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S Dusko Ehrlich

Publications and source records attributed to S Dusko Ehrlich.

54 records · Page 3Linked to original sources
PubMed2002

Replication fork reversal in DNA polymerase III mutants of Escherichia coli: a role for the beta clamp.

Certain replication mutations lead in Escherichia coli to a specific reaction named replication fork reversal: at blocked forks, annealing of the nascent strands and pairing of the template strands form a four-way junction. RuvABC-catalysed resolution of this Holliday junction causes chromosome double-strand breaks (DSBs) in a recBC context and therefore creates a requirement for the recombination proteins RecBC for viability. In the present work, two mutants were tested for replication fork reversal: a dnaEts mutant and a dnaNts mutant, affected in the alpha (polymerase) and beta (processivity clamp) subunits of DNA polymerase III holoenzyme respectively. In the dnaEts recB strain, RuvABC-dependent DSBs caused by the dnaEts mutation occurred at 37 degrees C or 42 degrees C, indicating the occurrence of replication fork reversal upon partial or complete inactivation of the DNA polymerase alpha subunit. DSB formation was independent of RecA, RecQ and the helicase function of PriA. In the dnaNts recB mutant, RuvABC-dependent DSB caused by the dnaNts mutation occurred only at semi-permissive temperature, 37 degrees C, indicating the occurrence of replication fork reversal in conditions in which the remaining activity of the beta clamp is sufficient for viability. In contrast, the dnaNts mutation did not cause chromosome breakage at 42 degrees C, a temperature at which DnaN is totally inactive and the dnaNts mutant is inviable. We propose that a residual activity of the DNA polymerase III beta clamp is required for replication fork reversal in the dnaNts mutant.

Bacterial Proteins↗
PubMed2002

Discovery of two novel families of proteins that are proposed to interact with prokaryotic SMC proteins, and characterization of the Bacillus subtilis family members ScpA and ScpB.

Structural maintenance of chromosomes (SMC) proteins are present in all eukaryotes and in many prokaryotes. Eukaryotic SMC proteins form complexes with various non-SMC subunits, which affect their function, whereas the prokaryotic homologues had no known non-SMC partners and were thought to act as simple homodimers. Here we describe two novel families of proteins, widespread in archaea and (Gram-positive) bacteria, which we denote 'segregation and condensation proteins' (Scps). ScpA genes are localized next to smc genes in nearly all SMC- containing archaea, suggesting that they belong to the same operon and are thus involved in a common process in the cell. The function of ScpA was studied in Bacillus subtilis, which also harbours a well characterized smc gene. Here we show that scpA mutants display characteristic phenotypes nearly identical to those of smc mutants, including temperature- sensitive growth, production of anucleate cells, formation of aberrant nucleoids, and chromosome splitting by the so-called guillotine effect. Thus, both SMC and ScpA are required for chromosome segregation and condensation. Interestingly, mutants of another B. subtilis gene, scpB, which is localized downstream from scpA, display the same phenotypes, which indicate that ScpB is also involved in these functions. ScpB is generally present in species that also encode ScpA. The physical interaction of ScpA and SMC was proven (i) by the use of the yeast two-hybrid system and (ii) by the isolation of a complex containing both proteins from cell extracts of B. subtilis. By extension, we speculate that interaction of orthologues of the two proteins is important for chromosome segregation in many archaea and bacteria, and propose that SMC proteins generally have non-SMC protein partners that affect their function not only in eukaryotes but also in prokaryotes.

Amino Acid Sequence↗
PubMed2002

A new mutation delivery system for genome-scale approaches in Bacillus subtilis.

In Bacillus subtilis, although many genetic tools have been developed, gene replacement remains labour-intensive and not compatible with large-scale approaches. We have developed a new one-step gene replacement procedure that allows rapid alteration of any gene sequence or multiple gene sequences in B. subtilis without altering the chromosome in any other way. This novel approach relies on the use of upp, which encodes uracil phosphoribosyl-transferase, as a counter-selectable marker. We fused the upp gene to an antibiotic-resistance gene to create an 'upp-cassette'. A polymerase chain reaction (PCR)-generated fragment, consisting of the target gene with the desired mutation joined to the upp-cassette, was integrated into the chromosome by homologous recombination, using positive selection for antibiotic resistance. Then, the eviction of the upp-cassette from the chromosome by recombination between short repeated chromosomal sequences, included in the design of the transforming DNA molecule, was achieved by counter-selection of upp. This procedure was successfully used to deliver a point mutation, to generate in-frame deletions with reduced polar effects, and to combine deletions in three paralogous genes encoding two-component sensor kinases. Also, two chromosome regions carrying previously unrecognized essential functions were identified, and large deletions in two dispensable regions were combined. This work outlines a strategy for identifying essential functions that could be used at genome scale.

Amino Acid Sequence↗
PubMed2002

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids.

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10(4) transformants per microg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii.

Bacteriophages↗
PubMed2002

The peptidyl-prolyl isomerase motif is lacking in PmpA, the PrsA-like protein involved in the secretion machinery of Lactococcus lactis.

The prsA-like gene from Lactococcus lactis encoding its single homologue to PrsA, an essential protein triggering the folding of secreted proteins in Bacillus subtilis, was characterized. This gene, annotated pmpA, encodes a lipoprotein of 309 residues whose expression is increased 7- to 10-fold when the source of nitrogen is limited. A slight increase in the expression of the PrsA-like protein (PLP) in L. lactis removed the degradation products previously observed with the Staphylococcus hyicus lipase used as a model secreted protein. This shows that PmpA either triggers the folding of the secreted lipase or activates its degradation by the cell surface protease HtrA. Unlike the case for B. subtilis, the inactivation of the gene encoding PmpA reduced only slightly the growth rate of L. lactis in standard conditions. However, it almost stopped its growth when the lipase was overexpressed in the presence of salt in the medium. Like PrsA of B. subtilis and PrtM of L. lactis, the L. lactis PmpA protein could thus have a foldase activity that facilitates protein secretion. These proteins belong to the third family of peptidyl-prolyl cis/trans-isomerases (PPIases) for which parvulin is the prototype. Almost all PLP from gram-positive bacteria contain a domain with the PPIase signature. An exception to this situation was found only in Streptococcaceae, the family to which L. lactis belongs. PLP from Streptococcus pneumoniae and Enterococcus faecalis possess this signature, but those of L. lactis, Streptococcus pyogenes, and Streptococcus mutans do not. However, secondary structure predictions suggest that the folding of PLP is conserved over the entire length of the proteins, including the unconserved signature region. The activity associated with the expression of PmpA in L. lactis and these genomic data show that either the PPIase motif is not necessary for PPIase activity or, more likely, PmpA foldase activity does not necessarily require PPIase activity.

Amino Acid Motifs↗
PubMed2002

Filamentous phage active on the gram-positive bacterium Propionibacterium freudenreichii.

We present the first description of a single-stranded DNA filamentous phage able to replicate in a gram-positive bacterium. Phage B5 infects Propionibacterium freudenreichii and has a genome consisting of 5,806 bases coding for 10 putative open reading frames. The organization of the genome is very similar to the organization of the genomes of filamentous phages active on gram-negative bacteria. The putative coat protein exhibits homology with the coat proteins of phages PH75 and Pf3 active on Thermus thermophilus and Pseudomonas aeruginosa, respectively. B5 is, therefore, evolutionarily related to the filamentous phages active on gram-negative bacteria.

Amino Acid Sequence↗
PubMed2001

Identity elements in tRNA-mediated transcription antitermination: implication of tRNA D- and T-arms in mRNA recognition.

tRNA-mediated transcription antitermination has been shown to control the expression of several amino acid biosynthesis operons and aminoacyl-tRNA-synthetase-encoding genes in Gram-positive bacteria. A model originally put forward by Grundy & Henkin describes the conserved structural features of the leader sequences of these operons and genes. Two sequences of 3 and 4 nt, respectively, take a central position in this model and are thought to be responsible for the binding of the system-specific uncharged tRNA, an interaction which would stabilize the antiterminator conformation of the leader. Here a further evolution of this model is presented based on an analysis of trp regulation in Lactococcus lactis in which a function is assigned to hitherto unexplained conserved structures in the leader sequence. It is postulated that the mRNA-tRNA interaction involves various parts of the tRNA in addition to the anticodon and the acceptor in the original model and that these additional interactions contribute to the recognition of a specific tRNA, and hence to the specificity and efficacy of the regulatory response.

5' Untranslated Regions↗
PubMed2000

Six putative two-component regulatory systems isolated from Lactococcus lactis subsp. cremoris MG1363.

The genetic elements specifying six putative two-component regulatory systems (2CSs) were identified on the chromosome of Lactococcus lactis MG1363. These 2CSs appear to represent distinct loci, each containing a histidine kinase and response-regulator-encoding gene pair. Transcriptional analysis of the six 2CSs was performed either by generating transcriptional fusions to a reporter gene or by primer extension. Two of the systems appeared to be expressed constitutively at a high level, whilst the remaining four exhibited growth-phase-dependent expression. Insertional mutagenesis established that the two constitutively expressed 2CSs are necessary for normal cell growth and/or survival. Mutational analysis of the remaining four systems revealed that they are implicated in susceptibility to extreme pH, osmotic or oxidative conditions, or the regulation of phosphatase activity in L. lactis.

Amino Acid Sequence↗
PubMed1999

sbcB sbcC null mutations allow RecF-mediated repair of arrested replication forks in rep recBC mutants.

We have proposed previously that, in Escherichia coli, blockage of replication forks can lead to the reversal of the fork. Annealing of the newly synthesized strands creates a double-stranded end adjacent to a Holliday junction. The junction is migrated away from the DNA end by RuvAB and can be cleaved by RuvC, while RecBCD is required for the repair of the double-stranded tail. Consequently, the rep mutant, in which replication arrests are frequent and fork reversal occurs, requires RecBCD for growth. We show here that the combination of sbcB sbcCD null mutations restores the viability to rep recBC mutants by activation of the RecF pathway of recombination. This shows that the proteins belonging to the RecF pathway are able to process the DNA ends made by the replication fork reversal into a structure that allows recombination-dependent replication restart. However, we confirm that, unlike sbcB null mutations, sbcB15, which suppresses all other recBC mutant defects, does not restore the viability of rep recBC sbcCD strains. We also show that ruvAB inactivation suppresses the lethality and the formation of double-stranded breaks (DSBs) in a rep recBC recF strain, totally deficient for homologous recombination, as well as in rep recBC mutants. This confirms that RuvAB processing of arrested replication forks is independent of the presence of recombination intermediates.

Bacterial Proteins↗
PubMed1998

A vector for systematic gene inactivation in Bacillus subtilis.

To study the functions of the uncharacterized open reading frames identified in the Bacillus subtilis genome, several vectors were constructed to perform insertional mutagenesis in the chromosome. All the pMUTIN plasmids carry a lacZ reporter gene and an inducible Pspac promoter, which is tightly regulated and can be induced about 1000-fold. The integration of a pMUTIN vector into the target gene has three consequences: (1) the target gene is inactivated; (2) lacZ becomes transcriptionally fused to the gene, allowing its expression pattern to be monitored; (3) the Pspac promoter controls the transcription of downstream genes in an IPTG-dependent fashion. This last feature is important because B. subtilis genes are often organized in operons. The potential polar effects generated by the integration of the vectors can be alleviated by addition of IPTG. Also, conditional mutants of essential genes can be obtained by integrating pMUTIN vectors upstream of the target gene. The vectors are currently being used for systematic inactivation of genes without known function within the B. subtilis European consortium. pMUTIN characteristics and the inactivation of eight genes in the resA-serA region of the chromosome are presented.

Bacillus subtilis↗
PubMed1998

The kdgRKAT operon of Bacillus subtilis: detection of the transcript and regulation by the kdgR and ccpA genes.

Transcription of a new catabolic operon in Bacillus subtilis, involved in the late stages of galacturonic acid utilization, has been studied. The operon consists of four genes: kdgR, encoding the putative regulator protein; kdgK, encoding 2-keto-3-deoxygluconate kinase; kdgA, encoding 2-keto-3-deoxygluconate-6-phosphate aldolase; and kdgT, encoding a transporter. These four genes are organized in one transcriptional unit and map at 198 degrees of the B. subtilis chromosome. Primer extension experiments and Northern blot analysis show that an active sigmaA-dependent promoter precedes kdgR and transcription is terminated at the putative p-independent terminator downstream of kdgT. The operon is negatively regulated by the kdgR and ccpA gene products, which belong to the LacI family of transcription regulators. The expression of the genes in this operon can be induced by galacturonate and strongly repressed when glucose is present in the growth medium. Knockout mutations in genes kdgR and ccpA remove, respectively, the effects of galacturonate and glucose on the transcription of this operon.

Bacillus subtilis↗
PubMed1998

Disruption of the sole ldhL gene in Lactobacillus sakei prevents the production of both L- and D-lactate.

A 7 kb DNA fragment was cloned from Lactobacillus sakei which contains the IdhL gene encoding the L(+)-lactate dehydrogenase (L-LDH). Analysis of the DNA sequence, Northern experiments and primer extension experiments showed that IdhL is transcribed from a single promoter, leading to a monocistronic 1.15 kb mRNA which yields the L-LDH. A stable mutant was constructed by chromosomal integration of a chloramphenicol cassette into IdhL by a double-crossover event. Both L- and D-lactate were produced by the wild-type strain whereas only residual amounts of both isomers were produced by the mutant. This demonstrates that L. sakei possesses an L-LDH producing L-lactate and a lactate racemase able to transform it to D-lactate, but is devoid of D-LDH activity. Moreover the ability to degrade L-lactate present in the medium that was observed with the mutant strain grown aerobically suggests that an L-lactate oxidase activity is also present in L. sakei.

Cloning, Molecular↗
PubMed1998

Transcription of the trp operon in Lactococcus lactis is controlled by antitermination in the leader region.

The regulatory functions of the leader region preceding the Lactococcus lactis trp operon have been studied by mutagenesis analysis. This leader presents striking similarity to 'T-box' leaders found upstream of many Gram-positive aminoacyl-tRNA synthetase genes and some amino acid biosynthesis operons, which are controlled by antitermination through interaction of the leader transcript with cognate uncharged tRNA. A region of the L. lactis leader transcript also contains a series of (G/U) AG repeats which, in Bacillus, are involved in the binding of the trp RNA-binding protein (TRAP) which controls trp transcription. A screen was developed for the isolation of regulatory mutants affected in the leader region. All spontaneous mutants contained deletions; point mutations were only obtained after UV-induced mutagenesis. All mutations affected the putative transcription terminator upstream of the trp operon, demonstrating that trp is indeed controlled by transcription antitermination.

Base Sequence↗
PubMed1997

Sequencing and functional annotation of the Bacillus subtilis genes in the 200 kb rrnB-dnaB region.

The 200 kb region of the Bacillus subtilis chromosome spanning from 255 to 275 degrees on the genetic map was sequenced. The strategy applied, based on use of yeast artificial chromosomes and multiplex Long Accurate PCR, proved to be very efficient for sequencing a large bacterial chromosome area. A total of 193 genes of this part of the chromosome was classified by level of knowledge and biological category of their functions. Five levels of gene function understanding are defined. These are: (i) experimental evidence is available of gene product or biological function; (ii) strong homology exists for the putative gene product with proteins from other organisms; (iii) some indication of the function can be derived from homologies with known proteins; (iv) the gene product can be clustered with hypothetical proteins; (v) no indication on the gene function exists. The percentage of detected genes in each category was: 20, 28, 20, 15 and 17, respectively. In the sequenced region, a high percentage of genes are implicated in transport and metabolic linking of glycolysis and the citric acid cycle. A functional connection of several genes from this region and the genes close to 140 degrees in the chromosome was also observed.

Bacillus subtilis↗
PubMed1997

Identification by PCR of genes encoding multiple response regulators.

Environmental sensing in bacteria often involves the concerted action of sensor kinases and response regulators. Degenerate oligonucleotide primers were designed on the basis of amino acid similarity in the response regulators of these two-component systems. The primers were used in PCR to specifically amplify an internal DNA segment corresponding to the receiver module domain from genes encoding response regulators. Amplification products of the expected size were obtained from 12 different Gram-positive and Gram-negative bacteria. Sequence analysis revealed that 22 DNA fragments, which clearly originated from response regulator genes, were amplified from Escherichia coli, Agrobacterium tumefaciens, Bacillus subtilis and Lactobacillus bulgaricus. In each of these four species the receiver module of putative response regulator genes, which do not seem to be related to any of the already characterized genes, was identified. This simple and powerful method is therefore particularly useful for discovering new signal transduction systems which cannot be revealed by usual genetic studies.

Agrobacterium tumefaciens↗
PubMed1997

Sequence of the Bacillus subtilis genome region in the vicinity of the lev operon reveals two new extracytoplasmic function RNA polymerase sigma factors SigV and SigZ.

Two regions with sizes 18,900 and 25,400 bp, which join previously known contigs containing levRDEFG, aadK and blt genes near 235 degrees of the Bacillus subtilis chromosome, were sequenced. Among others, two genes, which encode proteins homologous to RNA polymerase sigma-factors, were identified within this region. The gene products designated SigV and SigZ, show the highest homology with sigma-factors encoded by the gene carQ of Myxococcus xanthus and sigX (formerly orfX20) of B. subtilis, correspondingly. All sigma-factors which show statistically significant homology to SigV and SigZ, belong to the ECF (extracytoplasmic functions) subfamily. SigV and SigZ do not have N-terminal sequence which prevents such proteins from binding to DNA without RNA polymerase core enzyme.

Amino Acid Sequence↗
PubMed1995

The Bacillus subtilis dnaI gene is part of the dnaB operon.

The dnaI gene of Bacillus subtilis, previously identified through the isolation of the dnaI2 mutant, was found to be the second gene of the dnaB operon. The nucleotide substitution in the dnaI2 mutant gene was determined.

Bacillus subtilis↗