pyrC and pyrD mutations. In the present study, it was found that the E. faecalis pyrimidine biosynthetic genes in this clone (designated pKV48) are part of a larger cluster resembling that seen in Bacillus spp. Transposon insertions were isolated at a number of sites throughout the cluster and resulted in loss of the ability to complement E. coli auxotrophs. The DNA sequences of the entire pyrD gene of E. faecalis and selected parts of the rest of the cluster were determined, and computer analyses found these to be similar to genes from Bacillus subtilis and Bacillus caldolyticus pyrimidine biosynthesis operons. Five of the transposon insertions were introduced back into the E. faecalis chromosome, and all except insertions in pyrD resulted in pyrimidine auxotrophy. The prototrophy of pyrD knockouts was observed for two different insertions and suggests that E. faecalis is similar to Lactococcus lactis, which has been shown to possess two pyrD genes. A similar analysis was performed with the purL gene from E. faecalis, contained in another cosmid clone, and purine auxotrophs were isolated. In addition, a pool of random transposon insertions in pKV48, isolated in E. coli, was introduced into the E. faecalis chromosome en masse, and an auxotroph was obtained. These results demonstrate a new methodology for constructing defined knockout mutations in E. faecalis.     

L-alanine auxotrophy of Lactobacillus johnsonii as demonstrated by physiological, genomic, and gene complementation approaches

Using a chemically defined medium without L-alanine, Lactobacillus johnsonii was demonstrated to be strictly auxotrophic for that amino acid. A comparative genetic analysis showed that all known genes involved in L-alanine biosynthesis are absent from the genome of L. johnsonii. This auxotrophy was complemented by heterologous expression of the Bacillus subtilis L-alanine dehydrogenase.     

Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium.

Transposon Tn5 insertions causing anaerobic cysteine auxotrophy were isolated from a Salmonella typhimurium cysI parent (auxotrophic under aerobic but not anaerobic conditions). Insertions in one mutant group appeared to be in cysG. A second group of insertions, designated asr (anaerobic sulfite reduction), were located near map unit 53 on the S. typhimurium chromosome. They did not cause aerobic or anaerobic auxotrophy in a cys1+ background but did prevent dissimilatory sulfite reduction. Plasmids containing asr DNA cloned from wild-type S. typhimurium conferred anaerobic prototrophy and the ability to produce hydrogen sulfide from sulfite on an Escherichia coli cys1 mutant.     

Expression of the Bacillus subtilis glutamine synthetase gene in Escherichia coli.

The structural gene for glutamine synthetase (glnA) in Bacillus subtilis ( glnAB ) cloned in the lambda vector phage Charon 4A was used to transduce a lysogenic glutamine auxotrophic Escherichia coli strain to prototrophy. The defective E. coli gene ( glnAE ) was still present in the transductant since it could be transduced. In addition, curing of the prototroph resulted in the restoration of glutamine auxotrophy. Proteins in crude extracts of the transductant were examined by a "Western blotting" procedure for the presence of B. subtilis or E. coli glutamine synthetase antigen; only the former was detected. Growth of the strain in media without glutamine was not curtailed even when the bacteriophage lambda pL and pRM promoters were hyperrepressed . The specific activities and patterns of derepression of glutamine synthetase in the transductant were similar to those of B. subtilis, with no evidence for adenylylation. The information necessary for regulation of glnAB must be closely linked to the gene and appears to function in E. coli.     

In vivo random mutagenesis of Bacillus subtilis by use of TnYLB-1, a mariner-based transposon

This report describes the construction and characterization of a mariner-based transposon system designed to be used in Bacillus subtilis, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the mariner-Himar1 transposase gene, modified for expression in B. subtilis, under the control of either sigmaA- or sigmaB-dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kan r cassette bracketed by the Himar1-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Erm r gene of pE194ts. TnYLB-1 transposes into the B. subtilis chromosome with high frequency (10(-2)) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a "TA" dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo- clones, three with insertions in known sporulation genes (kinA, spoVT, and yqfD) and two in genes (ybaN and yubB) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in lysA and another in a gene (yjzB) whose function is unknown.     

Genetic Mapping of a Defective Bacteriophage on the Chromosome of Bacillus subtilis 168

A genetic marker responsible for the killing activity of PBSX, a defective phage carried by Bacillus subtilis 168, has been located on the bacterial chromosome. Two mutant strains of B. subtilis 168, which produced tailless phage particles upon mitomycin C induction, were shown to carry lesions, designated xtl-1 and xtl-2, which were linked by transformation and PBS1-mediated transduction to metC. The link-age relationship between xtl and adjacent auxotrophic markers was determined by three-factor PBS1 transduction, the suggested order of markers being argO 1 metA metC xtl.     

Genetic Analysis in Bacillus pumilus by PBS1-Mediated Transduction

Bacteriophage PBS1 mediates generalized transduction in Bacillus pumilus NRRL B-3275 (BpB1). Transduction frequencies for single auxotrophic markers are of the order of 10(-4) transductants per plaque-forming unit in crude phage lysates. The characteristics of PBS1 propagated on BpB1 and the properties of the system of transduction are similar to those reported for PBS1 propagated on Bacillus subtilis. By transduction, eight amino acid auxotrophic markers in BpB1 have been oriented into two linkage groups. One group contains the auxotrophic markers arginine A, leucine, and phenylalanine, and the other group contains the markers lysine, serine, tryptophan, isoleucine-valine, and isoleucine. The nature and relative order of the markers within each linkage group suggest that the arrangement of genes in these areas of the chromosome of BpB1 is similar to the arrangement of phenotypically comparable genes in two linkage groups (defined by PBS1 transduction) in B. subtilis. However, transduction of any of the above cited markers in BpB1 to prototrophy with PBS1 propagated on B. subtilis 168 could not be demonstrated. In addition to BpB1, seven other strains of B. pumilus can be infected with PBS1. Transduction has been demonstrated in three of these strains.     

A general method for the consecutive integration of single copies of a heterologous gene at multiple locations in the Bacillus subtilis chromosome by replacement recombination.

We have devised a two-step procedure by which multiple copies of a heterologous gene can be consecutively integrated into the Bacillus subtilis 168 chromosome without the simultaneous integration of markers (antibiotic resistance). The procedure employs the high level of transformability of B. subtilis 168 strains and makes use of the observation that thymine-auxotrophic mutants of B. subtilis are resistant to the folic acid antagonist trimethoprim (Tmpr), whereas thymine prototrophs are sensitive. First, a thymine-auxotrophic B. subtilis mutant is transformed to prototrophy by integration of a thymidylate synthetase-encoding gene at the desired chromosomal locus. In a second step, the mutant strain is transformed with a DNA fragment carrying the heterologous gene and Tmpr colonies are selected. Approximately 5% of these appear to be thymine auxotrophic and contain a single copy of the heterologous gene at the chromosomal locus previously carrying the thymidylate synthetase-encoding gene. Repetition of the procedure at different locations on the bacterial chromosome allows the isolation of strains carrying multiple copies of the heterologous gene. The method was used to construct B. subtilis strains carrying one, two, and three copies of the Bacillus stearothermophilus branching enzyme gene (glgB) in their genomes.     

Integration of various plasmids into the Bacillus subtilis chromosome

Some features of integration of temperature-sensitive pE194, pGG10 and pGG20 plasmids into the Bacillus subtilis chromosome were studied. Several auxotrophic mutations were obtained using insertion of these plasmids into the chromosome. The sites of plasmids for illegitimate recombination were determined. It was shown that the integration into the Bac. subtilis chromosome is characteristic not only for the plasmid pE194 but is the property of Staphylococcus aureus plasmid pC194 and Escherichia coli pBR322 plasmid. The influence of different Bac. subtilis rec mutations on the frequency of integration was studied.     

A new method for multiple gene inactivations in Bacillus subtilis 168, producing a strain free of selectable markers

This study describes a novel method for repeated gene inactivation in Bacillus subtilis 168. A B. subtilis strain (BS-PS) that is conditionally auxotrophic for lysine was obtained by replacing the PlysA promoter with the Pspac promoter. The homologous recombination integration vector PLC-T was constructed to contain lacI, which encodes a Pspac promoter repressor, and the chloromycetin resistance gene. Target genes were manipulated by generating an insertion sequence with two homologous arms and the target gene in PLC-T to create a specific integrating vector. Integration into the BS-PS chromosome occurred by a single crossover at either of the two homologous arms. The resulting transitional strain (BS-PS-PI) was chloromycetin resistant and lysine auxotrophic and had an unstable genome structure because of the duplication. Excision of lacI and chloromycetin resistance gene was achieved by a second single crossover at the duplication. Recovery of a lysine prototroph functioned as counter-selection and was identified by PCR. In this work, we inactivated nprE and aprE, two protease genes secreted by B. subtilis 168 free of selectable markers.     

Leaky pantothenate and thiamin mutations of Salmonella typhimurium conferring suphometuron methyl sensitivity

The herbicide suphometuron methyl inhibits the utilization of pyruvate and 2-ketobutyrate by the branched-chain amino acid biosynthetic enzyme acetolactate synthase. Eighteen insertions of the transposon Tn10 into the genome of Salmonella typhimurium LT2 caused hypersensitivity to this herbicide. Five of these insertions conferred a partial auxotrophic requirement. Concurrent herbicide sensitivity and heat-labile pantothenate auxotrophy was due to panD::Tn10 mutations, while coincident sulphometuron methyl sensitivity and thiamin auxotrophy was attributable to thiA::Tn10 mutations. The phenotypes of these mutations suggested that coenzyme A and thiamin pyrophosphate availability modulated the cells' response to sulphometuron methyl. A model suggesting a key role for 2-ketobutyrate accumulation in herbicide action is supported by the function of thiamin pyrophosphate in 2-ketoacid metabolism and the known role of a 2-ketoacid in coenzyme A synthesis.     

A Bacillus subtilis malate dehydrogenase gene.

A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation.     

Intergenotic Transformation of the Bacillus subtilis Genospecies

A multiple auxotrophic derivative of Bacillus subtilis 168 (strain BR151 carrying lys-3, trpC2, metB10) was transformed with deoxyribonucleic acid (DNA) isolated from B. subtilis 168, Bacillus amyloliquefaciens H, B. subtilis HSR, Bacillus pumilus, and Bacillus licheniformis. Transformation with heterologous DNA occurred at a very low frequency for the three auxotrophic markers. Heterologous transformation to rifampin resistance was 100 to 1,000 times more efficient than transformation to prototrophy. Transformants from the various heterologous exchanges were used to prepare donor DNA. The fragment of integrated DNA from the heterologous (foreign) species, termed the "intergenote," was capable of transforming BR151 with an efficiency almost equal to that of homologous DNA. When BR151 DNA contained the Rfm(R) (rifampin resistance) intergenote from B. amyloliquefaciens H, the frequency of transformation was frequently greater than that of the homologous DNA. Accompanying this increased efficiency was a marked change in the physiology of the cells. The growth rate of the transformants carrying this intergenote was approximately one-half that of either parental strain. Thus, in a prokaryotic transformation system, adverse side effects can occur after incorporation of a segment of foreign DNA.     

Tn10-derived transposons active in Bacillus subtilis.

Small derivatives of the Escherichia coli transposon Tn10, comprising IS10 ends and a chloramphenicol resistance gene, were introduced in Bacillus subtilis on a thermosensitive plasmid, pE194. In the presence of the Tn10 transposase gene fused to signals functional in B. subtilis, these derivatives transposed with a frequency of 10(-6) per element per generation. They had no highly preferred insertion site or region, as judged by restriction analysis of the chromosomal DNA, and generated auxotrophic and sporulation-deficient mutants with a frequency of about 1%. These results suggest that Tn10 derivatives might be a useful genetic tool in B. subtilis and possibly other gram-positive microorganisms.     

Genetic design of conditional D-glutamate auxotrophy for Bacillus subtilis: use of a vector-borne poly-gamma-glutamate synthetic system

Bacillus subtilis possesses two glutamate racemase isozymes, RacE and YrpC. For the first time, we succeeded in constructing glutamate racemase-gene disruptants of B. subtilis. Phenotypic analysis of their D-glutamate auxotrophy indicated that the RacE-type glutamate racemase is important for ensuring maximum growth rate but dispensable. The YrpC-type glutamate racemase probably operates as an anaplerotic enzyme for RacE, especially under liquid culture conditions. We found novel applicability of RacE-less mutants inheriting only a marginal activity for endogenous D-glutamate supply, viz. the employment for the in vivo identification of D-glutamate-consuming systems. In fact, the genetic induction of a poly-gamma-glutamate synthetic system led a RacE-less mutant to severe growth suppression, which was overcome in the presence of a high concentration of exogenous D-glutamate. The results indicate that a significant amount of D-glutamate is consumed during poly-glutamate biosynthesis. To our knowledge, this is the first report of conditional D-glutamate auxotrophy for B. subtilis.     

Cloning of an unstable spoIIA-tyrA fragment from Bacillus subtilis

A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spoVA or spoIVA (spoIIA+) strains, subclones of pRC12, lacking a functional spoIIA gene, did complement these mutations. pRC12 inhibited sporulation in a spo+ recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.     

The transposition properties of Tn2 in E. coli

We report the characterization of Tn2 transpositions into the chromosome and into an F' lacproB episome of E. coli. When Tn2 transposes into the chromosome a proportion of the insertions result in auxotrophy. These insertions can revert to prototrophs and lose ampicillin resistance concomitantly. However, Tn2 insertions in F' lacproB are often associated with deletions, and it seems probable that insertion and deletion occur simultaneously.