Harnessing the intragenomic variability of rRNA operons to improve differentiation of Vibrio species.

Although the 16S rRNA gene is frequently used as a phylogenetic marker in analysis of environmental DNA, this marker often fails to distinguish closely related species, including those in the genus Vibrio. Here, we investigate whether inclusion and analysis of 23S rRNA sequence can help overcome the intrinsic weaknesses of 16S rRNA analyses for the differentiation of Vibrio species. We construct a maximum likelihood 16S rRNA gene tree to assess the use of this gene to identify clades of Vibrio species. Within the 16S rRNA tree, we identify the putative informative bases responsible for polyphyly, and demonstrate the association of these positions with tree topology. We demonstrate that concatenation of 16S and 23S rRNA genes increases the number of informative nucleotide positions, thereby overcoming ambiguities in 16S rRNA-based phylogenetic reconstructions. Finally, we experimentally demonstrate that this approach considerably improves the differentiation and identification of Vibrio species in environmental samples.

Analysis of laccase-like enzymes secreted by fungi isolated from a cave in northern Spain.

Laccases belong to a family of multicopper enzymes able to oxidize a broad spectrum of organic compounds. Despite the well-known property of laccases to carry out bleaching and degradation of industrial dyes and polyphenolic compounds, their industrial use is often limited by the high cost, low efficiency, or instability of these enzymes. To look for new microorganisms which produce laccases that are potentially suitable for industrial applications, we have isolated several fungal strains from a cave in northern Spain. Their phenotypic analysis on agar plates supplemented with ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) disclosed two laccase-positive strains. Further genotyping revealed that they belonged to the Gliomastix murorum and Conidiobolus thromboides species. The secretion of G. murorum and C. thromboides laccase-like enzymes was then confirmed by zymography. Further identification of these polypeptides by mass-spectroscopy revealed the nature of the laccases and made it possible to predict their functional domains and other features. In addition, plate assays revealed that the laccases secreted by both G. murorum and C. thromboides were capable of degrading industrial dyes (Congo Red, Indigo, and Eriochrome Black T). Homology modeling and substrate docking predicted the putative structure of the currently uncrystallized G. murorum enzyme as well as its amino acid residues potentially involved in interactions with these dyes. In summary, new biochemical and structural insights into decolorization mediated by G. murorum laccase as well as identification of laccase-like oxidase in C. thromboides point to a promising future for these enzymes in biotechnology.

Defining the transcription landscape of the Gram-negative marine bacterium Vibrio harveyi.

Vibrio harveyi is a Gram-negative pathogenic bacterium ubiquitously present in natural aquatic systems. Although environmental adaptability in V. harveyi may be enabled by profound reprogramming of gene expression previously observed during responses to starvation, suboptimal temperatures and other stress factors, the key characteristics of V. harveyi transcripts and operons, such as their boundaries and size as well as location of small RNA genes, remain largely unknown. To reveal the main features of the V. harveyi transcriptome, total RNA of this organism was analyzed by differential RNA sequencing (dRNA-seq). Analysis of the dRNA-seq data made it possible to define the primary transcriptome of V. harveyi along with cis-acting regulatory elements (riboswitches and leader sequences) and new genes. The latter encode a number of putative polypeptides and new phylogenetically conserved antisense RNAs potentially involved in the post-transcriptional control of gene expression.

Quaternary structure and biochemical properties of mycobacterial RNase E/G.

The RNase E/G family of endoribonucleases plays the central role in numerous post-transcriptional mechanisms in Escherichia coli and, presumably, in other bacteria, including human pathogens. To learn more about specific properties of RNase E/G homologues from pathogenic Gram-positive bacteria, a polypeptide comprising the catalytic domain of Mycobacterium tuberculosis RNase E/G (MycRne) was purified and characterized in vitro. In the present study, we show that affinity-purified MycRne has a propensity to form dimers and tetramers in solution and possesses an endoribonucleolytic activity, which is dependent on the 5'-phosphorylation status of RNA. Our data also indicate that the cleavage specificities of the M. tuberculosis RNase E/G homologue and its E. coli counterpart are only moderately overlapping, and reveal a number of sequence determinants within MycRne cleavage sites that differentially affect the efficiency of cleavage. Finally, we demonstrate that, similar to E. coli RNase E, MycRne is able to cleave in an intercistronic region of the putative 9S precursor of 5S rRNA, thus suggesting a common function for RNase E/G homologues in rRNA processing.

Mycobacterial RNase E-associated proteins.

RNase E and its complex with other proteins ('degradosome') play an important role in RNA processing and decay in Escherichia coli and in many other bacteria. To identify the proteins which can potentially interact with this enzyme in mycobacteria, Mycobacterium tuberculosis H37Rv RNase E was cloned and expressed as a 6HisFLAG-tagged fusion protein. Analysis of the mycobacterial RNase E overexpressed and purified from M. bovis BCG revealed the presence of GroEL and two other copurified proteins, products of the Mb1721 (inorganic polyphosphate/ATP-NAD kinase) and Mb0825c (acetyltransferase) genes. Identical copies of these two genes can be found in M. tuberculosis H37Rv.

Determination of the catalytic parameters of the N-terminal half of Escherichia coli ribonuclease E and the identification of critical functional groups in RNA substrates.

Ribonuclease E is required for the rapid decay and correct processing of RNA in Escherichia coli. A detailed understanding of the hydrolysis of RNA by this and related enzymes will require the integration of structural and molecular data with quantitative measurements of RNA hydrolysis. Therefore, an assay for RNaseE that can be set up to have relatively high throughput while being sensitive and quantitative will be advantageous. Here we describe such an assay, which is based on the automated high pressure liquid chromatography analysis of fluorescently labeled RNA samples. We have used this assay to optimize reaction conditions, to determine for the first time the catalytic parameters for a polypeptide of RNaseE, and to investigate the RNaseE-catalyzed reaction through the modification of functional groups within an RNA substrate. We find that catalysis is dependent on both protonated and unprotonated functional groups and that the recognition of a guanosine sequence determinant that is upstream of the scissile bond appears to consist of interactions with the exocyclic 2-amino group, the 7N of the nucleobase and the imino proton or 6-keto group. Additionally, we find that a ribose-like sugar conformation is preferred in the 5'-nucleotide of the scissile phosphodiester bond and that a 2'-hydroxyl group proton is not essential. Steric bulk at the 2' position in the 5'-nucleotide appears to be inhibitory to the reaction. Combined, these observations establish a foundation for the functional interpretation of a three-dimensional structure of the catalytic domain of RNaseE when solved.

Expanding the use of zymography by the chemical linkage of small, defined substrates to the gel matrix.

In the postgenomic era, the comprehensive proteomic analysis of metabolic and signaling pathways is inevitably faced with the challenge of large-scale identification and characterization of polypeptides with a particular enzymatic activity. Previous work has shown that a wide variety of enzymatic activities of microbial, plant, and animal origin can be assigned to individual polypeptides using in-gel activity staining (zymography). However, a number of limitations, such as special substrate requirements, the lack of a standard procedure, and difficulties in distinguishing enzymes with overlapping activities have precluded the widespread use of zymography as a routine laboratory method. Here we demonstrate that, by employing small-defined substrates that are covalently attached to the gel matrix, we can largely overcome the aforementioned problems and assay readily a number of different classes of enzymatic activities within gels after standard SDS-polyacrylamide electrophoresis. Moreover, this development is compatible with the two-dimensional separation of proteins and thus has great potential in the high-throughput screening and characterization of complex biological and clinical samples.