RNA Regulatory Networks as a Control of Stochasticity in Biological Systems.

The discovery that the non-protein coding part of human genome, dismissed as "junk DNA," is actively transcripted and carries out crucial functions is probably one of the most important discoveries of the past decades. These transcripts are becoming the rising stars of modern biology. In this review, we have casted a new light on RNAs. We have placed these molecules in the context of life origins, evolution with a big emphasize on the "RNA networks" concept. We discuss how this view can help us to understand the global role of RNA networks in modern cells, and can change our perception of the cell biology and therapy. Finally, although high-throughput methods as well as traditional case-to-case studies have laid the groundwork for our current knowledge of transcriptomes, we would like to discuss new strategies that are better suited to uncover and tackle these integrated and complex RNA networks.

Synthesis and Physicochemical Characterization of D-Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis.

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated.

A pathway closely related to the (D)-tagatose pathway of gram-negative enterobacteria identified in the gram-positive bacterium Bacillus licheniformis.

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus.

IND-6, a highly divergent IND-type metallo-beta-lactamase from Chryseobacterium indologenes strain 597 isolated in Burkina Faso.

The genus Chryseobacterium and other genera belonging to the family Flavobacteriaceae include organisms that can behave as human pathogens and are known to cause different kinds of infections. Several species of Flavobacteriaceae, including Chryseobacterium indologenes, are naturally resistant to beta-lactam antibiotics (including carbapenems), due to the production of a resident metallo-beta-lactamase. Although C. indologenes presently constitutes a limited clinical threat, the incidence of infections caused by this organism is increasing in some settings, where isolates that exhibit multidrug resistance phenotypes (including resistance to aminoglycosides and quinolones) have been detected. Here, we report the identification and characterization of a new IND-type variant from a C. indologenes isolate from Burkina Faso that is resistant to beta-lactams and aminoglycosides. The levels of sequence identity of the new variant to other IND-type metallo-beta-lactamases range between 72 and 90% (for IND-4 and IND-5, respectively). The purified enzyme exhibited N-terminal heterogeneity and a posttranslational modification consisting of the presence of a pyroglutamate residue at the N terminus. IND-6 shows a broad substrate profile, with overall higher turnover rates than IND-5 and higher activities than IND-2 and IND-5 against ceftazidime and cefepime.

Structure of the archaeal pab87 peptidase reveals a novel self-compartmentalizing protease family.

Self-compartmentalizing proteases orchestrate protein turnover through an original architecture characterized by a central catalytic chamber. Here we report the first structure of an archaeal member of a new self-compartmentalizing protease family forming a cubic-shaped octamer with D(4) symmetry and referred to as CubicO. We solved the structure of the Pyrococcus abyssi Pab87 protein at 2.2 A resolution using the anomalous signal of the high-phasing-power lanthanide derivative Lu-HPDO3A. A 20 A wide channel runs through this supramolecular assembly of 0.4 MDa, giving access to a 60 A wide central chamber holding the eight active sites. Surprisingly, activity assays revealed that Pab87 degrades specifically d-amino acid containing peptides, which have never been observed in archaea. Genomic context of the Pab87 gene showed that it is surrounded by genes involved in the amino acid/peptide transport or metabolism. We propose that CubicO proteases are involved in the processing of d-peptides from environmental origins.

Specificity and reversibility of the transpeptidation reaction catalyzed by the Streptomyces R61 D-Ala-D-Ala peptidase.

The specificity of the Streptomyces R61 penicillin-sensitive D-Ala-D-Ala peptidase has been re-examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with Gly-L-Xaa dipeptides as acceptor substrates are themselves poor substrates of the enzyme. This is in apparent contradiction with the classically accepted specificity rules for D-Ala-D-Ala peptidases. The Gly-L-Xaa dipeptide is regenerated by both the hydrolysis and transpeptidation reactions. The latter reaction is observed when another Gly-L-Xaa peptide or D-Alanine are supplied as acceptors. Utilization of substrates in which the terminal -COO(-) group has been esterified or amidated shows that a free carboxylate is not an absolute prerequisite for activity. The results are discussed in the context of the expected reversibility of the transpeptidation reaction.

Specificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-carboxypeptidase with new penicillin binding activity by directed mutagenesis.

The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to beta-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed.

Inactivation of bacterial DD-peptidase by beta-sultams.

N-Acyl-beta-sultams are time-dependent, irreversible active site-directed inhibitors of Streptomyces R61 DD-peptidase. The rate of inactivation is first order with respect to beta-sultam concentration, and the second-order rate constants show a dependence on pH similar to that for the hydrolysis of a substrate. Inactivation is due to the formation of a stable 1:1 enzyme-inhibitor complex as a result of the active site serine being sulfonylated by the beta-sultam as shown by ESI-MS analysis and by X-ray crystallography. A striking feature of the sulfonyl enzyme is that the inhibitor is not bound to the oxyanion hole but interacts extensively with the "roof" of the active site where the Arg 285 is located.

The fate of the BlaI repressor during the induction of the Bacillus licheniformis BlaP beta-lactamase.

The induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis 749/I BlaP beta-lactamases by beta-lactam antibiotics occurs according to similar processes. In both bacteria, the products of the blaI and blaRl genes share a high degree of sequence homology and act as repressors and penicillin-sensory transducers respectively. It has been shown in S. aureus that the BlaI repressor, which controls the expression of BlaZ negatively, is degraded after the addition of the inducer. In the present study,we followed the fate of BlaI during beta-lactamase induction in B. licheniformis 749/I and in a recombinant Bacillus subtilis 168 strain harbouring the pDML995 plasmid, which carries the B. licheniformis blaP, blaI and blaRl genes. In contrast to the situation in B. licheniformis 749/I, beta-lactamase induction in B.subtilis 168/pDML995 was not correlated with the proteolysis of BlaI. To exclude molecular variations undetectable by SDS-PAGE, two-dimensional gel electrophoresis was performed with cellular extracts from uninduced or induced B. subtilis 168/pDML995cells. No variation in the Blal isoelectric point was observed in induced cells, whereas the DNA-binding property was lost. Cross-linking experiments with dithiobis(succimidylpropionate) confirmed that, in uninduced recombinant B. subtilis cells, BlaI was present as a homodimer and that this situation was not altered in induced conditions. This latter result is incompatible with a mechanism of inactivation of BlaI by proteolysis and suggests that the inactivation of BlaI results from a non-covalent modification by a co-activator and that the subsequent proteolysis of BlaI might be a secondary phenomenon. In addition to the presence of this co-activator, our results show that the presence of penicillin stress is also required for full induction of beta-lactamase biosynthesis.