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.

Cloning and expression of a Streptococcus mutans glucosyltransferase gene in Bacillus subtilis.

The gtfA gene of Streptococcus mutans GS-5, which encodes a 55-kDa glucosyltransferase has been isolated from a genetic library in an Escherichia coli-Bacillus subtilis shuttle vector, pMK3. The construction containing the gene enables E. coli JM83 and a sucrase-deficient mutant of B. subtilis to grow on sucrose as the sole carbohydrate source. The gene is expressed under its own control in both organisms. The level of biochemical activity detectable in B. subtilis carrying the clone is approx. 50% of that found in E. coli harboring the same construction. In Bacillus, the gene is expressed through exponential and stationary phases of growth with a decrease in activity as the culture enters stationary phase, corresponding to increases in intracellular protease levels. The enzyme produced in E. coli or B. subtilis harboring the cloned gene is identical to the enzyme produced by S. mutans GS-5 as determined by migration in native polyacrylamide gels.

Identification and initial characterization of glucose-repressible promoters of Streptococcus mutans.

Three catabolite-repressible promoters from Streptococcus mutans have been isolated. These promoters were identified by utilizing the vector pRQ200 which contains a promoterless amylase-encoding gene, a Gram- origin of replication, and an erythromycin-resistance determinant. A library of S. mutans DNA was constructed in pRQ200, amplified in Escherichia coli and integrated by Campbell-type insertion into the S. mutans chromosome following transformation. Colonies exhibiting amylase production on media lacking an extraneous carbohydrate source were screened for diminished amylase production on media containing glucose. The effect of glucose on these promoters has been characterized using a quantitative spectrophotometric assay of amylase activity.

New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments.

Two new shuttle vectors have been constructed by fusing the Escherichia coli plasmid pUC9 with the Staphylococcus aureus plasmids pU110 and pC194. The resulting hybrids replicate in both E. coli and Bacillus subtilis and contain seven restriction sites within a part of the lacZ gene. Insertion of foreign DNA into those sites can be easily detected in E. coli and hybrid plasmids can subsequently be transformed into B. subtilis.

Properties of Bacillus subtilis 168 derivatives freed of their natural prophages.

An isogenic set of Bacillus subtilis 168 strains which are non-inducible for prophage PBSX and are cured of prophage SP beta has been constructed. By utilizing these strains, prophage SP beta has been shown to control the inducible DNA modification system which exists in this bacterium. However, neither the PBSX nor the SP beta prophages alter the ability of the bacterium to undergo genetic recombination, to repair damaged DNA or to sporulate. Prophageless B. subtilis would be a useful host for the phi 3T cloning vector, because of the absence of vector--prophage interactions.

A genetic and molecular characterization of the recA gene from Staphylococcus aureus.

Previous studies have identified mutant strains of Staphylococcus aureus that have deficiencies in genetic recombination and DNA repair. Although these phenotypes were tentatively attributed to mutations within the S. aureus recA gene, experimental evidence to confirm this has never been reported. To characterize recA from S. aureus, we first isolated transposon insertion mutations that were in close proximity to the recA-like mutation (uvs-568) in strain 112 UVS-1. This allowed for the mobilization of the uvs-568 mutation into strain RN4220, the common laboratory strain of S. aureus. Next, using Bacillus subtilis recA as a probe, we cloned S. aureus recA and determined its nucleotide sequence. The deduced amino acid (aa) sequence of RecA contained 347 aa and was 74% identical to B. subtilis RecA. Using a cloned DNA fragment originating from within S. aureus recA, we then constructed a recA null mutant strain, designated KB103, which exhibited the same phenotypic characteristics imposed by the uvs-568 mutation in the same background. Furthermore, genetic and physical mapping of S. aureus recA placed it in the same region as the uvs-568 mutation. These data strongly suggest that these mutations represent different alleles of the same recA gene.

The recE(A)+ gene of B subtilis and its gene product: further characterization of this universal protein.

Although the SOS system of E coli and the SOB system of B subtilis share many similarities, there are distinct differences with respect to the regulation and specificity of the phenomena that constitute these global regulons. One of these differences resides in the regulation of the respective RecA and RecA-like proteins. In B subtilis the RecA-like protein, the RecE protein, shares 60% amino acid homology with its E coli counterpart. The E coli recA gene can complement most, but not all, of the functions that are lost in strains of B subtilis that do not produce a functional RecE protein. The DNA sequence of the recE+ gene as well as the sequence of the recE4 allele and the recA73 allele of B subtilis has demonstrated that mutants of the recE and recA loci of this bacterium actually represent alleles of the same complex gene. Accordingly, the major recombination protein of B subtilis should be referred to as RecA and the gene that encodes this protein as recA+.

Molecular characterization of regulatory elements controlling expression of the Bacillus subtilis recA+ gene.

Expression of the Bacillus subtilis recA gene is induced following DNA damage as well as during the development of the competent state. DNA damage-induction of the recA gene occurs by a RecA-dependent mechanism, whereas competence-induction occurs by a RecA-independent mechanism. To examine the molecular mechanisms that control the expression of the recA gene, a deletion analysis of the recA promoter region was performed. A regulatory region that is required for repression of recA expression was identified upstream of the recA promoter. Deletion of this regulatory region derepressed expression and abolished damage-induction of the recA promoter. Within this region are sequences similar to the consensus sequence that has been identified within DNA damage-inducible promoter regions of other B subtilis genes. Another regulatory region was identified that is required for the RecA-independent, competence-specific induction of the recA gene. Deletion of these sequences significantly reduced competence-induction of the recA promoter.

Tight genetic linkage of a glucosyltransferase and dextranase of Streptococcus mutans GS-5.

A genetic library consisting of over 5000 clones with an average insert size of 6.9 kilobasepairs (kbp) of Streptococcus mutans GS-5 has been constructed in a bivalent plasmid vector pMK3, which is capable of replicating in Escherichia coli and Bacillus subtilis. The recombinant plasmid pSUCRI, containing a 6.0 kbp fragment of S. mutans GS-5 DNA, was the focus of this study. Using Southern hybridization, in vitro and in vivo gene expression techniques, and biochemical analysis, this clone was shown to encode the 55 kiloDalton (kDal) GS-5 gtfA gene product, as well as a 38 and a 66 kDal polypeptide. In addition to the gtfA gene, pSUCRI encodes a dextranase activity with specificity for alpha(1----6)-linked glucans, and with no detectable activity on mutan. The dextranase enzyme had an apparent molecular weight of 66 kDal as demonstrated by SDS-PAGE analysis of the proteins produced by a dextranase-negative deletion derivative. The pH optimum of the enzyme was approximately 6.0, and there was no detectable activity below pH 5.0. By subcloning various combinations of DNA fragments from pSUCRI, it was demonstrated that the dextranase gene (designated dexB) can be separated from the gtfA gene and still be efficiently expressed in both E. coli and B. subtilis. The dexB gene contained its own promoter and ribosome-binding site. The genetic linkage of the gtfA and dexB genes in the S. mutans GS-5 chromosome was confirmed by Southern hybridization and by the independent isolation of four distinct clones containing the gtfA gene and common flanking sequences. In addition to a glucosyltransferase and dextranase, an invertase-like activity is also encoded on pSUCRI, indicating that there is a cluster of genes on the S. mutans GS-5 chromosome which is devoted to the dissimilation of sucrose and concomitant synthesis or modification of glucans into a water-insoluble form, perhaps constituting an operon for glucan modification which can be coordinately regulated in response to environmental alterations.

Mutagenic and toxic effects of ruthenium.

Selected platinum and ruthenium complexes were tested for their ability to cause Salmonella typhimurium strains TA98 and TA100 to revert to histidine independence. The results indicate that ruthenium compounds are capable of reverting both strains while cis-Cl2(NH3)2Pt primarily causes reversions in strain TA100. In addition, cis-platinum is an order of magnitude more mutagenic and toxic than are the ruthenium complexes. Selected compounds were also tested for their ability to induce the bacterial SOS system in the Bacillus subtilis Comptest. In this system, cis-platinum similarly showed greater inducing ability than did the ruthenium complexes. These results also demonstrated that the nature of the sixth ligand in the ruthenium compounds has a significant effect on the mutagenic capacity of these agents.

Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway. I: DNA base and nucleolytic specificity.

Cells of Bacillus subtilis can enter a natural physiological state, termed competence, that is permissive for uptake of DNA from the surrounding medium. In the B. subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, followed by intracellular processing that may ultimately lead to productive infection. Previous investigations have shown that transfecting DNA is usually far less infectious (on a molar basis) than is the DNA injected by phage particles; this result is apparently due to inactivating events suffered by transfecting DNA during its metabolism by competent cells. Earlier studies also demonstrated that, in some cases, the infectivity of transfecting DNA can be increased by ultraviolet (UV) irradiation of the competent cells prior to transfection, or by cotransfection of UV-irradiated heterologous DNAs; collectively, these phenomena have been termed transfection enhancement (TE). We propose here that some transfecting B. subtilis phage DNAs are attacked by a novel host DNA repair system, and that TE reflects inhibition of this by a competing substrate in UV-irradiated DNA. In support of this model, we show that UV-DNA cotransfection leads to a reduced rate of intracellular endonucleolytic breakdown of transfecting DNA. We also demonstrate that TE displays marked specificity of a kind frequently observed for repair enzymes. Thus, phages that contain hydroxymethyl uracil (HMU), but not thymine, in their genomes are susceptible to this process. In addition, we show that the photoproduct(s) in UV-irradiated DNA that produces TE by cotransfection is specific, and is not uracil, a pyrimidine dimer, thymine glycol, HMU, or a substrate for the E. coli thymine glycol DNA N-glycosylase. This photoproduct is derivable from thymine or HMU. The implications of these results are discussed.

Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway. II: Host constitutive expression, repair DNA synthesis, and in vitro activity.

In the Bacillus subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, sometimes followed by productive cell infection. Previous studies have shown that ultraviolet (UV)-irradiation of the competent host cells, or cotransfection of UV-irradiated heterologous DNA, can increase the efficiency of transfection in some cases; these latter two phenomena have been called transfection enhancement (TE). In an accompanying paper, we show that TE is apparently confined to the B. subtilis phages that contain hydroxymethyluracil (HMU) in their DNA, and that the photoproduct in UV-irradiated DNA that mediates TE is specific, and different than the pyrimidine dimer, thymine glycol, uracil, or HMU. We also show that TE is due to reduced intracellular endonucleolytic attack of transfecting DNA. Based on this DNA base and nucleolytic specificity, we hypothesized that TE reflects the incidental action of a host DNA repair system on transfecting HMU phage DNA. In continuing these studies, we show here that duplex infecting HMU phage DNA is apparently inactivated by this same putative repair system when phage protein synthesis is blocked. We find, too, that this inactivation of infecting HMU phage DNA can be inhibited by UV-irradiated DNA, and that this process has a similar DNA base specificity as for TE. The survival of infecting HMU phage DNA is dependent on host DNA polymerase activity. We can detect specific DNA synthesis consistent with formation of repair patches when inactivation of infecting HMU phage DNA is ongoing, but not when it is inhibited by the presence of UV DNA or by allowing phage gene expression. Each of these results is consistent with the hypothesis that TE reflects the action of a novel DNA repair pathway. We show that a candidate TE-associated enzymatic activity can be detected in cell free extracts of uninfected, but not HMU phage-infected, B. subtilis cells. Correspondingly, the extracts of phage-infected cells appear to contain a diffusible factor that acts as an inhibitor of this host enzyme.

DNA repair in Bacillus subtilis. I. The presence of an inducible system.

Following UV irradiation of Bacillus subtilis there is a coordinate induction of: 1) a new protein, 2) a W-reactivation system, 3) a DNA modification system, and 4) prophages. These functions are induced following UV irradiation of repair proficient bacteria and mutants deficient in excision repair (UVR-1) and DNA polymerase I activity (polA5). However, they are not induced, or are impaired in their ability to be induced in bacteria containing the recA1 and the recG13 mutations. This inducible system is compared to the SOS system observed in E. coli.

DNA repair in Bacillus subtilis. II. Activation of the inducible system in competent bacteria.

Competent B. subtilis are more UV sensitive than the non-competent population of the culture. This increased sensitivity is lose in mutants unable to induce the 'SOS system' (recA1,, recG13), in mutants defective in the induction of prophage PBSX (xin), and in late stage competent cells. Moreover, bacteriophage phi 105 produced from transfected cells are less restricted on strain YB880 than bacteriophage produced from infected cells. Therefore, competent cells (those capable of being transfected) have a DNA modification system, whereas the average log phase cell does not. These data support the hypothesis that the development of competence is correlated with the activation of derepression of the "SOS" system in B. subtilis.

Transient growth requirement in Bacillus subtilis following the cessation of exponential growth.

During an investigation of the parameters controlling mutations in Bacillus subtilis we observed that this bacterium exhibits a transient growth requirement for two nonessential amino acids (glutamic acid and isoleucine) during a type of postexponential growth on a minimal medium.

A DNA excision repair system for Neisseria gonorrhoeae.

The removal of pyrimidine dimers from deoxyribonucleic acid of ultraviolet irradiated cultures of Neisseria gonorrhoeae can not be readily ascertained by using radioactively labeled thymidine precursors. However, by adapting the alkaline agarose gel technique of Achey et al. (Photochem Photobiol 29, 305-310, 1979), it was possible to demonstrate that this human pathogen does possess an active excision repair system that functions on pyrimidine dimers.

Cloning of the Bacillus subtilis recE+ gene and functional expression of recE+ in B. subtilis.

By use of the Bacillus subtilis bacteriophage cloning vehicle phi 105J23, B. subtilis chromosomal MboI fragments have been cloned that alleviate the pleiotropic effects of the recE4 mutation. The recombinant bacteriophages phi 105Rec phi 1 (3.85-kilobase insert) and phi 105Rec phi 4 (3.3-kilobase insert) both conferred on the recE4 strain YB1015 resistance to ethylmethane sulfonate, methylmethane sulfonate, mitomycin C, and UV irradiation comparable with the resistance observed in recE+ strains. While strain YB1015 (recE4) and its derivatives lysogenized with bacteriophage phi 105J23 were not transformed to prototrophy by B. subtilis chromosomal DNA, strain YB1015 lysogenized with either phi 105Rec phi 1 or phi 105Rec phi 4 was susceptible to transformation with homologous B. subtilis chromosomal DNA. The heteroimmune prophages phi 105 and SPO2 were essentially uninducible in strain YB1015. Significantly, both recombinant prophages phi 105Rec phi 1 and phi 105Rec phi 4 were fully inducible and allowed the spontaneous and mitomycin C-dependent induction of a coresident SPO2 prophage in a recE4 host. The presence of the recombinant prophages also restored the ability of din genes to be induced in strains carrying the recE4 mutation. Finally, both recombinant bacteriophages elaborated a mitomycin C-inducible, 45-kilodalton protein that was immunoreactive with Escherichia coli recA+ gene product antibodies. Collectively, these data demonstrate that the recE+ gene has been cloned and that this gene elaborates the 45-kilodalton protein that is involved in SOB induction and homologous recombination.

Transduction in Bacillus subtilis by bacteriophage SPP1.

Lysates of the virulent bacteriophage SPP1 were shown to be capable of mediating generalized transduction. Suppressible mutants of this bacteriophage (sus) were capable of transduction at a lower multiplicity of infection than virulent SPP1. Linkage analysis demonstrated that bacteriophage SPP1 transduced segments of the genome equal in size to that transferred by SP10. This bacteriophage should be useful in analyzing the regions of the genome where PBS1 appears to give anomalous results.

Deoxyribonucleic Acid Repair in Bacillus subtilis: Development of Competent Cells into a Tester for Carcinogens.

The development of competent transformed Bacillus subtilis into a tester system for carcinogens is described. Precocious or noninduced activation of SOS functions occur in competent cells. Thus, lower doses or concentrations of SOS inducing agents are needed to cause cell death due to indigenous prophage activation and lysis of bacteria. The two known defective prophages in B. subtilis enhance the sensitivity of competent cells to the carcinogens ultraviolet light, mitomycin C, and methyl methanesulfonate. However, these same cells have no enhanced sensitivity for the non-carcinogenic ethyl methanesulfonate or for nalidixic acid. Therefore, competent B. subtilis appear to be a sensitive tester for carcinogens.