<Emphasis Type="Italic">Thermus thermophilus</Emphasis> genome analysis: benefits and implications

The genome sequence analysis of Thermus thermophilus HB27, a microorganism with high biotechnological potential, has recently been published. In that report, the chromosomal and the megaplasmid sequence were compared to those of other organisms and discussed on the basis of their physiological and metabolic features. Out of the 2,218 putative genes identified through the large genome sequencing project, a significant number has potential interest for biotechnology. The present communication will discuss the accumulating information on molecules participating in fundamental biological processes or having potential biotechnological importance.     

Novel aspects of RNA regulation in Staphylococcus aureus

A plethora of RNAs with regulatory functions has been discovered in many non-pathogenic and pathogenic bacteria. In Staphylococcus aureus, recent findings show that a large variety of RNAs control target gene expression by diverse mechanisms and many of them are expressed in response to specific internal or external signals. These RNAs comprise trans-acting RNAs, which regulate gene expression through binding with mRNAs, and cis-acting regulatory regions of mRNAs. Some of them possess multiple functions and encode small but functional peptides. In this review, we will present several examples of RNAs regulating pathogenesis, antibiotic resistance, and host-pathogen interactions and will illustrate how regulatory proteins and RNAs form complex regulatory circuits to express the virulence factors in a dynamic manner.     

Phosphorylation activity of the response regulator of the two-component signal transduction system AtoS-AtoC in E. coli

Antizyme, long known to be a non-competitive inhibitor of ornithine decarboxylase, is encoded by the atoC gene in Escherichia coli. The present study reveals another role for AtoC, that of a response regulator of the AtoS-AtoC two component system regulating the expression of the atoDAEB operon upon acetoacetate induction. This operon encodes enzymes involved in short-chain fatty acid catabolism in E. coli. Evidence is presented to show that AtoS is a sensor kinase that together with AtoC constitutes a two-component signal transduction system. AtoS is a membrane protein which can autophosphorylate and then transfer that phosphoryl group to AtoC. This process can also be reproduced in vitro. AtoC contains in its amino acid sequence a conserved aspartic acid (D55), which is the putative phosphorylation site, as well as an unexpected "H box" consensus sequence (SHETRTPV), common to histidine kinases, with the histidine contained therein (H73) being a second potential target for phosphorylation. Substitution of either D55 or H73 in His10-AtoC diminished but did not abrogate AtoC phosphorylation suggesting that either both residues can be phosphorylated independently or that the phosphate group can be transferred between them. However, the D55 mutation in comparison to H73 had a more pronounced effect in vivo, on the activation of atoDAEB promoter after acetoacetate induction, although it was the presence of both mutations that rendered AtoC totally unresponsive to induction. These data provide evidence that the gene products of atoS and atoC constitute a two-component signal transduction system, with some unusual properties, involved in the regulation of the atoDAEB operon.     

The essential YycFG two-component system controls cell wall metabolism in Bacillus subtilis

Adaptation of bacteria to the prevailing environmental and nutritional conditions is often mediated by two-component signal transduction systems (TCS). The Bacillus subtilis YycFG TCS has attracted special attention as it is essential for viability and its regulon is poorly defined. Here we show that YycFG is a regulator of cell wall metabolism. We have identified five new members of the YycFG regulon: YycF activates expression of yvcE, lytE and ydjM and represses expression of yoeB and yjeA. YvcE(CwlO) and LytE encode endopeptidase-type autolysins that participate in peptidoglycan synthesis and turnover respectively. We show that a yvcE lytE double mutant strain is not viable and that cells lacking LytE and depleted for YvcE exhibit defects in lateral cell wall synthesis and cell elongation. YjeA encodes a peptidoglycan deacetylase that modifies peptidoglycan thereby altering its susceptibility to lysozyme digestion and YdjM is also predicted to have a role in cell wall metabolism. A genetic analysis shows that YycFG essentiality is polygenic in nature, being a manifestation of disrupted cell wall metabolism caused by aberrant expression of a number of YycFG regulon genes.     

Peptidoglycan metabolism is controlled by the WalRK (YycFG) and PhoPR two‐component systems in phosphate‐limited Bacillus subtilis cells

In Bacillus subtilis, the WalRK (YycFG) two‐component system controls peptidoglycan metabolism in exponentially growing cells while PhoPR controls the response to phosphate limitation. Here we examine the roles of WalRK and PhoPR in peptidoglycan metabolism in phosphate‐limited cells. We show that B. subtilis cells remain viable in a phosphate‐limited state for an extended period and resume growth rapidly upon phosphate addition, even in the absence of a PhoPR‐mediated response. Peptidoglycan synthesis occurs in phosphate‐limited wild‐type cells at ～27% the rate of exponentially growing cells, and at ～18% the rate of exponentially growing cells in the absence of PhoPR. In phosphate‐limited cells, the WalRK regulon genes yocH, cwlO(yvcE), lytE and ydjM are expressed in a manner that is dependent on the WalR recognition sequence and deleting these genes individually reduces the rate of peptidoglycan synthesis. We show that ydjM expression can be activated by PhoP～P in vitro and that PhoP occupies its promoter in phosphate‐limited cells. However, iseA(yoeB) expression cannot be repressed by PhoP～P in vitro, but can be repressed by non‐phosphorylated WalR in vitro. Therefore, we conclude that peptidoglycan metabolism is controlled by both WalRK and PhoPR in phosphate‐limited B. subtilis cells.     

Copper(II) complexes of diclofenac: Spectroscopic studies and DNA strand breakage

Copper(II) complexes of diclofenac with interesting anti-inflammatory profiles have been prepared and studied by infrared and electronic spectroscopy. In the solid state and in polar and coordinating solvents, all the complexes are solvated binuclear carboxylato-bridged complexes, [Cu(L)²(S)]², where L is monodeprotonated diclofenac and S is the axially bonded solvent. The effect of the copper(II) complexes on the in vitro DNA strand breakeage was studied by agarose gel electrophoresis. Relaxation or double stranded scissions of pDNA were observed leading to the formation of linear pDNA. Treatment of pDNA with high concentrations of these compounds caused a disappearance of pDNA. For the parent drug, sodium diclofenac, no effect on the pDNA was observed. This study presents some indications that the binuclear copper(II) complexes, [Cu(L)²(S)]², could have some relevance in the treatment of tumor cell lines.     

Molecular modeling and functional analysis of the AtoS-AtoC two-component signal transduction system of Escherichia coli

The AtoS-AtoC two-component signal transduction system positively regulates the expression of the atoDAEB operon in Escherichia coli. Upon acetoacetate induction, AtoS sensor kinase autophosphorylates and subsequently phosphorylates, thereby activating, the response regulator AtoC. In a previous work we have shown that AtoC is phosphorylated at both aspartate 55 and histidine73. In this study, based on known three-dimensional structures of other two component regulatory systems, we modeled the 3D-structure of the receiver domain of AtoC in complex with the putative dimerization/autophosphorylation domain of the AtoS sensor kinase. The produced structural model indicated that aspartate 55, but not histidine 73, of AtoC is in close proximity to the conserved, putative phosphate-donor, histidine (H398) of AtoS suggesting that aspartate 55 may be directly involved in the AtoS-AtoC phosphate transfer. Subsequent biochemical studies with purified recombinant proteins showed that AtoC mutants with alterations of aspartate 55, but not histidine 73, were unable to participate in the AtoS-AtoC phosphate transfer in support of the modeling prediction. In addition, these AtoC mutants displayed reduced DNA-dependent ATPase activity, although their ability to bind their target DNA sequences in a sequence-specific manner was found to be unaltered.