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Biomedical subjects

S D Ehrlich

Publications and source records attributed to S D Ehrlich.

At least 109 records · Page 6Linked to original sources
PubMed1996

Countertranscript-driven attenuation system of the pAM beta 1 repE gene.

The plasmid-encoded RepE protein is absolutely essential and rate-limiting for replication of the promiscuous plasmid pAM beta 1 originating from Enterococcus faecalis. We previously showed that the rep gene is transcribed from a promoter that is negatively regulated (approximately 10-fold reduction) by the CopF repressor. In this report, we show that this transcription is decreased a further approximately 10-times by a countertranscript-driven transcriptional attenuation system. Extensive mutagenesis revealed that this system operates by a mechanism similar to that previously described for the unrelated repC gene of plasmid pT181.

Bacterial Proteins↗
PubMed1996

Carbohydrate Utilization in Lactobacillus sake.

The ability of Lactobacillus sake to use various carbon sources was investigated. For this purpose we developed a chemically defined medium allowing growth of L. sake and some related lactobacilli. This medium was used to determine growth rates on various carbohydrates and some nutritional requirements of L. sake. Mutants resistant to 2-deoxy-d-glucose (a nonmetabolizable glucose analog) were isolated. One mutant unable to grow on mannose and one mutant deficient in growth on mannose, fructose, and sucrose were studied by determining growth characteristics and carbohydrate uptake and phosphorylation rates. We show here that sucrose, fructose, mannose, N-acetylglucosamine, and glucose are transported and phosphorylated by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS permease specific for mannose, enzyme II(supMan), was shown to be responsible for mannose, glucose, and N-acetylglucosamine transport. A second, non-PTS system, which was responsible for glucose transport, was demonstrated. Subsequent glucose metabolism involved an ATP-dependent phosphorylation. Ribose and gluconate were transported by PTS-independent permeases.

Journal Article↗
PubMed1996

Imbalance of leucine flux in Lactococcus lactis and its use for the isolation of diacetyl-overproducing strains.

Diacetyl is a by-product of pyruvate metabolism in Lactococcus lactis, where pyruvate is first converted to alpha-acetolactate, which is slowly decarboxylated to diacetyl in the presence of oxygen. L. lactis usually converts alpha-acetolactate to acetoin enzymatically, by alpha-acetolactate decarboxylase encoded by the aldB gene. We took advantage of the fact that this enzyme also has a central role in the regulation of branched-chain amino acids, to select spontaneous aldB mutants in an unbalanced concentration of leucine versus those of valine and isoleucine in the medium. Industrial dairy strains of L. lactis subsp. lactis biovar diacetylactis containing point mutations and deletions of aldB were isolated and characterized. Their growth in milk was not affected, but they rapidly accumulated a large amount of alpha-acetolactate instead of acetoin from citrate in milk. Under aerated condition, strains devoid of AldB produced about 10 times more diacetyl than did the parental strains.

Amino Acid Sequence↗
PubMed1996

Genetic manipulation of the pathway for diacetyl metabolism in Lactococcus lactis.

Diacetyl is an important food flavor compound produced by certain strains of citrate-metabolizing lactic acid bacteria. Citrate is converted to pyruvate, from which diacetyl is produced via intermediate alpha-acetolactate. This paper reports the cloning and analysis of the gene (aldB) encoding alpha-acetolactate decarboxylase from Lactococcus lactis MG1363. Deletion of the MG1363 chromosomal aldB gene was achieved by double crossover homologous recombination. The mutant strain was found to produce diacetyl; alpha-acetolactate decarboxylase activity was eliminated. Overexpression of the cloned ilvBN genes (encoding an alpha-acetolactate synthase) in the aldB deletion strain produced even higher levels of alpha-acetolactate, acetoin, and diacetyl.

Acetolactate Synthase↗
PubMed1996

Gene organization and transcription of a late-expressed region of a Lactococcus lactis phage.

The lactococcal phage bIL41 belongs to the small isometric-headed phages of the 936 quasi-species and is resistant to the abortive infection determined by abiB. A 10.2-kb segment from this phage, in which late transcription is initiated, has been sequenced. Thirteen open reading frames (ORFs) organized in one transcriptional unit have been identified. The location of two of them and the structural features of the proteins they code for are evocative of terminase subunits. Five other ORFs specify proteins which are highly homologous to structural proteins from the closely related phage F4-1. By comparing the phage bIL41 sequence with partial sequences available for four related phages, we were able to deduce a chimerical phage map covering the middle- and a large part of the late-expressed regions. Phages from this quasi-species differ by the insertion or deletion of either 1 to about 400 bp in noncoding regions or an entire ORF. Transcription was initiated 9 min after infection at a promoter with a -10 but no -35 consensus sequence. Synthesis of a phage activator protein was needed for initiation of transcription. A large 16-kb transcript covering all of the late-expressed region of the genome was synthesized. This transcript gave rise to smaller units. One of these units most probably resulted from a RNase E processing.

Bacteriophages↗
PubMed1996

Efficient insertional mutagenesis in lactococci and other gram-positive bacteria.

In lactococci, the study of chromosomal genes and their regulation is limited by the lack of an efficient transposon mutagenesis system. We associated the insertion sequence ISS1 with the thermosensitive replicon pG+ host to generate a mutagenic tool that can be used even in poorly transformable strains. ISS1 transposition is random in different lactococcal strains as well as in Enterococcus faecalis and Streptococcus thermophilus. High-frequency random insertion (of about 1%) obtained with this system in Lactococcus lactis allows efficient mutagenesis, with typically one insertion per cell. After ISS1 replicative transposition, the chromosome contains duplicated ISS1 sequences flanking pG+ host. This structure allows cloning of the interrupted gene. In addition, efficient excision of the plasmid leaves a single ISS1 copy at the mutated site, thus generating a stable mutant strain with no foreign markers. Mutants obtained by this transposition system are food grade and can thus be used in fermentation processes.

Base Sequence↗
PubMed1996

Regulators of aerobic and anaerobic respiration in Bacillus subtilis.

Two Bacillus subtilis genes, designated resD and resE, encode proteins that are similar to those of two-component signal transduction systems and play a regulatory role in respiration. The overlapping resD-resE genes are transcribed during vegetative growth from a very weak promoter directly upstream of resD. They are also part of a larger operon that includes three upstream genes, resABC (formerly orfX14, -15, and -16), the expression of which is strongly induced postexponentially. ResD is required for the expression of the following genes: resA, ctaA (required for heme A synthesis), and the petCBD operon (encoding subunits of the cytochrome bf complex). The resABC genes are essential genes which encode products with similarity to cytochrome c biogenesis proteins. resD null mutations are more deleterious to the cell than those of resE. resD mutant phenotypes, directly related to respiratory function, include streptomycin resistance, lack of production of aa3 or caa3 terminal oxidases, acid accumulation when grown with glucose as a carbon source, and loss of ability to grow anaerobically on a medium containing nitrate. A resD mutation also affected sporulation, carbon source utilization, and Pho regulon regulation. The data presented here support an activation role for ResD, and to a lesser extent ResE, in global regulation of aerobic and anaerobic respiration i B.subtilis.

Aerobiosis↗
PubMed1995

Primosome assembly site in Bacillus subtilis.

A single-strand initiation site was detected on the Enterococcus faecalis plasmid pAM beta 1 by its ability to prevent accumulation of single stranded DNA of a rolling circle plasmid, both in Bacillus subtilis and Staphylococcus aureus. This site, designated ssiA, is located on the lagging strand template, approximately 150 bp downstream from the replication origin. ssiA priming activity requires the DnaE primase, the DnaC replication fork helicase, as well as the products of the dnaB, dnaD and dnaI genes of B.subtilis, but not the RNA polymerase. The primase and the replication fork helicase requirements indicate that ssiA is a primosome assembly site. Interestingly, the pAM beta 1 lagging strand synthesis is inefficient when any of the proteins involved in ssiA activity is mutated, but occurs efficiently in the absence of ssiA. This suggests that normal plasmid replication requires primosome assembly and that the primosome can assemble not only at ssiA but also elsewhere on the plasmid. This work for the first time describes a primosome in a Gram-positive bacterium. Involvement of the B.subtilis proteins DnaB, DnaD and DnaI, which do not have any known analogue in Escherichia coli, raises the possibility that primosome assembly and/or function in B.subtilis differs from that in E.coli.

Bacillus subtilis↗
PubMed1995

A 7-base-pair sequence protects DNA from exonucleolytic degradation in Lactococcus lactis.

Linear DNA molecules are subject to degradation by various exonucleases in vivo unless their ends are protected. It has been demonstrated that a specific 8-bp sequence, 5'-GCTGGTGG-3', named Chi, can protect linear double-stranded DNA from the major Escherichia coli exonuclease RecBCD. Chi protects linear replication products of rolling-circle plasmids from RecBCD degradation in vivo, in agreement with observations in vitro. A unique 7-bp sequence, 5'-GCGCGTG-3', is shown to protect similar replication products from degradation in Lactococcus lactis strains but not in more distantly related Gram-positive bacteria. The properties of this sequence in L. lactis correspond to those of a Chi site. Linear plasmid replication products have been detected in numerous prokaryotes, suggesting the widespread existence of short species-specific sequences that preserve linear DNA from extensive degradation by host cell exonucleases.

Base Composition↗
PubMed1995

Competition between parental and recombinant plasmids affects the measure of recombination frequencies.

Recombination frequencies in multicopy plasmids are generally deduced from the rate of appearance of cells expressing a recombinant phenotype (i.e., "recombinant cells"). Detection of these cells requires not only the formation of a recombinant molecule but also the establishment of this molecule in the presence of the resident incompatible parental plasmid. Differences in fitness between parental and recombinant molecules will affect this establishment and could have great consequences for plasmid recombination measurements. To test this hypothesis, we compared recombination frequencies when the recombinant plasmid has or does not have a replication advantage over the parental plasmid. We used pBR322-derived plasmids which carry or lack the replication terminator TerB; recombination took place between directly repeated sequences of 16 bp and deleted TerB from the plasmid. The rate of appearance of recombinant cells strongly increased when the Tus/Ter system was active; however, we found no evidence for direct stimulation of recombination between direct repeats by replication fork stalling. The main factor responsible for the increase in the rate of appearance of recombinant cells when the parental plasmid carries TerB is the facilitated establishment of the recombinant plasmid since: (i) the transformation efficiency of the recombinant plasmid is higher in cells containing the Ter+ parent than in cells containing the Ter- parent; (ii) most recombinant plasmids did not lead to the appearance of recombinant cells when pBR322 was was not blocked by Tus, whereas the presence of TerB allows the detection of most events; and (iii) decreasing the parental plasmid copy number without modification of the recombinant plasmid leads to an exponential increase in the rate of appearance of recombinant cells. Our results show that the level of competition between parent and recombinant plasmids can greatly affect plasmid recombination frequencies deduced from the measure of recombinant cells. This effect can be as high as several orders of magnitude.

Base Sequence↗
PubMed1995

The Bacillus subtilis chromosome region encoding homologues of the Escherichia coli mssA and rpsA gene products.

A gene was found in Bacillus subtilis which encodes a protein highly homologous to the Escherichia coli rpsA gene product, the S1 ribosomal protein. The B. subtilis protein contains the domain responsible for binding to ribosomes and two S1 motifs, instead of four as found in the E. coli protein. The B. subtilis protein is similar in this way to the equivalent protein of plant chloroplast ribosomes, supposed to be the counterpart of E. coli S1. The gene is expressed during vegetative growth in B. subtilis at the transcriptional and translational levels, as judged by Northern hybridization and expression in a translational fusion with a reporter gene. In contrast to the E. coli situation, it can be inactivated without dramatic effects on cell viability. Southern hybridization of the B. subtilis DNA fragment encoding this gene revealed specific homologous fragments in all other Gram-positive bacteria tested. The hybridization pattern with B. stearothermophilus suggests the presence of at least two homologous genes in this bacterium. We show that in B. subtilis the ORF preceding the rpsA homologue encodes a protein which is highly similar to the product of the E. coli mssA gene which is located upstream of rpsA. Again, in contrast to the E. coli situation, where these genes are co-transcribed, in B. subtilis they are separated by a transcription terminator and the mssA homologue is transcribed during sporulation. We suggest that during the evolution very similar structures and genetic organization of these two genes were conserved but acquired different functions in Gram-negative and Gram-positive bacteria.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence of the Bacillus subtilis dnaD gene.

The dnaD gene of Bacillus subtilis was identified within a 104 kb DNA segment cloned into a yeast artificial chromosome. The nucleotide sequence of the wild type and dnaD23 mutant genes were determined. dnaD is predicted to encode a protein of 232 amino acids with no similarity to proteins in the data banks.

Amino Acid Sequence↗
PubMed1995

Repair of oxidative DNA damage in gram-positive bacteria: the Lactococcus lactis Fpg protein.

The formamidopyrimidine DNA glycosylase gene (fpg-L) of the Gram-positive microaerophilic bacterium Lactococcus lactis subsp. cremoris ML3 has been cloned, characterized and sequenced. The fpg-L gene is composed of 819 bp encoding a protein of 31.3 kDa (Fpg-L). The deduced amino acid sequence of the Fpg-L protein shows 59% similarity and 38% identity with the Escherichia coli Fpg protein (Fpg-E). Polyclonal antibodies against Fpg-E react with the Fpg-L protein. The Fpg-L protein was purified to apparent homogeneity from the overproducing E. coli strain BH410 hosting plasmid pVE1064, which carries fpg-L under the control of the E. coli lac promoter. In its active form, Fpg-L is a 30 kDa monomeric enzyme with a measured isoelectric point of 9.0. It contains one zinc per molecule and has a zinc finger motif localized at the carboxyterminal end (Cys-X2-Cys-X16-Cys-X2-Cys-X3-COOH). The Fpg-L protein has two enzyme activities: DNA glycosylase, which excises 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine and 7,8-dihydro-8-oxoguanine, and DNA nicking at abasic sites. Furthermore, the expression of the fpg-L gene in fpg and mutY mutants of E. coli suppresses their spontaneous GC-->TA mutator phenotype. The similarity of the activity of the two Fpg proteins and its conversation in evolutionarily distant bacteria may reflect the importance of its role in protecting bacterial DNA against oxidative free radicals.

Amino Acid Sequence↗
PubMed1995

Transcription-induced deletions in Escherichia coli plasmids.

Characterization of functions that render DNA susceptible to rearrangement is important for a better understanding of genome instability. In a previous work, we showed that sequences located downstream of a strong promoter are particularly prone to deletion. In this paper, the parameters that influence transcription-induced deletions were studied. pBR322 derivatives carrying the M13 (+) replication origin and a PTac-dependent transcription region were used. Deletion formation was analysed in the presence of the replication protein of M13, which introduces a nick at the phage replication origin, and in a rep- strain to avoid M13-driven replication. Our study showed that: (i) 4 h after induction of transcription, a few per cent of the plasmids have experienced a deletion; (ii) these deletions result in joining of the M13 replication origin to a nucleotide located in or downstream of the transcribed region; (iii) deletion formation strongly depends on the orientation of transcription, on promoter strength and transcript length, but is independent of translation; (iv) formation of transcription-induced supercoiling domains does not induce deletion formation. We propose that deletions in the transcribed region result from collisions between converging replication and transcription machineries.

Base Sequence↗
PubMed1995

The recA gene of Lactococcus lactis: characterization and involvement in oxidative and thermal stress.

The role of recA in Lactococcus lactis, a microaerophilic fermenting organism, was examined by constructing a recA-disrupted strain. This single alteration had a surprisingly pleiotropic effect. In addition to its roles in homologous recombination and DNA repair, recA is also involved in responses to oxygen and heat stresses. We found that oxygen stress induced by aeration causes reductions in growth and stationary-phase survival of the recA strain. Toxicity is a consequence of hydroxyl radical production via the Fenton Reaction and is alleviated by catalase or Ferrozine addition. These results suggest that oxygen radicals are not efficiently eliminated and accumulate in lactococcal cultures, and that RecA is needed to deal with the damage they incur. Unexpectedly, thermal stress arrested growth of the recA strain. Immunological data indicate that the recA mutant is deficient in heat-shock proteins DnaK, GroEL, and GrpE. Poor growth at elevated temperature is therefore due to a diminished heat-shock response in the recA strain. In contrast, levels of a novel heat-shock protein, HfIB, are elevated. In Escherichia coli, HfIB downregulates the heat-shock response by promoting degradation of the transcription factor sigma 32. We propose that recA regulates the heat-shock response via HfIB. This work provides the first evidence showing that two major pathways of stress response, induced by heat shock and DNA damage, are interactive.

ATP-Dependent Proteases↗
PubMed1995

Lethality of rep recB and rep recC double mutants of Escherichia coli.

A rep mutation in combination with a recB or a recC mutation renders Escherichia coli non-viable. This conclusion is based on the following lines of evidence: (i) double mutants cannot be constructed by P1 transduction; (ii) induction of the lambda Gam protein, which inactivates most of the RecBCD activities, is lethal in rep mutants; (iii) rep recBts recCts mutants are not viable at high temperature. The resons for a requirement for the RecBCD enzyme in rep strains were investigated. Initiation of chromosome replication, elongation and chromosomal segregation do not seem impaired in the rep recBtsrecCts mutant at the non-permissive temperature. The viability of other rep derivatives was tested. rep recA recD triple mutants are not viable, whereas rep recD and rep recA double mutants are. Inactivation of both exoV activity and recBC-dependent homologous recombination is therefore responsible for the non-viability of rep recBC strains. However, sbcA and sbcB mutations, which render recBC mutants recombination proficient, do not restore viability of rep recBC mutants, indicating that recombination via the RecF or the RecE pathways cannot functionally replace RecBCD-mediated recombination. The specific requirement for RecBCD suggests the occurrence of double-strand DNA breaks in rep strains. Additional arguments in favour of the presence of DNA lesions in rep mutants are as follows: (i) expression of SOS repair functions delays lethality of rep derivatives after inactivation of RecBCD; (ii) sensitivity of rep strains to ultraviolet light is increased by partial inactivation of RecBCD. A model for the recovery of cells from double-strand breaks in rep mutants is discussed.

Bacterial Proteins↗
PubMed1995

DNA restriction-modification systems mediate plasmid maintenance.

Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 of Bacillus sp. strain RFL6), enhance plasmid segregational stability in E. coli and Bacillus subtilis, respectively. Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems. We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free cells. Plasmid-encoded methyltransferase modifies host DNA and thus prevents its digestion by the restriction endonuclease. Plasmid loss entails degradation and/or dilution of the methylase during cell growth and appearance of unmethylated sites in the chromosome. Double-strand breaks, introduced at these sites by the endonuclease, eventually cause the death of the plasmid-free cells. Contribution to plasmid stability is a previously unrecognized biological role of the R-M systems.

Bacillus subtilis↗