ClpP/ClpX-mediated degradation of the bacteriophage λ O protein and regulation of λ phage and λ plasmid replication

The O protein is a replication initiator that binds to the oriλ region and promotes assembly of the bacteriophage λ replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage λ is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, λ plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium. Contrary to λ plasmid replication, the efficiency of lytic growth of bacteriophage λ was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major λ promoters operating during the lytic development, pR and pL, were found to be slightly dependent on the host growth rate. However, when pR activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of λ plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage λ lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells

DnaA-stimulated transcriptional activation of orilambda: Escherichia coli RNA polymerase beta subunit as a transcriptional activator contact site

We present evidence that Escherichia coli RNA polymerase beta subunit may be a transcriptional activator contact site. Stimulation of the activity of the pR promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage lambda. We found that DnaA activates the pR promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of lambda plasmids; this suppression was allele-specific. When a potential DnaA-binding sequence located several base pairs downstream of the pR promoter was scrambled by in vitro mutagenesis, the pR promoter was no longer activated by DnaA both in vivo and in vitro. Therefore, we conclude that DnaA may contact the beta subunit of RNA polymerase during activation of the pR promoter. A new classification of prokaryotic transcriptional activators is proposed

Molecular mechanism of heat shock-provoked disassembly of the coliphage lambda replication complex

We have found previously that, in contrast to the free O initiator protein of lambda phage or plasmid rapidly degraded by the Escherichia coli ClpP/ClpX protease, the lambdaO present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43 degrees C resulted in the decay of the lambdaO fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of lambda plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from lambda DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroELS and ClpP/ClpX proteins. In contrast to lambda cro+, in lambda cro- plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the lambda crots plasmid-harboring cells, a Cro repressor-independent control of lambda DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to lambda DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the lambda cro- plasmid replication contro

The cbpA chaperone gene function compensates for dnaJ in lambda plasmid replication during amino acid starvation of Escherichia coli

We found previously that lambda plasmid DNA replication in amino acid-starved Escherichia coli relA mutants (i.e., during the relaxed response), which is carried out by the inherited replication complex, is dependent on functions of DnaK and GrpE molecular chaperones but proceeds in a dnaj mutant at a nonpermissive temperature. Here we demonstrate that this replication is inhibited when functions of both dnaJ and cbpA are impaired. In complete media, the growth of the lambda pi A66 phage (capable of replicating in E. coli dnaJ, dnaK, and grpE missense mutants at 30 degrees C), as well as efficiency of transformation by the lambda pi A66 plasmid, is significantly decreased in a dnaJ259 cbpA::kan double mutant. These results strengthen the proposal of other authors (C. Ueguchi, M. Kakeda, H. Yamada, and T. Mizuno, Proc. Natl. Acad. Sci. USA 91:1054-1058, 1994; C. Ueguchi, T. Shiozawa, M. Kakeda, H. Yamada, and T. Mizuno, J. Bacteriol. 177:3894-3896, 1995; and T. Yamashino, M. Kakeda, C. Ueguchi, and T. Mizuno, Mol. Microbiol. 13:475-483, 1994) that the cbpA gene product is a functional analog of the DnaJ chaperone in E. coli

Dataset (sourdough and bread analysis) "Developing lactic acid bacteria starter cultures for wholemeal rye flour bread with improved functionality, nutritional value, taste, appearance and safety"

Dataset including: - Chemical composition - Organic acids - Phytate - Alcohols and sugars - Quality - Texture and moisture - Organoleptic assesment - Active and potential acidity changes during sourdough fermentatio

High-Fat Diet with Normal Caloric Intake Elevates TMA and TMAO Production and Reduces Microbial Diversity in Rats

Background/Objectives: Trimethylamine (TMA), produced by gut microbiota, and its derivative trimethylamine N-oxide (TMAO) are both associated with cardiometabolic diseases. While the effects of high-fat diets (HFDs) and high-disaccharide diets (HDDs) on gut microbiota in the context of obesity have been well studied, their impact on TMA/TMAO production, particularly alongside physiological caloric intake, remains obscure. This study investigates how standard HFDs and HDDs alongside physiological caloric intake influence gut microbiota composition and TMA/TMAO production in rats. Methods: Sprague Dawley rats were fed one of three diets a standard diet, an HFD, or an HDD for 12 weeks, with chow availability adjusted by age to maintain physiological caloric intake. Gut bacterial diversity was analyzed using 16S rRNA gene sequencing, and metabolites were quantified via High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) in urine and plasma. Results: The HFD group had significantly higher urinary levels of TMA and TMAO compared to the control and HDD groups. Gut bacterial diversity in the HFD group was markedly reduced, displaying the lowest species richness and phylogenetic diversity among all the groups. Notably, Pasteurellaceae (within the order Pasteurellales) and S24-7 (within the order Bacteroidales) were positively correlated with TMAO levels. The demonstrated HDD group increased microbial diversity compared to both the control and HFD groups. Conclusions: A high-fat diet during controlled and physiological caloric intake increases TMA/TMAO production and reduces gut microbial diversity. This underscores the role of diet composition, beyond caloric excess, in shaping gut microbiota and the related cardiometabolic biomarkers

Biology, systematics, and clinical manifestations of Zygomycota infections

Fungi cause opportunistic, nosocomial, and community-acquired infections. Among fungal infections (mycoses) zygomycoses are exceptionally severe, with a mortality rate exceeding 50 %. Immunocompromised hosts, transplant recipients, and diabetic patients with uncontrolled keto-acidosis and high iron serum levels are at risk. Zygomycota are capable of infecting hosts immune to other filamentous fungi. The infection often follows a progressive pattern, with angioinvasion and metastases. Moreover, current antifungal therapy frequently has an unfavorable outcome. Zygomycota are resistant to some of the routinely used antifungals, among them azoles (except posaconazole) and echinocandins. The typical treatment consists of surgical debridement of the infected tissues accompanied by amphotericin B administration. The latter has strong nephrotoxic side effects, which make it unsuitable for prophylaxis. Delayed administration of amphotericin and excision of mycelium-containing tissues worsens survival prognoses. More than 30 species of Zygomycota are involved in human infections, among them Mucorales is the most abundant. Prognosis and treatment suggestions differ for each species, which makes fast and reliable diagnosis essential. Serum sample PCR-based identification often gives false-negative results; culture-based identification is time-consuming and not always feasible. With the dawn of Zygomycota sequencing projects significant advancement is expected, as in the case of treatment of Ascomycota infections

Genetic Characterization of the CcpA-Dependent, Cellobiose-Specific PTS System Comprising CelB, PtcB and PtcA that Transports Lactose in Lactococcus lactis IL1403.

Lactose metabolism is one of the most important areas of research on Lactic Acid Bacteria (LAB). In rapidly acidifying industrial Lactococcus lactis strains, lactose is transported by a lactose-specific phosphotransferase system (PTS) encoded by a plasmid. However, an alternative lactose catabolic pathway was evidenced in the plasmid-cured, and thus initially lactose-negative L. lactis IL1403. We showed that in this strain the chromosomally-encoded cellobiose-specific PTS system comprising the celB, ptcB and ptcA genes is also able to transport lactose. By expression studies in the wild type IL1403 strain and IBB550, its ccpA-deficient derivative, we demonstrated that celB, ptcB and ptcA are tightly regulated by the general catabolite repression system, whereas celB additionally requires the presence of cellobiose to be fully induced. The comparison of expression levels of sugar catabolic genes indicated that the efficiency of CcpA-mediated catabolic repression depends on conservation of the cre sequence, and that in the case of perfect matching with the cre consensus, CcpA still drives a strong repression even under non-repressing conditions