Biomedical subjects

Dusko S Ehrlich

Publications and source records attributed to Dusko S Ehrlich.

7 recordsLinked to original sources

PubMed2006

Genetic structure and transcriptional analysis of the arginine deiminase (ADI) cluster in Lactococcus lactis MG1363.

In a recent proteomic analysis, we showed the overproduction of the ArcA and ArcB proteins in Lactococcus lactis MG1363 at low pH. The corresponding genes belong to the arcABD1C1C2TD2 cluster that encodes components of the arginine deiminase pathway. In this study, we characterized this cluster at the genetic level. Northern blot experiments showed the expression of at least seven transcripts, all induced by acidity. Transcript analysis using 5'RACE PCR (rapid amplification of cDNA ends polymerase chain reaction) in the arcB-arcD1 intergenic region. In silico analysis identified nine stem-loop structures, all located in intergenic regions. Collectively, these data suggest a role for RNA processing and (or) premature termination in the differential expression of genes within the arcABD1C1C2TD2 cluster.

Bacterial Proteins↗

PubMed2005

Proteomic characterization of the acid tolerance response in Lactococcus lactis MG1363.

Exponentially growing cells of Lactococcus lactis MG1363 are able to develop an Acid Tolerance Response (ATR) when incubated at pH 5, in both rich (M17)--and chemically defined (SA)--culture media. Physiological and proteomic characterization of this adaptive response indicated that L. lactis reorganizes its metabolism in response to acid stress to a great extent and quite differently in the two media. The development of ATR was fully dependent on protein de novo synthesis in SA and only partly dependent in M17. 2D gel electrophoresis revealed a total of 90 spots induced by acidity, 80 of which were identified by mass spectrometry. Only 10 proteins (BglA, PycA, GlmS, HasC, ArgS, GatA, AtpA, ArcB, Cfa, and SodA) were overproduced in the two media. A transcriptional analysis of the corresponding genes suggested that for half of them the mode of regulation may differ in the two media. Among the protein spots upregulated during the ATR in SA but not in M17, 13 already displayed an elevated rate of synthesis in M17 at neutral pH. These proteins could play an important role in the development of the protein de novo synthesis-independent ATR observed in M17.

Adaptation, Physiological↗

PubMed2005

Fructose utilization in Lactococcus lactis as a model for low-GC gram-positive bacteria: its regulator, signal, and DNA-binding site.

In addition to its role as carbon and energy source, fructose metabolism was reported to affect other cellular processes, such as biofilm formation by streptococci and bacterial pathogenicity in plants. Fructose genes encoding a 1-phosphofructokinase and a phosphotransferase system (PTS) fructose-specific enzyme IIABC component reside commonly in a gene cluster with a DeoR family regulator in various gram-positive bacteria. We present a comprehensive study of fructose metabolism in Lactococcus lactis, including a systematic study of fru mutants, global messenger analysis, and a molecular characterization of its regulation. The fru operon is regulated at the transcriptional level by both FruR and CcpA and at the metabolic level by inducer exclusion. The FruR effector is fructose-1-phosphate (F1P), as shown by combined analysis of transcription and measurements of the intracellular F1P pools in mutants either unable to produce this metabolite or accumulating it. The regulation of the fru operon by FruR requires four adjacent 10-bp direct repeats. The well-conserved organization of the fru promoter region in various low-GC gram-positive bacteria, including CRE boxes as well as the newly defined FruR motif, suggests that the regulation scheme defined in L. lactis could be applied to these bacteria. Transcriptome profiling of fruR and fruC mutants revealed that the effect of F1P and FruR regulation is limited to the fru operon in L. lactis. This result is enforced by the fact that no other targets for FruR were found in the available low-GC gram-positive bacteria genomes, suggesting that additional phenotypical effects due to fructose metabolism do not rely directly on FruR control, but rather on metabolism.

Amino Acid Sequence↗

PubMed2005

Sulfur amino acid metabolism and its control in Lactococcus lactis IL1403.

Cysteine and methionine availability influences many processes in the cell. In bacteria, transcription of the specific genes involved in the synthesis of these two amino acids is usually regulated by different mechanisms or regulators. Pathways for the synthesis of cysteine and methionine and their interconversion were experimentally determined for Lactococcus lactis, a lactic acid bacterium commonly found in food. A new gene, yhcE, was shown to be involved in methionine recycling to cysteine. Surprisingly, 18 genes, representing almost all genes of these pathways, are under the control of a LysR-type activator, FhuR, also named CmbR. DNA microarray experiments showed that FhuR targets are restricted to this set of 18 genes clustered in seven transcriptional units, while cysteine starvation modifies the transcription level of several other genes potentially involved in oxidoreduction processes. Purified FhuR binds a 13-bp box centered 46 to 53 bp upstream of the transcriptional starts from the seven regulated promoters, while a second box with the same consensus is present upstream of the first binding box, separated by 8 to 10 bp. O-Acetyl serine increases FhuR binding affinity to its binding boxes. The overall view of sulfur amino acid metabolism and its regulation in L. lactis indicates that CysE could be a master enzyme controlling the activity of FhuR by providing its effector, while other controls at the enzymatic level appear to be necessary to compensate the absence of differential regulation of the genes involved in the interconversion of methionine and cysteine and other biosynthesis genes.

Amino Acids↗

PubMed2004

Recent genetic transfer between Lactococcus lactis and enterobacteria.

The genome sequence of Lactococcus lactis revealed that the ycdB gene was recently exchanged between lactococci and enterobacteria. The present study of ycdB orthologs suggests that L. lactis was probably the gene donor and reveals three instances of gene transfer to enterobacteria. Analysis of ycdB gene transfer between two L. lactis subspecies, L. lactis subsp. lactis and L. lactis subsp. cremoris, indicates that the gene can be mobilized, possibly by conjugation.

Base Sequence↗

PubMed2003

Genome engineering reveals large dispensable regions in Bacillus subtilis.

Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.

Bacillus Phages↗

PubMed2003

Lethality of bypass polymerases in Escherichia coli cells with a defective clamp loader complex of DNA polymerase III.

Escherichia coli DNA polymerase III (Pol III) is one of the best studied replicative DNA polymerases. Here we report the properties of an E. coli mutant that lacks one of the subunits of the Pol III clamp loader complex, Psi (psi), as a result of the complete inactivation of the holD gene. We show that, in this mutant, chronic induction of the SOS response in a RecFOR-dependent way leads to lethality at high temperature. The SOS-induced proteins that are lethal in the holD mutant are the specialized DNA polymerases Pol II and Pol IV, combined with the division inhibitor SfiA. Prevention of SOS induction or inactivation of Pol II, Pol IV and SfiA encoding genes allows growth of the holD mutant, although at a reduced rate compared to a wild-type cell. In contrast, the SOS-induced Pol V DNA polymerase does not participate to the lethality of the holD mutant. We conclude that: (i) Psi is essential for efficient replication of the E. coli chromosome; (ii) SOS-induction of specialized DNA polymerases can be lethal in cells in which the replicative polymerase is defective, and (iii) specialized DNA polymerases differ in respect to their access to inactivated replication forks.

Bacterial Proteins↗