Search PubMed⌕ Search

Biomedical subjects

S D Ehrlich

Publications and source records attributed to S D Ehrlich.

At least 73 records · Page 4Linked to original sources
PubMed1998

A five-nucleotide sequence protects DNA from exonucleolytic degradation by AddAB, the RecBCD analogue of Bacillus subtilis.

Homologous recombination in Bacillus subtilis requires the product of the addA and addB genes, the AddAB enzyme. This enzyme, which is both a helicase and a powerful nuclease, is thought to be the counterpart of the Escherichia coli RecBCD enzyme. From this analogy, it is expected that the nuclease activity of AddAB can be downregulated by a specific DNA sequence, which would correspond to the chi site in E. coli. Using protection of linear double-stranded DNA as a criterion, we identified the five-nucleotide sequence 5'-AGCGG-3', or its complement 5'-CCGCT-3', as being sufficient for AddAB nuclease attenuation. We have shown further that this attenuation occurs only if the sequence is properly oriented with respect to the translocating AddAB enzyme. Finally, inspection of the complete B. subtilis genome revealed that this five-nucleotide sequence is over-represented and is, in a majority of cases, co-oriented with DNA replication. Based on these observations, we propose that 5'-AGCGG-3', or its complement, is the B. subtilis analogue of the E. coli chi sequence.

Bacillus subtilis↗
PubMed1998

Transcription-driven DNA replication of plasmid pAMbeta1 in Bacillus subtilis.

pAMbeta1 is a plasmid isolated from Enterococcus faecalis which replicates in Bacillus subtilis by a unidirectional theta mechanism. It has been shown previously that initiation of pAMbeta1 replication requires a plasmid-encoded protein (RepE) and a short origin and is carried out by the host DNA polymerase I. It is not known which primer is used by this polymerase for initiating replication. Here, we report that a transcription fork passing through the origin is a limiting factor for plasmid replication. Transcription that activates the origin is initiated at the repE promoter and is thus regulated by the plasmid copy-number control system. Two lines of evidence suggest that the transcription generates the primer for the DNA polymerase I. First, the transcription must start upstream from the origin and progress in the direction of replication to be effective. Second, 3' ends of RNA transcripts initiated upstream of the origin map within the origin, provided that the Rep protein and an intact origin are present. This is the first report for simultaneous requirement of a transcription fork, a replication protein and the DNA polymerase I in initiation of DNA replication.

3' Untranslated Regions↗
PubMed1998

Use of luciferase genes as biosensors to study bacterial physiology in the digestive tract.

A method based on the use of the bacterial luciferase genes was developed in order to investigate Lactococcus lactis gene expression in the mouse digestive tract. Germfree mice were monoassociated with different strains containing transcriptional fusions of promoters with the luciferase genes. Our results demonstrate that this method is readily applicable to the study of promoter strength and physiology of bacteria in the digestive tract.

Animals↗
PubMed1998

Multiple transcriptional control of the Lactococcus lactis trp operon.

The Lactococcus lactis trpEGDCFBA operon is preceded by a noncoding leader region. Transcriptional studies of the trp operon revealed three transcripts with respective sizes of 8 kb (encompassing the entire operon), 290 bases, and 160 bases (corresponding to parts of the leader region). These transcripts most likely result from initiation at the unique Ptrp promoter, transcription termination at either T1 (upstream of the trp operon) or T2 (downstream of the trp operon), and/or processing. Three parameters were shown to differentially affect the amount of these transcripts: (i) following tryptophan depletion, the amount of the 8-kb transcript increases 300- to 500-fold; (ii) depletion in any amino acid increased transcription initiation about fourfold; and (iii) upon entry into stationary phase the amount of the 8-kb transcript decreases abruptly. The tryptophan-dependent transcription control is exerted through transcription antitermination.

Base Sequence↗
PubMed1998

A type IC restriction-modification system in Lactococcus lactis.

Three genes coding for the endonuclease, methylase, and specificity subunits of a type I restriction-modification (R-M) system in the Lactococcus lactis plasmid pIL2614 have been characterized. Plasmid location, sequence homologies, and inactivation studies indicated that this R-M system is most probably of type IC.

Amino Acid Sequence↗
PubMed1998

A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis.

Lactococcus lactis, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins. We examined the secretion efficiency and capacity of L. lactis by using the Staphylococcus aureus nuclease (Nuc) as a heterologous model protein. When expressed in L. lactis from an efficient lactococcal promoter and its native signal peptide, only approximately 60% of total Nuc was present in a secreted form at approximately 5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency was reduced further to approximately 30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). We identified a modification which improved secretion efficiency of both native Nuc and NucT. A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site. In the case of Nuc, secretion efficiency was increased to approximately 80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to approximately 20 mg per liter). The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90%. Similarly, the secretion efficiency of a third protein, the alpha-amylase of Bacillus stearothermophilus, was also improved by LEISSTCDA. These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in L. lactis.

Amino Acid Sequence↗
PubMed1998

An export-specific reporter designed for gram-positive bacteria: application to Lactococcus lactis.

The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of the Staphylococcus aureus secreted nuclease (Nuc) as a reporter for gram-positive bacteria. Nuc devoid of its export signal (called delta(SP)Nuc) was used to create two fusions whose locations could be differentiated. Nuclease activity was shown to require an extracellular location in Lactococcus lactis, thus demonstrating the suitability of delta(SP)Nuc to report protein export. The shuttle vector pFUN was designed to construct delta(SP)Nuc translational fusions whose expression signals are provided by inserted DNA. The capacity of delta(SP)Nuc to reveal and identify exported proteins was tested by generating an L. lactis genomic library in pFUN and by screening for Nuc activity directly in L. lactis. All delta(SP)Nuc fusions displaying a strong Nuc+ phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches. The function of some of the predicted signals was confirmed by cell fractionation studies. The fusions analyzed included long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in L. lactis. Homology searches indicate that several of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing, and protein folding. Our results with L. lactis show that delta(SP)Nuc is well suited to report both protein export and membrane protein topology.

Amino Acid Sequence↗
PubMed1997

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Bacillus subtilis↗
PubMed1997

Recombination-dependent repair of DNA double-strand breaks with purified proteins from Escherichia coli.

We have developed an in vitro system in which repair of DNA double-strand breaks is performed by purified proteins of Escherichia coli. A segment was deleted from a circular duplex DNA molecule by restriction at two sites. 3' single-stranded overhangs were introduced at both ends of the remaining linear fragment. In a first step, RecA protein catalyzed the formation of a D-loop between one single-stranded tail and a homologous undeleted supercoiled DNA molecule. In a second step, E. coli DNA polymerase II or III used the 3' end in the D-loop as a primer to copy the missing sequences of the linear substrate on one strand of the supercoiled template. Under proper conditions, the integrity of the deleted substrate was restored, as shown by analysis of the products by electrophoresis, restriction, and transformation. In this reaction, DNA synthesis is strictly dependent on recombination, and repair is achieved without formation of a Holliday junction.

Bacterial Proteins↗
PubMed1997

Deletions at stalled replication forks occur by two different pathways.

Replication blockage induces non-homologous deletions in Escherichia coli. The mechanism of the formation of these deletions was investigated. A pBR322-mini-oriC hybrid plasmid carrying two E. coli replication terminators (Ter sites) in opposite orientations was used. Deletions which remove at least the pBR322 blocking site (named Ter1) occurred at a frequency of 2 x 10(-6) per generation. They fall into two equally large classes: deletions that join sequences with no homology, and others that join sequences of 3-10 bp of homology. Some 95% of the deletions in the former class resulted from the fusion of sequences immediately preceding the two Ter sites, indicating a direct role for blocked replication forks in their formation. These deletions were not found in a topA10 mutant, suggesting a topoisomerase I-mediated process. In contrast, deletions joining short homologous sequences were not affected by the topA10 mutation. However, the incidence of this second class of deletions increased 10-fold in a recD mutant, devoid of exonuclease V activity. This indicates that linear molecules are intermediates in their formation. In addition, approximately 50% of these deletions were clustered in the region flanking the Ter1 site. We propose that they are produced by repair of molecules broken at the blocked replication forks.

Base Sequence↗
PubMed1997

DNA double-strand breaks caused by replication arrest.

We report here that DNA double-strand breaks (DSBs) form in Escherichia coli upon arrest of replication forks due to a defect in, or the inhibition of, replicative DNA helicases. The formation of DSBs was assessed by the appearance of linear DNA detected by pulse-field gel electrophoresis. Processing of DSBs by recombination repair or linear DNA degradation was abolished by mutations in recBCD genes. Two E. coli replicative helicases were tested, Rep, which is essential in recBC mutants, and DnaB. The proportion of linear DNA increased up to 50% upon shift of rep recBTS recCTS cells to restrictive temperature. No increase in linear DNA was observed in the absence of replicating chromosomes, indicating that the formation of DSBs in rep strains requires replication. Inhibition of the DnaB helicase either by a strong replication terminator or by a dnaBTS mutation led to the formation of linear DNA, showing that blocked replication forks are prone to DSB formation. In wild-type E. coli, linear DNA was detected in the absence of RecBC or of both RecA and RecD. This reveals the existence of a significant amount of spontaneous DSBs. We propose that some of them may also result from the impairment of replication fork progression.

Bacterial Proteins↗
PubMed1997

Isolation of a dnaE mutation which enhances RecA-independent homologous recombination in the Escherichia coli chromosome.

The mechanism of recombination of tandem repeats in the chromosome of Escherichia coli was investigated by genetic means. Tandem repeats 624 bp long were introduced into the lacZ gene of E. coli and the efficiency of deletion of one repeat was compared in different recombination mutants. No effects of the recA, recBC, recF, ruvA or ruvA recG mutations were detected. Hence, tandem repeat deletion appears to not proceed via the RecBCD or RecF homologous recombination pathways. A new mutant in which RecA-independent recombination is increased 15-fold was isolated. The mutation lies in the dnaE gene coding for the alpha subunit of polymerase III: it is a Gly to Asp change at codon 133. Another dnaE mutation, dnaE486, was tested and also shown to stimulate RecA-independent recombination. It is proposed that tandem-repeat recombination occurs by a replication slippage mechanism. RecA-independent recombination is also enhanced in a rep mutant, in which chromosomal replication is slowed down by the absence of the Rep helicase, suggesting that replication pausing may facilitate slippage.

Chromosomes, Bacterial↗
PubMed1997

uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway.

It has previously been shown that recombination between tandem repeats is not significantly affected by a recA mutation in Escherichia coli. Here, we describe the activation of a RecA-dependent recombination pathway in a hyper-recombination mutant. In order to analyse how tandem repeat deletion may proceed, we searched for mutants that affect this process. Three hyper-recombination clones were characterized and shown to be mutated in the uvrD gene. Two of the mutations were identified as opal mutations at codons 130 and 438. A uvrD::Tn5 mutation was used to investigate the mechanism of deletion formation in these mutants. The uvrD-mediated stimulation of deletion was abolished by a lexAind3 mutation or by inactivation of either the recA, recF, recQ or ruvA genes. We conclude that (i) this stimulation requires SOS induction and (ii) tandem repeat recombination in uvrD mutants occurs via the RecF pathway. In uvrD+ cells, constitutive expression of SOS genes is not sufficient to stimulate deletion formation. This suggests that the RecF recombination pathway activated by SOS induction is antagonized by the UvrD protein. Paradoxically, we observed that the overproduction of UvrD from a plasmid also stimulates tandem repeat deletion. However, this stimulation is RecA independent, as is deletion in a wild-type strain. We propose that the presence of an excess of the UvrD helicase favours replication slippage. This work suggests that the UvrD helicase controls a balance between different routes of tandem repeat deletion.

Adenosine Triphosphatases↗
PubMed1997

Blocking rolling circle replication with a UV lesion creates a deletion hotspot.

UV light irradiation increases genetic instability by causing mutations and deletions. The mechanism of UV-induced rearrangements was investigated making use of deletion-prone plasmids. Chimeric plasmids carrying pBR322 and M13 replication origins undergo deletions that join the M13 replication origin to a random nucleotide. A restriction fragment was UV irradiated, introduced into such a hybrid plasmid and deletions formed at the M13 origin were analysed. In most of the deletant molecules, the M13 replication nick site was linked to a nucleotide in the irradiated fragment, showing that UV lesions are deletion hotspots. These deletions were independent of the UvrABC excision repair proteins, suggesting that the deletogenic structure is the lesion itself and not a repair intermediate. They were not found in the absence of M13 replication, indicating that they result from the encounter of the M13 replication fork with the UV lesion. Furthermore, UV-induced deletions occurred independently of pBR322 replication. We conclude that, in contrast to pBR322 replication forks, M13 replication forks blocked by UV lesions are deletion prone. We propose that the deletion-prone properties of a UV-arrested polymerase depend on the associated helicase.

Adenosine Triphosphatases↗
PubMed1997

Replication terminus for DNA polymerase I during initiation of pAM beta 1 replication: role of the plasmid-encoded resolution system.

Replication of plasmid pAM beta 1 is initiated by DNA polymerase I (Pol I) and completed by DNA polymerase III holoenzyme contained in the replisome machinery. In this study we report that initiation of DNA replication generates D-loop structures containing the nascent leading strand paired to its template, and that D-loop extension is arrested approximately 230 bp from the initiation site of DNA synthesis in the presence of the plasmid-encoded resolvase. In vitro and in vivo data suggest that this arrest is caused by a collision between Pol I and the resolvase bound to its target. As the arrested D-loop replication intermediates carry a single-stranded primosome-assembly site, we hypothesize that the biological role of the replication arrest is to limit the region replicated by Pol I and to promote the replacement of Pol I by the replisome in order to initiate concerted synthesis of the leading and lagging strands.

Bacillus subtilis↗
PubMed1997

Molecular cloning and analysis of the ptsHI operon in Lactobacillus sake.

The ptsH and ptsI genes of Lactobacillus sake, encoding the general enzymes of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), were cloned and sequenced. HPr (88 amino acids), encoded by ptsH, and enzyme I (574 amino acids), encoded by ptsI, are homologous to the corresponding known enzymes of other bacteria. Nucleotide sequence and mRNA analysis showed that the two genes are cotranscribed in a large transcript encoding both HPr and enzyme I. The transcription of ptsHI was shown to be independent of the carbon source. Four ptsI mutants were constructed by single-crossover recombination. For all mutants, growth on PTS carbohydrates was abolished. Surprisingly, the growth rates of mutants on ribose and arabinose, two carbohydrates which are not transported by the PTS, were accelerated. This unexpected phenotype suggests that the PTS negatively controls ribose and arabinose utilization in L. sake by a mechanism different from the regulation involving HPr described for other gram-positive bacteria.

Amino Acid Sequence↗