Nucleotide sequence and expression of the beta-lactamase gene from Staphylococcus aureus plasmid pI258 in Escherichia coli, Bacillus subtilis, and Staphylococcus aureus.

The structural gene for beta-lactamase in the Staphylococcus aureus plasmid pI258 was cloned into a Staphylococcus aureus-Bacillus subtilis-Escherichia coli shuttle vector, pWN101, and the nucleotide sequence of the gene was determined. pWN101 was structurally stable and the beta-lactamase gene was expressed efficiently from its native promoter and ribosome-binding site in all three hosts.     

Crude lysates of Staphylococcus aureus can transform Bacillus subtilis

Plasmids can be transferred from Staphylococcus aureus to Bacillus subtilis by crude lysates prepared with penicillin or lysostaphin. These lysates mediate drug-resistance plasmid transformation in competent B. subtilis at an efficiency paralleling that of purified DNA.     

Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis.

Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into Bacillus subtilis. Four of these plasmids (pUB110, pCM194, pSA2100, and pSA0501) have been selected for further study. These plasmids replicate as multicopy autonomous replicons in both Rec+ and Rec- B. subtilis strains. They may be transduced between B. subtilis strains or transformed at a frequency of 10(4) to 10(5) transformants per microgram of DNA. The molecular weights of these plasmids were estimated, and restriction endonuclease cleavage site maps are presented. Evidence is given that pSA2100, an in vivo recombinant of pSA0501 and pCM194 (S. Iord?nescu, J. Bacteriol. 124:597-601, 1975), arose by a fusion of the latter plasmids, possibly by insertion of one element into another as a translocatable element. Genetic information from three other S. aureus plasmids (pK545, pSH2, and pUB101) has also been introduced into B. subtilis, although no covalently closed circular plasmid DNA was recovered.     

Promoter-probe plasmid for Bacillus subtilis.

We have constructed a promoter-probe expression vector for Bacillus subtilis. This plasmid, pCED6, can be used to fuse various DNA sequences to the structural gene of Escherichia coli beta-galactosidase, permitting analysis of the promoter activity of such sequences. pCED6 replicates and confers drug resistances in both E. coli and B. subtilis.     

Prophage-dependent plasmid integration in Staphylococcus aureus.

A study has been done of reversion to thermostability of thermosensitive, replication-defective (TSR) mutant penicillinase plasmids. All three of the expected classes of reversions were encountered: back mutation, suppression, and integration. The latter class was examined in some detail and it was found that the presence of the phi 11 phophage enhance the frequency of reversion by integration some 103-fold. Prophage-dependent integration resulted in inactivation of plasmid-linked arsenate and arsenite resistance; these revertant strains gave rise to high frequency tranducing lysates where the plasmid was restored upon transduction to its original TSR state including recovery of these resistances. The integrated plasmid-prophage complexes were stable at high temperatures (43 C) but slow growing and unstable at low (32 C); loss of either plasmid or prophage restored normal growth and stability. Sometimes restoration of the plasmid to its autonomous TSR state was observed and molecular studies showed that in most cases the plasmid was essentially the same size as before integration. In some cases an excision complex was recovered that was more than twice the size of the plasmid and could have been a plasmid-phage co-integrate. Integration also took place in the absence of the ? 11 prophage. These integrations retained all plasmid-linked resistances, were stable at all temperatures, and gave rise to low frequency transducing lysates in which the integrated state was retained upon transduction. On the basis of these results it is suggested that the prophage promotes integration at or near its attachment site.     

Introduction of a Streptococcus cremoris plasmid in Bacillus subtilis.

Streptococcus cremoris Wg2 plasmid pWV01 was introduced in Bacillus subtilis by protoplast transformation. The yield of pWV01 isolated from B. subtilis was low. pWV01 contains a unique site for the restriction endonuclease MboI.     

Replication properties of pIM13, a naturally occurring plasmid found in Bacillus subtilis, and of its close relative pE5, a plasmid native to Staphylococcus aureus.

A naturally occurring plasmid from Bacillus subtilis, pIM13, codes for constitutively expressed macrolide-lincosamide-streptogramin B (MLS) resistance, is stably maintained at a high copy number, and exists as a series of covalent multimers. The complete sequence of pIM13 has been reported (M. Monod, C. Denoya, and D. Dubnau, J. Bacteriol. 167:138-147, 1986) and two long open reading frames have been identified, one of which (ermC') is greater than 90% homologous to the ermC MLS resistance determinant of the Staphylococcus aureus plasmid pE194. The second reading frame (repL) shares homology with the only long open reading frame of the cryptic S. aureus plasmid pSN2 and is probably involved in plasmid replication. The map of pIM13 is almost a precise match with that of pE5, a naturally occurring, stable, low-copy-number, inducible MLS resistance plasmid found in S. aureus. pIM13 is unstable in S. aureus but still multimerizes in that host, while pE5 is unstable in B. subtilis and does not form multimers in either host. The complete sequence of pE5 is presented, and comparison between pIM13 and pE5 revealed two stretches of sequence present in pE5 that were missing from pIM13. It is likely that a 107-base-pair segment in the ermC' leader region missing from pIM13 accounts for the constitutive nature of the pIM13 MLS resistance and that the lack of an additional 120-base-pair segment in pIM13 that is present on pE5 gives rise to the high copy number, stability, and multimerization in B. subtilis. The missing 120 base pairs occur at the carboxyl-terminal end of the putative replication protein coding sequence and results in truncation of that protein. It is suggested either that the missing segment contains a site involved in resolution of multimers into monomers or that the smaller replication protein causes defective termination of replication. It is concluded that pIM13 and pE5 are coancestral plasmids and it is probable that pIM13 arose from pE5.     

Specific pattern of instability of Escherichia coli HisG gene cloned in Bacillus subtilis via the Staphylococcus aureus plasmid pCS194

The plasmid pCS194, generated in vivo by recombination of two Staphylococcus aureus plasmids, pC194 and pS194, coding, respectively, for chloramphenicol (Cm) and streptomycin (Sm) resistance, can be replicated also in Bacillus subtilis in the presence of either of the two antibiotics. In their absence, no segregation of the individual components is observed, but the whole plasmid is lost at a rate of about 10% per generation. The unique EcoRI site of pCS194 is located in the Sm^R determinant. EcoRI-cleaved pCS194 has been joined to an EcoRI-linearized Escherichia coli replicon, the in vitro recombinant pHisG plasmid, composed of the vector pBR313 plus a BglII-segment of E. coli chromosomal DNA, containing a functional hisG gene. The ligation mixture has been used to transform either E. coli or B. subtilis. Following E. coli transformation and selection for Ap^R and Cm^R (the latter is expressed in E. coli by the pC194 determinant), two his⁺ clones were picked at random and the plasmids extracted. These appear identical and contain the original segments. Conversely, after transformation of B. subtilis and selection for Cm^R, only his^? clones have been obtained. From them, deleted plasmids have been extracted. They have lost part or, more frequently, all of the E. coli DNA insert. In the latter case also most of the bracketing pS194 sequence has been lost, and the resulting plasmids are almost identical to pC194, the Cm^R parent of pCS194. When the intact recombinant plasmids, isolated from his⁺ Ap^R Cm^RE. coli clones, have been used to transform B. subtilis cells for Cm^R, again deleted plasmids almost identical to pC194 have been obtained. The events causing these rearrangements occur after in vitro ligation, during either transformation or early propagation of the plasmids, and are probably caused by a translocatable element present in pCS194. A detailed physical map of pC194, carrying the restriction sites for HindIII, HaeIII, HpaII, MboII, AluI, HhaI, and BglI, is presented.     

Cell wall-DNA association in Bacillus subtilis.

Autolysis of cell walls of Bacillus subtilis 168 resulted in solubilization of wall-associated DNA. Most of the DNA was solubilized only in the later stages of autolysis. Solubilization of up to 70% of the wall by autolysins resulted in only 25 to 30% solubilization of wall-associated DNA. When the wall fragments remaining after 70% autolysis were analyzed by electron microscopy, it was observed that the preparations were highly enriched for completed septa, or poles. Partial autolysis at pH 5.2 or pH 8.6, both of which reflect hydrogen ion levels that permit either N-acetylglucosaminidase or N-acetylmuramyl-L-alanine amidase, but not both, to act, gave rise to enrichment of cell poles. When walls were incubated with subtilisin, DNase, or RNase, release of DNA (or DNA fragments) was accelerated. Density gradient centrifugation patterns of lysates of cells pulse-labeled with N-[3H]acetylglucosamine and then chased revealed that a small, but significant, proportion of the radioactivity sedimented to a density position equivalent to that of DNA-membrane complexes. Because the pulse-chase sequence enriched for radioactivity in cell poles, the results suggest that at least some molecules from polar cell walls have an affinity for DNA-membrane complexes. We suggest that DNA binds strongly, possibly via a DNA-membrane complex, to cell poles of B. subtilis. The results provide support for a view offered previously (Koch et al., FEMS Microbiol. Lett. 12:201-208, 1981) that some special structure in or very near the poles of gram-positive bacilli is involved in the segregation of DNA during cell division.     

Characterization of BshA, bacillithiol glycosyltransferase from Staphylococcus aureus and Bacillus subtilis

The first step during bacillithiol (BSH) biosynthesis involves the formation of N-acetylglucosaminylmalate from UDP-N-acetylglucosamine and l-malate and is catalyzed by a GT4 class glycosyltransferase enzyme (BshA). Recombinant Staphylococcus aureus and Bacillus subtilis BshA were highly specific and active with l-malate but the former showed low activity with d-glyceric acid and the latter with d-malate. We show that BshA is inhibited by BSH and similarly that MshA (first enzyme of mycothiol biosynthesis) is inhibited by the final product MSH.     

Analysis of plasmid deletional instability in Bacillus subtilis.

Using a model system, we have studied deletion formation in Bacillus subtilis. When the staphylococcal plasmids pSA2100 (7.1 kilobases) and pUB110 (4.5 kilobases) were ligated to one another at their unique XbaI sites and transformed into either rec+ or recE4 strains of B. subtilis, an intramolecular recombination event usually occurred. Two plasmids, one of 2.6 kilobases and the other of 9.0 kilobases, were consistently isolated and shown by restriction enzyme analysis to be derived by recombination occurring in the pSA2100-pUB110 cointegrate. Analysis of the sequence of the junctions of the recombinant plasmids and of the crossover regions of the parental plasmids suggested that a reciprocal, conservative, intramolecular recombination event had occurred between short 18-base-pair homologous sequences that were oriented as direct repeats and bounded by regions of dyad symmetry. Evidence is presented that the above illegitimate recombination event is biased to occur intramolecularly and that randomly chosen direct repeats of either 22 or 29 base pairs are not sufficient to support recombination. The recombination event occurs in recA1, recB2, recD3, recE5, recL16, recM13, polA59, polA13, uvr-22, uvr-13, and stb mutants of B. subtilis and does not require that the competent state be established.     

Replication of a Bacillus subtilis oriC plasmid in vitro

We constructed an in vitro replication system specific for a Bacillus subtilis oriC plasmid using a soluble fraction derived from cell extracts of B. subtilis. DNA polymerase III and two initiation proteins, DnaA and DnaB, were required for in vitro replication as observed in vivo. Both upstream and downstream DnaA box regions of the dnaA gene were required as cis-elements for in vitro synthesis of the B. subtilis oriC plasmid as well as for in vivo activity. The replication was semi-conservative and only one round of replication occurred within 15min. These results indicate that in vitro replication faithfully reproduced in vivo replication. To elucidate the site of initiation and the direction of replication, we analysed replicative intermediates generated in vitro in the presence of various concentrations of ddGTP by two methods. First, analysis of restriction fragments around the dnaA gene showed a high level of incorporation of the radioactive substrate, indicating that replication began within the vicinity of the dnaA gene. Second, using 2-dimensional gel electrophoresis, bubble arcs were detected only on fragments containing the DnaA box region downstream of the dnaA gene, indicating that the initiation site resided within this region. The distribution of the bubble arcs suggested that both bidirectional and unidirectional replication occurred in vitro.