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S D Ehrlich

Publications and source records attributed to S D Ehrlich.

At least 91 records · Page 5Linked to original sources
PubMed1997

Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes.

Single-crossover homologous integration in Lactobacillus sake was studied. Integration was conducted with nonreplicative delivery vector pRV300. This vector is composed of a pBluescript SK- replicon for propagation in Escherichia coli and an erythromycin resistance marker. Random chromosomal DNA fragments of L. sake 23K ranging between 0.3 and 3.4 kb were inserted into pRV300. The resulting plasmids were able to integrate into the chromosome by homologous recombination as single copies and were maintained stably. The single cross-over integration frequency was logarithmically proportional to the extent of homology between 0.3 and 1.2 kb and reached a maximum value of 1.4 x 10(3) integrants/micrograms of DNA. We used this integration strategy to inactivate the ptsI gene, encoding enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system, and the lacL gene, which is one of the two genes required for the synthesis of a functional beta-galactosidase. The results indicated that our method facilitates genetic analysis of L. sake.

Crossing Over, Genetic↗
PubMed1997

Characterization of Lactococcus lactis UV-sensitive mutants obtained by ISS1 transposition.

Studies of cellular responses to DNA-damaging agents, mostly in Escherichia coli, have revealed numerous genes and pathways involved in DNA repair. However, other species, particularly those which exist under different environmental conditions than does E. coli, may have rather different responses. Here, we identify and characterize genes involved in DNA repair in a gram-positive plant and dairy bacterium, Lactococcus lactis. Lactococcal strain MG1363 was mutagenized with transposition vector pG+host9::ISS1, and 18 mutants sensitive to mitomycin and UV were isolated at 37 degrees C. DNA sequence analyses allowed the identification of 11 loci and showed that insertions are within genes implicated in DNA metabolism (polA, hexB, and deoB), cell envelope formation (gerC and dltD), various metabolic pathways (arcD, bglA, gidA, hgrP, metB, and proA), and, for seven mutants, nonidentified open reading frames. Seven mutants were chosen for further characterization. They were shown to be UV sensitive at 30 degrees C (the optimal growth temperature of L. lactis); three (gidA, polA, and uvs-75) were affected in their capacity to mediate homologous recombination. Our results indicate that UV resistance of the lactococcal strain can be attributed in part to DNA repair but also suggest that other factors, such as cell envelope composition, may be important in mediating resistance to mutagenic stress.

Bacterial Proteins↗
PubMed1997

Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis.

The alpha-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp. lactis. It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter. The product of aldB is responsible for leucine sensibility under valine starvation. In the presence of more than 10 microM leucine, the alpha-acetolactate produced by the biosynthetic acetohydroxy acid synthase IlvBN is transformed to acetoin by AldB and, consequently, is not available for valine synthesis. AldB is also involved in acetoin formation in the 2,3-butanediol pathway, initiated by the catabolic acetolactate synthase, AlsS. The differences in the genetic organization, the expression, and the kinetics parameters of these enzymes between L. lactis and Klebsiella terrigena, Bacillus subtilis, or Leuconostoc oenos suggest that this pathway plays a different role in the metabolism in these bacteria. Thus, the alpha-acetolactate decarboxylase from L. lactis plays a dual role in the cell: (i) as key regulator of valine and leucine biosynthesis, by controlling the acetolactate flux by a shift to catabolism; and (ii) as an enzyme catalyzing the second step of the 2,3-butanediol pathway.

Acetoin↗
PubMed1997

Apparent and real recombination frequencies in multicopy plasmids: the need for a novel approach in frequency determination.

Recombination studies of bacteria are often carried out with multicopy plasmids, and recombination frequencies are often deduced from the proportion of cells in the population that express a recombinant phenotype. These frequencies should however be called apparent frequencies, since detection of the recombinant cells requires not only the formation of a rearranged plasmid but also its establishment in the cell. The establishment of the recombinant plasmid can possibly be affected by its interaction with the parental plasmids. To test this hypothesis, we have used a plasmid system enabling the study of deletion formation between short direct repeats (18 bp) in Bacillus subtilis and developed a method by which deletion frequencies are measured under conditions under which interaction is abolished. Real deletion frequencies were thus determined and compared with apparent deletion frequencies. Real frequencies were underestimated by a factor ranging from 4- to 500-fold, depending upon the plasmid under study. This implies that a large majority of the recombinant molecules that are formed are generally not detected. We show that apparent deletion frequencies strongly depend upon (i) the parental plasmid copy number, (ii) the ability of the recombinant molecules to form heterodimeric plasmids, and (iii) the fitness of the recombinant molecules relative to that of parental molecules. Finally, we show that under conditions under which all recombinant molecules are scored, transcription can inhibit the deletion process 10-fold.

Bacillus subtilis↗
PubMed1997

Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria.

The M6 protein from Streptococcus pyogenes is the best-characterized member of a family of cell envelope-associated proteins. Based on the observation that the C-terminal sorting signals of these proteins can drive cell wall anchoring of heterologous unanchored proteins, we have cloned and expressed the emm6 structural gene for the M6 protein in various lactic acid bacteria (LAB). The emm6 gene was successfully expressed from lactococcal promoters in several Lactococcus lactis strains, an animal-colonizing Lactobacillus fermentum strain, Lactobacillus sake, and Streptococcus salivarius subsp. thermophilus. The M6 protein was efficiently anchored to the cell wall in all strains tested. In lactobacilli, essentially all detectable M6 protein was cell wall associated. These results suggest the feasibility of using the C-terminal anchor moiety of M6 for protein surface display in LAB.

Antigens, Bacterial↗
PubMed1996

Plasmid vectors for gram-positive bacteria switching from high to low copy number.

A set of vectors for Gram-positive bacteria was constructed with a new feature which enables the switching down of their copy number per cell. These vectors carry the replication region of pAM beta 1, containing a gene essential for replication, repE, and its regulator, copF. The latter gene was inactivated by inserting a linker into its unique KpnI site. Since copF downregulates the expression of repE, its inactivation leads to an increase in the plasmid copy number per cell. The original low copy state can be restored by removal of the linker via KpnI cleavage and ligation. The new replicon was used to build (i) vectors for studying gene regulation by transcriptional or translational fusion with the bacterial luciferase gene, (ii) vectors for gene expression, and (iii) cassettes of the replicon with different multiple cloning sites, which would facilitate construction of vectors for novel purposes.

Amino Acid Sequence↗
PubMed1996

Change of a catalytic reaction carried out by a DNA replication protein.

The RepA protein of plasmid pC194 initiates and terminates rolling circle replication. At initiation, it forms a 5'-phosphotyrosyl DNA link, whereas at termination, a glutamate residue directs hydrolytic cleavage of the newly synthesized origin, and the resulting 3'-hydroxyl group undergoes transesterification with the phosphotyrosine link. The protein is thus released from DNA, and the termination is uncoupled from reinitiation of replication. Replacement of the glutamate with tyrosine in RepA altered this mechanism, so that termination occurred by two successive transesterifications and became coupled to reinitiation. This result suggests that various enzymes involved in DNA cleavage and rejoining may have similar mechanistic and evolutionary roots.

Bacteriophage phi X 174↗
PubMed1996

Transcription-induced deletions in plasmid vectors: M13 DNA replication as a source of instability.

We have previously shown that concurrent progression of pBR322 replication and pTac-directed transcription in opposite orientations induces illegitimate recombination events. We tested here the effects of M13 rolling circle replication on the incidence of plasmid deletions. The progression of the M13 replication fork leads to an increase of more than 300-fold in the frequency of transcription-dependent deletion events. pBR322 derivatives carrying the M13 replication origin and a 511 bp transcribed region under the control of the pTac promoter were used. Up to 12% of the plasmid population has sustained deletions within 4 h following the induction of pTac-directed transcription and M13 DNA replication, provided that the two proceed in opposite orientations. We observed that induction of transcription of the whole Escherichia coli lacZ gene (3244 bp) in the direction opposite to M13 replication leads to a fivefold decrease in plasmid copy number within 2 h, which is consistent with the proposal that deletions arise because replication fork progression is impeded. This decrease in parental plasmid copy number leads in turn to an enrichment in deleted plasmid forms. Our data confirm and extend the notion that simultaneous transcription and replication in opposite directions can efficiently promote deletion formation. In addition, this instability may be amplified when the rearranged molecules acquire a replicative advantage.

Adenosine Triphosphatases↗
PubMed1996

In vivo analysis of the plasmid pAM beta 1 resolution system.

The promiscuous plasmid pAM beta 1 from Gram-positive bacteria encodes a resolution system which differs from that of Tn3 in that (i) it requires a histone-like protein and an unusual resolvase-DNA interaction to promote recombination and (ii) it mediates in vivo DNA inversion in plasmid substrates. In this in vivo analysis, the pAM beta 1 resolution site is narrowed down to a 99 bp segment, the strand exchange is mapped within 10 bp and the serine residue at position 10 of the resolvase is shown to be essential for enzyme activity. In addition, data showing that the resolution system does not promote DNA inversion in the Bacillus subtilis chromosome are presented. Implications of this observation are discussed.

Bacillus subtilis↗
PubMed1996

Copy-choice recombination mediated by DNA polymerase III holoenzyme from Escherichia coli.

Formation of deletions by recombination between short direct repeats is thought to involve either a break-join or a copy-choice process. The key step of the latter is slippage of the replication machinery between the repeats. We report that the main replicase of Escherichia coli, DNA polymerase III holoenzyme, slips between two direct repeats of 27 bp that flank an inverted repeat of approximately equal 300bp. Slippage was detected in vitro, on a single-stranded DNA template, in a primer extension assay. It requires the presence of a short (8 bp) G+C-rich sequence at the base of a hairpin that can form by annealing of the inverted repeats. It is stimulated by (i) high salt concentration, which might stabilize the hairpin, and (ii) two proteins that ensure the processivity of the DNA polymerase III holoenzyme: the single-stranded DNA binding protein and the beta subunit of the polymerase. Slippage is rather efficient under optimal reaction conditions because it can take place on >50% of template molecules. This observation supports the copy-choice model for recombination between short direct repeats.

Base Sequence↗
PubMed1996

Dramatic decay of phage transcripts in lactococcal cells carrying the abortive infection determinant AbiB.

The abortive infection determinant AbiB prevents growth of the sensitive phage bIL170, but not of the resistant phage bIL41, on Lactococcus lactis strain IL1403. Here we show that AbiB promotes a dramatic degradation of sensitive phage transcripts, starting 10-15 min after infection. The decay of the transcripts is the probable cause of the arrest of the sensitive phage development. Mapping of the 5' end of degradation products established that they result from endonucleolytic cleavage preferentially at U/U, A/U and U/A sites. We propose that an early product of the sensitive phage either induces the synthesis or stimulates the activity of an RNase in an AbiB+ cell.

Bacterial Proteins↗
PubMed1996

Theta-type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome.

To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of approximately 4 kb. The replicon was derived from a broad-host-range plasmid, pAM beta 1, which replicates by a unidirectional theta mechanism and its thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and approximately 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.

Bacillus subtilis↗
PubMed1996

Bacillus subtilis can modulate its capacity and specificity for protein secretion through temporally controlled expression of the sipS gene for signal peptidase I.

Bacillus subtilis contains three chromosomally encoded type I signal peptidases (SipS, SipT and SipU), which remove signal peptides from secretory precursor proteins. In the present study the biological function of SipS and the regulation of its synthesis were analysed. Unlike the type I signal peptidase of Escherichia coli, SipS was essential neither for protein secretion nor viability of the cell. However, in the absence of SipS the rate of processing of several preproteins was reduced, and four of the seven major secreted proteins of B. subtilis were hardly detectable in the growth medium. Surprisingly, the processing of Bacillus amyloliquefaciens alpha-amylase and the secretion of at least two endogenous B. subtilis proteins was improved in the absence of SipS. These findings indicate that the substrate preference of SipS differs from that of SipT and SipU, and that SipS is an important factor determining the efficiency of protein secretion in B. subtilis. SipS is transcribed in a growth phase- and medium-dependent manner. In minimal medium, the growth phase-dependent transcription of sipS is controlled by the DegS-DegU two-component regulatory system, indicating that the expression of sipS is regulated by the same factors that control the expression of most genes for secreted degradative enzymes. Our observations suggest that B. subtilis can modulate its capacity and specificity for protein secretion through the controlled expression of sipS.

Bacillus subtilis↗
PubMed1996

Organization of the Bacillus subtilis 168 chromosome between kdg and the attachment site of the SP beta prophage: use of Long Accurate PCR and yeast artificial chromosomes for sequencing.

Within the Bacillus subtilis genome sequencing project, the region between lysA and ilvA was assigned to our laboratory. In this report we present the sequence of the last 36 kb of this region, between the kdg operon and the attachment site of the SP beta prophage. A two-step strategy was used for the sequencing. In the first step, total chromosomal DNA was cloned in phage M13-based vectors and the clones carrying inserts from the target region were identified by hybridization with a cognate yeast artificial chromosome (YAC) from our collection. Sequencing of the clones allowed us to establish a number of contigs. In the second step the contigs were mapped by Long Accurate (LA) PCR and the remaining gaps closed by sequencing of the PCR products. The level of sequence inaccuracy due to LA PCR errors appeared to be about 1 in 10,000, which does not affect significantly the final sequence quality. This two-step strategy is efficient and we suggest that it can be applied to sequencing of longer chromosomal regions. The 36 kb sequence contains 38 coding sequences (CDSs), 19 of which encode unknown proteins. Seven genetic loci already mapped in this region, xpt, metB, ilvA, ilvD, thyB, dfrA and degR were identified. Eleven CDSs were found to display significant similarities to known proteins from the data banks, suggesting possible functions for some of the novel genes: cspD may encode a cold shock protein; bcsA, the first bacterial homologue of chalcone synthase; exol, a 5' to 3' exonuclease, similar to that of DNA polymerase I of Escherichia coli; and bsaA, a stress-response-associated protein. The protein encoded by yplP has homology with the transcriptional NifA-like regulators. The arrangement of the genes relative to possible promoters and terminators suggests 19 potential transcription units.

Amino Acid Sequence↗
PubMed1996

Mapping of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome using Long Accurate PCR and three yeast artificial chromosomes.

We constructed a PCR map of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome. It was established using known sequences of the spoIIIC, blt, aadK, sacC, spoVB and pheA loci and eight random sequence tags. The tags were generated using PFGE-purified DNA of yeast artificial chromosome (YAC) 11-17 from the yeast clone which carries the major part of this region. The ends of two other YACs were positioned on the map using total DNA extracted from yeast cells carrying them. The procedure allowed the placement of precisely known and new (putative) genes on the physical chromosome map and the generation of sufficient amounts of DNA for sequencing this region. Apart from allowing correction of the genetic map in this region, these results demonstrate how a collection of long segments of bacterial chromosome and Long Accurate PCR can be used for reliable high-resolution physical mapping of an extended chromosome area.

Bacillus subtilis↗
PubMed1996

Sequence analysis of the Bacillus subtilis chromosome region between the serA and kdg loci cloned in a yeast artificial chromosome.

The standard strategies of genome sequencing based on lambda-vector or cosmid libraries are only partially applicable to AT-rich Gram-positive bacteria because of the problem of instability of their chromosomal DNA in heterologous hosts like Escherichia coli. One complete collection of ordered clones known for such bacteria is that of Bacillus subtilis, established by using yeast artificial chromosomes (YACs). This paper reports the results of the direct use of one of the YAC clones from the above collection for the sequencing of the region cloned in it. The strategy applied consisted of the following: (i) construction of M13 banks of the partially purified YAC DNA and sequencing of 800 M13 clones chosen at random; (ii) directed selection of M13 clones to sequence by using marginal contig fragments as hybridization probes; (iii) direct sequencing of joining PCR fragments obtained by combinations of primers corresponding to the ends of representative contigs. The complete 104,109 bp insert sequence of this YAC clone was thus established. The strategy used allowed us to avoid resequencing the two largest, previously sequenced, contigs (13,695 and 20,303 bp) of the YAC insert. We propose that the strategy used can be applied to the sequencing of the whole bacterial genome without intermediate cloning, as well as for larger inserts of eukaryotic origin cloned in YACs. Sequencing of the insert of the YAC clone 15-6B allowed us to establish the contiguous sequence of 127 kb from spollA to kdg. The organization of the newly determined region is presented. Of the 138 ORFs identified in the spollA-kdg region, 57 have no clear putative function from their homology to proteins in the databases.

Amino Acid Sequence↗
PubMed1996

A new approach using multiplex long accurate PCR and yeast artificial chromosomes for bacterial chromosome mapping and sequencing.

An efficient approach for structural studies on bacterial chromosomes is presented. It is based on high-resolution PCR map construction by using a multiplex long accurate PCR (MLA PCR) protocol and a YAC clone carrying the region to be mapped as indicator. The high-resolution PCR map of the bacillus subtilis rrnB-dnaB region is presented as an example. Data are also presented on the use of DNA generated by LA PCR for sequencing; they are relevant to LA PCR induced mutations and justify the application of such mapping for sequencing long stretches of bacterial chromosomes.

Bacillus subtilis↗