Biomedical subjects

Dusko Ehrlich

Publications and source records attributed to Dusko Ehrlich.

4 recordsLinked to original sources

PubMed2006

Control of EpsE, the phosphoglycosyltransferase initiating exopolysaccharide synthesis in Streptococcus thermophilus, by EpsD tyrosine kinase.

We studied the roles of Streptococcus thermophilus phosphogalactosyltransferase (EpsE) (the priming enzyme), tyrosine kinase (EpsD), phosphatase (EpsB), and a membrane-associated protein with no known biochemical function (EpsC) in exopolysaccharide (EPS) synthesis. These proteins are well-conserved among bacteria and are usually encoded by clustered genes. Exopolysaccharide synthesis took place in the wild-type strain and a mutant lacking EpsB but not in mutants lacking EpsC, EpsD, or EpsE. The three mutants unable to synthesize EPS lacked the EpsE phosphogalactosyltransferase activity, while the two EPS-synthesizing strains possessed this activity, showing that EpsC and EpsD are required for EpsE function. An EpsD phosphorylated form was found in all strains except the epsC mutant, indicating that EpsC is necessary for EpsD phosphorylation. Moreover, the phosphorylated form of EpsD, a supposedly cytoplasmic protein, was found to be associated with the plasma membrane, possibly due to interaction with EpsC. Finally, the EpsD and EpsE elution profiles in a gel filtration chromatography assay were similar, suggesting that these two proteins colocalize in the membrane. Mutation of Tyr200, predicted to be a phosphorylation site and present in a conserved motif in bacterial phosphoglycosyltransferases, led to EpsE inactivation. In contrast, mutation of Tyr162 or Tyr199 had no effect. Taken together, these data show that EpsD controls EpsE activity. Possible mechanisms for this control are discussed.

Amino Acid Sequence↗

PubMed2004

Cells defective for replication restart undergo replication fork reversal.

We have studied the fate of blocked replication forks with the use of the Escherichia coli priA mutant, in which spontaneously arrested replication forks persist owing to the lack of the major replication restart pathway. Such blocked forks undergo a specific reaction named replication fork reversal, in which newly synthesized strands anneal to form a DNA double-strand end adjacent to a four-way junction. Indeed, (i) priA recB mutant chromosomes are linearized by a reaction that requires the presence of the Holliday junction resolvase RuvABC, and (ii) RuvABC-dependent linearization is prevented by the presence of RecBC. Replication fork reversal in a priA mutant occurs independently of the recombination proteins RecA and RecR. recBC inactivation does not affect priA mutant viability but prevents priA chronic SOS induction. We propose that, in the absence of PriA, RecBC action at reversed forks does not allow replication restart, which leads to the accumulation of SOS-inducing RecA filaments. Our results suggest that types of replication blockage that cause replication fork reversal occur spontaneously.

Adenosine Triphosphatases↗

PubMed2004

Intracellular effectors regulating the activity of the Lactococcus lactis CodY pleiotropic transcription regulator.

CodY is a pleiotropic transcriptional regulator conserved in low-G+C Gram-positive bacteria. Two distinct signals have been shown independently to influence the activity of this regulator: the level of intracellular GTP in Bacillus subtilis and the level of intracellular branched-chain amino acids (BCAA) isoleucine, leucine and valine in Lactococcus lactis. Measurement of BCAA and GTP levels in several environmental conditions showed that L. lactis CodY responded to the intracellular BCAA concentrations but not to physiological fluctuations in intracellular GTP. In addition, we demonstrated that CodY from L. lactis did not respond to intracellular GTP even when complementing CodY activity in B. subtilis. However, L. lactis CodY activity could still be modulated in B. subtilis by adding a rich nitrogen source to the growth media. This finding suggests that only BCAA are sensed by L. lactis CodY, whereas both GTP and BCAA signals may be integrated by B. subtilis CodY. The difference in the function of CodY from B. subtilis and L. lactis seems to reflect the difference in the physiology of these two bacteria.

Adaptation, Physiological↗

PubMed2002

Essential bacterial helicases that counteract the toxicity of recombination proteins.

PcrA, Rep and UvrD are three closely related bacterial helicases with a DExx signature. PcrA is encoded by Gram-positive bacteria and is essential for cell growth. Rep and UvrD are encoded by Gram-negative bacteria, and mutants lacking both helicases are also not viable. To understand the non-viability of the helicase mutants, we characterized spontaneous extragenic suppressors of a Bacillus subtilis pcrA null mutation. Here we report that one of these suppressors maps in recF and that previously isolated mutations in B.subtilis recF, recL, recO and recR, which belong to the same complementation group, all suppress the lethality of a pcrA mutation. Similarly, recF, recO or recR mutations suppress the lethality of the Escherichia coli rep uvrD double mutant. We conclude that RecFOR proteins are toxic in cells devoid of PcrA in Gram-positive bacteria, or Rep and UvrD in Gram-negative bacteria, and propose that the RecFOR proteins interfere with an essential cellular process, possibly replication, when DExx helicases PcrA, or Rep and UvrD are absent.

Adenosine Triphosphatases↗