Biomedical subjects

S D Ehrlich

Publications and source records attributed to S D Ehrlich.

At least 145 records · Page 8Linked to original sources

PubMed1993

A fourth class of theta-replicating plasmids: the pAM beta 1 family from gram-positive bacteria.

Plasmid pAM beta 1 from Enterococcus faecalis uses a unidirectional theta mode of replication. We show here that this replication (i) is dependent on a plasmid-encoded replication protein (Rep) but not on a DNA structure typical for origins of most Rep-dependent plasmids and (ii) is initiated by DNA polymerase I (PolI). pAM beta 1 minimal replicon shares no homology with highly conserved ColE1-type replicons, which use PolI for initiation but do not encode a Rep, or with ColE2 and ColE3 replicons, which require PolI for replication and encode a Rep. We propose that pAM beta 1 and a number of other naturally occurring and closely related plasmids from a distinct plasmid class.

Base Sequence↗

PubMed1993

Antipairing and strand transferase activities of E. coli helicase II (UvrD).

The product of the uvrD gene of Escherichia coli, UvrD (helicase II), is known to be involved in methyl-directed mismatch repair, transposon excision and uvrABC excision repair. In conjugational crosses, various uvrD mutants have been reported to result in higher, lower or unaffected recombination frequencies. In an attempt to clarify the role of UvrD in recombination, we have studied in vitro its effects on two key reactions driven by RecA, homologous pairing and strand exchange. We show here that UvrD efficiently prevents or reverses RecA-mediated homologous pairing. Unexpectedly, we also found that it can stimulate RecA-driven branch migration and even catalyze strand exchange in the absence of RecA. A possible in vivo role for these antagonistic activities is discussed.

Adenosine Triphosphatases↗

PubMed1993

An ordered collection of Bacillus subtilis DNA segments cloned in yeast artificial chromosomes.

A collection of 772 Bacillus subtilis DNA segments was obtained by cloning in yeast artificial chromosomes. The B. subtilis inserts of 288 clones were mapped by hybridization using as probes 65 cloned genes and 188 isolated insert ends. In this way, 59 inserts were ordered in four contigs that cover > 98% of the B. subtilis chromosome. This ordered collection is now available for further genetic and physical analysis of the B. subtilis genome.

Bacillus subtilis↗

PubMed1993

Biochemical and genetic analysis of the unidirectional theta replication of the S. agalactiae plasmid pIP501.

pIP501 is a broad-host-range plasmid originating from Streptococcus agalactiae. In this report we show that (i) it replicates by a theta mechanism initiating at the 3' end of the gene encoding the replication protein RepR and progressing in the direction of transcription of this gene; (ii) its replication origin lies within or a few nucleotides downstream from ORF R and not upstream from it as suggested in the literature (Brantl et al. (1990) Nucleic Acids Res. 18, 4783-4789); (iii) the RepR protein positively regulates pIP501 copy number; and (vi) the main function ensured by the sequences located upstream from ORF R is to express this ORF. Since the replication properties of pIP501 are indistinguishable from those of the highly related Enterococcus faecalis plasmid pAM beta 1, we conclude that these elements form the first family of theta replicating plasmids in gram-positive bacteria. Based on sequence similarities, we extend this family to the S. pyogenes plasmid pSM19035 and to 12 other plasmids isolated from streptococci or enterococci.

Bacterial Proteins↗

PubMed1993

The organization of the Bacillus subtilis 168 chromosome region between the spoVA and serA genetic loci, based on sequence data.

Three different lambda phage clones with overlapping inserts of Bacillus subtilis DNA, which cover the region from spoIIAA to serA, have been isolated. The nucleotide sequence of their inserts, starting after spoVAF and ending at serA, has been determined. A contiguous sequence of 35,354 bp was established, including previously analysed overlapping adjacent regions. Within the newly determined sequence 31 open reading frames (ORFs) with putative ribosome-binding sites have been found. Nine of them correspond to previously sequenced and characterized genes: spo-VAF, lysA, sipS, ribG, ribB, ribA, ribH, ribTD and dacB. Comparison of the amino acid sequences of the products encoded by the other ORFs to known proteins allowed putative functions to be assigned to seven of these ORFs. Among these are the following: (i) the ppiB gene, encoding a cytoplasmic peptidylprolyl isomerase; (ii) two pairs of signal-transducers, one homologous to phoR-phoP of B. subtilis, encoding regulators of phosphatase biosynthesis, and the second to the fecI-fecR of Escherichia coli, which is responsible for the regulation of the citrate-dependent iron (III) transport system; (iii) aroC and serA genes, involved in the biosynthesis of aromatic amino acids and serine, respectively, the function of which has been confirmed by constructing corresponding mutants with disrupted ORFs. The organization of putative operons has been postulated on the basis of the sequences of their transcription terminators, promoters and regulatory elements.

Amino Acid Sequence↗

PubMed1993

The transcriptional organization of the Bacillus subtilis 168 chromosome region between the spoVAF and serA genetic loci.

The genetic organization of the spoVAF-serA area of the Bacillus subtilis chromosome and its putative transcription map have been derived from analysis of the nucleotide sequence. In order to confirm this transcription map as regards size of transcripts and to determine growth conditions for their appearance, we undertook Northern hybridization analysis of total RNA from vegetatively growing and sporulating cells. Twenty-three distinct transcripts were thus identified, 14 of which were predicted from sequence analysis and nine of which were not predicted. Eight of the latter are homologous to open reading frames identified by sequence analysis but were not expected, since no obvious promoter or terminator was found in the sequence. The last unexpected transcript does not correspond to an ORF and might identify a novel gene. Three predicted transcripts were not detected. The transcripts were classified in four groups as (i) constitutive, (ii) regulated by nutritional depletion, (iii) specific for sporulation, and (iv) possibly regulated temporally. These studies demonstrate that systematic Northern analysis of a bacterial chromosome region is a useful complement to sequence analysis.

Bacillus subtilis↗

PubMed1993

High-efficiency gene inactivation and replacement system for gram-positive bacteria.

A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.

DNA Replication↗

PubMed1993

Gene inactivation in Lactococcus lactis: branched-chain amino acid biosynthesis.

The Lactococcus lactis subsp. lactis strains isolated from dairy products are auxotrophs for branched-chain amino acids (leucine, isoleucine, and valine), while most strains isolated from nondairy media are prototrophs. We have cloned and sequenced the leu genes from one auxotroph, IL1403. The sequence is 99% homologous to that of the prototroph NCDO2118, which was determined previously. Two nonsense mutations and two small deletions were found in the auxotroph sequence, which might explain the branched-chain amino acid auxotrophy. Nevertheless, the leu genes from the auxotroph appear to be transcribed and regulated similarly to those from the prototroph.

Amino Acid Sequence↗

PubMed1993

Gene inactivation in Lactococcus lactis: histidine biosynthesis.

Lactococcus lactis strains from dairy and nondairy sources were tested for the ability to grow in the absence of histidine. Among 60 dairy strains tested, 56 required histidine, whereas only 1 of 11 nondairy strains had this requirement. Moreover, 10 of the 56 auxotrophic strains were able to grow in the presence of histidinol (Hol+), the immediate histidine precursor. This indicates that adaptation to milk often results in histidine auxotrophy. The histidine operon was detected by Southern hybridization in eight dairy auxotrophic strains tested. A large part of the histidine operon (8 kb, containing seven histidine biosynthetic genes and three unrelated open reading frames [ORFs]) was cloned from an auxotroph, which had an inactive hisD gene, as judged by its inability to grow on histidinol. Complementation analysis of three genes, hisA, hisB, and hisG, in Escherichia coli showed that they also were inactive. Sequence analysis of the cloned histidine region, which revealed 98.6% overall homology with that of the previously analyzed prototrophic strain, showed the presence of frameshift mutations in three his genes, hisC, hisG, and hisH, and two genes unrelated to histidine biosynthesis, ORF3 and ORF6. In addition, several mutations were detected in the promoter region of the operon. Northern (RNA) hybridization analysis showed a much lower amount of the his transcript in the auxotrophic strain than in the prototrophic strain. The mutations detected account for the histidine auxotrophy of the analyzed strain. Certain other dairy auxotrophic strains carry a lower number of mutations, since they were able to revert either to a Hol+ phenotype or to histidine prototrophy.

Amino Acid Sequence↗

PubMed1993

Mutation in the plasmid pUB110 Rep protein affects termination of rolling circle replication.

We isolated a mutant of plasmid pUB110 that has the following properties in Bacillus subtilis: (i) it is toxic for recA and add cells, particularly at elevated temperature; (ii) it has a copy number threefold higher than that of the parental plasmid, and the extra copies are present as multimers; and (iii) it can efficiently complement replication of a cmp- satellite plasmid, despite being cmp+. All these properties are due to a single change in the plasmid replication protein, i.e., Gly at position 148 to Glu. These properties of the mutant Rep protein reflect a diminished ability to terminate rolling circle replication. We propose that the Rep protein may have a diminished affinity for the plasmid origin; alternatively, it may be impaired for recognition of the plasmid conformations which distinguish initiation and termination.

Amino Acid Sequence↗

PubMed1993

Efficient plasmid mobilization by pIP501 in Lactococcus lactis subsp. lactis.

pIP501 is a streptococcal conjugative plasmid which can be transmitted among numerous gram-positive strains. To identify a minimal mobilization (mob) locus of pIP501, DNA fragments of pIP501 were cloned into nonconjugative target plasmids and tested for mobilization by pIP501. We show that nonmobilizable plasmids containing a specific fragment of pIP501 are transmitted at high frequencies between Lactococcus lactis subsp. lactis strains if transfer (tra) functions are provided in trans by a pIP501 derivative. Independent transfer of the mobilized plasmid was observed in up to 44% of transconjugants. A 2.2-kb segment containing mob was sequenced. This DNA segment is characterized by three palindromes (palI, palII, and palIII) and a 202-amino-acid open reading frame (ORFX) of unknown function. The smallest DNA fragment conferring high frequency mobilization was localized to a 1.0-kb region (extending from pIP501 coordinates 3.60 to 4.60 on the 30.2-kb map) which contains palI (delta G = -27 kcal/mol [ca. -110,000 J/mol]). A 26-bp sequence identical to palI is present on pIP501, upstream of the plasmid copy control region. Further homologies with the palI sequence are also found with the related Enterococcus faecalis conjugative plasmid pAM beta 1. The region containing mob maps outside the previously described segment mediating pIP501 conjugation. Our results with recA strains indicate that the mob site is a hot spot for cointegrate formation.

Amino Acid Sequence↗

PubMed1992

Chi sequence protects against RecBCD degradation of DNA in vivo.

RecBCD is a multifunctional enzyme involved in DNA degradation and homologous recombination. It also produces an endonucleolytic cleavage near properly oriented chi sites (5'-GCTGGTGG-3'). Plasmids are not known to be affected by either RecBCD enzyme or the presence of a chi site. We report here that plasmids that replicate by a rolling circle mechanism accumulate large amounts of high molecular weight linear multimers (HMW), either if they contain a chi site or if RecBCD is absent. An in vivo inducible system for rolling circle replication was constructed to study RecBCD and its interactions with chi. Results show that (i) HMW accumulation is chi orientation dependent, and (ii) a succession of chi sites prevents degradation of HMW by RecBCD enzyme. These results demonstrate chi activity in plasmids. The rolling circle mechanism produces a sigma structure during plasmid replication; we propose that the double-stranded DNA tail of this sigma form allows RecBCD entry; the tail is degraded unless it is protected by a chi site. By analogy, a principal role of chi in the survival of lambda red-gam- mutants in wild-type strains may be to protect rolling circle concatemers (in late replication) from degradation by RecBCD.

Base Sequence↗

PubMed1992

Induction of DNA amplification in the Bacillus subtilis chromosome.

A system allowing the induction of DNA amplification in Bacillus subtilis was developed, based on a thermosensitive plasmid, pE194, stably integrated in the bacterial chromosome. An amplification unit, comprising an antibiotic resistance marker flanked by directly repeated sequences, was placed next to the integrated plasmid. Activation of pE194 replication led to DNA amplification. Two different amplification processes appeared to take place: one increased the copy number of all sequences in the vicinity of the integrated plasmid and was possibly of the onion skin type, while the other increased the copy number of the amplification unit only and generated long arrays of amplification units. These arrays were purified and shown to consist mainly of directly repeated amplification units but to also contain non-linear regions, such as replication forks and recombination intermediates. They were attached to the chromosome at one end only, and were, in general, not stably inherited, which suggests that they are early amplification intermediates. Longer arrays were detected before the shorter ones during amplification. When the parental amplification unit contained repeats which differed by a restriction site the arrays which derived thereof contained in a majority of cases only a single type of repeat. We propose that the amplified DNA is generated by rolling circle replication, and that such a process might underlie a number of amplification events.

Bacillus subtilis↗

PubMed1992

DNA transcription and repressor binding affect deletion formation in Escherichia coli plasmids.

Chimeric plasmids containing phage M13 and plasmid pBR322 sequences undergo deletions in Escherichia coli with a high frequency. In all plasmids one deletion endpoint is the M13 replication origin nick site. We examined the effects of transcription on the position of the other deletion end-point, by inserting in the plasmids an inducible promoter followed by a transcription terminator. Transcription dramatically affected deletions in an orientation-dependent way, such that greater than 95% of end-points were localized downstream from the inserted promoter when it faced the major plasmid transcripts. The end-points were not constrained to the transcribed region and were not affected by the orientation of pBR322 DNA replication. We propose that deletion events occur preferentially in a plasmid domain which is rendered positively supercoiled by convergent transcription. We also show that interaction of LacI repressor with the cognate operator generates a localized deletion hot spot. This hot spot is dependent on pBR322 replication, and therefore probably acts by arresting progression of DNA replication.

Bacteriophage M13↗

PubMed1992

Divergence of Genomic Sequences between Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris.

Relatedness between Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis was assessed by Southern hybridization analysis, with cloned chromosomal genes as probes. The results indicate that strains of the two subspecies form two distinct groups and that the DNA sequence divergence between L. lactis subsp. lactis and L. lactis subsp. cremoris is estimated to be between 20 and 30%. The previously used phenotypic criteria do not fully discriminate between the groups; therefore, we propose a new classification which is based on DNA homology. In agreement with this revised classification, the L. lactis subsp. lactis and L. lactis subsp. cremoris strains from our collection have distinct phage sensitivities.

Journal Article↗

PubMed1992

Use of degenerate primers for polymerase chain reaction cloning and sequencing of the Lactococcus lactis subsp. lactis recA gene.

Two particularly well-conserved stretches in the RecA protein sequences were chosen as templates to synthesize degenerate oligonucleotides, which were used in polymerase chain reaction to amplify an internal recA DNA fragment of Lactococcus lactis subsp. lactis ML3. Using this fragment, we recovered and sequenced the entire lactococcal recA gene. The end of an open reading frame present upstream of the recA gene shows strong homology with formamidopyrimidine-DNA-glycosylase, a protein involved in DNA repair.

Amino Acid Sequence↗

PubMed1992

High-molecular-weight linear multimer formation by single-stranded DNA plasmids in Escherichia coli.

We inserted foreign DNA segments into plasmids which replicate by a rolling-circle mechanism in Escherichia coli and observed the appearance of high-molecular-weight plasmid multimers (HMW). This phenomenon, which occurs more frequently with GC-rich segments, depends on the mode of replication of the plasmid and on host homologous recombination functions. We found that (i) HMW are formed upon insertion of a foreign DNA segment into a single-stranded DNA plasmid, whereas the same DNA insert has no such effect on a theta replicon, and (ii) HMW are not present in a recA mutant strain but are found in a lexA (Ind-) mutant. Enzymatic studies allowed us to define the HMW structure as linear double-stranded tandem head-to-tail plasmid repeats. Use of heteroplasmid strains showed that HMW production by one plasmid does not affect another resident plasmid, indicating that no host functions are phenotypically inactivated. This distinguishes our system from the HMW observed with various replicons in the absence of RecBCD enzyme activity. We propose that the role of the foreign insert is to protect the DNA from RecBCD exonuclease attack.

DNA Mutational Analysis↗

PubMed1992

A general method for cloning recA genes of gram-positive bacteria by polymerase chain reaction.

An internal fragment of the recA gene from eight gram-positive organisms has been amplified by using degenerate primers in a polymerase chain reaction. The internal 348- or 360-bp recA DNA segments from Bacillus subtilis, Clostridium acetobutylicum, Lactobacillus bulgaricus, Lactobacillus helveticus, Leuconostoc mesanteroides, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus salivarus subsp. thermophilus were amplified, cloned, and sequenced. The G + C contents of the DNA from these species range from 28 to 52%. The sequences of the bacterial recA genes show strong relatedness. This method is particularly useful for the recovery of the recA genes of gram-positive bacteria and avoids the difficulties of using a genetic complementation test for cloning.

Amino Acid Sequence↗