Jiří Vohradský

Biocomputational prediction of small non-coding RNAs in Streptomyces

BackgroundThe first systematic study of small non-coding RNAs (sRNA, ncRNA) in Streptomyces is presented. Except for a few exceptions, the Streptomyces sRNAs, as well as the sRNAs in other genera of the Actinomyces group, have remained unstudied. This study was based on sequence conservation in intergenic regions of Streptomyces, localization of transcription termination factors, and genomic arrangement of genes flanking the predicted sRNAs.ResultsThirty-two potential sRNAs in Streptomyces were predicted. Of these, expression of 20 was detected by microarrays and RT-PCR. The prediction was validated by a structure based computational approach. Two predicted sRNAs were found to be terminated by transcription termination factors different from the Rho-independent terminators. One predicted sRNA was identified computationally with high probability as a Streptomyces 6S RNA. Out of the 32 predicted sRNAs, 24 were found to be structurally dissimilar from known sRNAs.ConclusionStreptomyces is the largest genus of Actinomyces, whose sRNAs have not been studied. The Actinomyces is a group of bacterial species with unique genomes and phenotypes. Therefore, in Actinomyces, new unique bacterial sRNAs may be identified. The sequence and structural dissimilarity of the predicted Streptomyces sRNAs demonstrated by this study serve as the first evidence of the uniqueness of Actinomyces sRNAs.

Point pattern matching in the analysis of two‐dimensional gel electropherograms

In the automation of proteome analysis, matching of two‐dimensional (2‐D) electropherograms represents a bottleneck in the process. Here we present a point pattern recognition approach to the matching of spots in 2‐D electropherograms. The algorithm is based on a comparison of spot neighborhoods, converted to point patterns between reference and compared gels. The neighborhood was characterized by a syntactic descriptor which minimized the influence of spot displacements. A combined criterion utilizing the similarity of point patterns and a metric definition of position similarity was derived. The efficiency and accuracy of the algorithm was tested on a set of 69 gels with different levels of similarity. For a typical gel the accuracy of matching was higher than 98% and the number of correctly identified spots was higher than 95%.

Major proteins related to chlortetracycline biosynthesis in a Streptomyces aureofaciens production strain studied by quantitative proteomics

Abstract. Changes in synthesis and abundance of proteins associated with chlortetracycline (CTC) production in Streptomyces aureofaciens were investigated by two-dimensional polyacrylamide gel electrophoresis of proteins pulse-labelled in vivo with L-[35S]methionine. Eleven individual protein spots were selected as being related to formation of the antibiotic. Expression of these prominent proteins was not observed in the non-producing mutant; moreover, they were overexpressed in cultures grown in the presence of benzyl thiocyanate, a specific stimulator of CTC biosynthesis used in industrial fermentations. The expression kinetics of the selected proteins was assessed using the technique of computer-assisted image analysis with the EQIAS software and the elongation factor Tu as an internal standard. Interestingly, the kinetic profiles were generally not identical, including those of anhydrotetracycline monooxygenase and the 13-kDa subunit of tetracycline dehydrogenase, two enzymes involved in the terminal sequential steps of the CTC biosynthetic pathway. The presence of more forms of these enzymes with different charge characteristics was observed. The data presented demonstrated how dramatically the industrial microorganism can change its protein repertoire during the production phase; at least five proteins were nearly comparable in level to the most prominent proteins, exemplified by elongation factor Tu.

An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5′ UTRs and its implications for eukaryotic gene translation regulation

There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA–rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5′ untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5′ UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5′ UTRs of mRNAs.

The suboptimal structures find the optimal RNAs: homology search for bacterial non-coding RNAs using suboptimal RNA structures

Non-coding RNAs (ncRNAs) are regulatory molecules encoded in the intergenic or intragenic regions of the genome. In prokaryotes, biocomputational identification of homologs of known ncRNAs in other species often fails due to weakly evolutionarily conserved sequences, structures, synteny and genome localization, except in the case of evolutionarily closely related species. To eliminate results from weak conservation, we focused on RNA structure, which is the most conserved ncRNA property. Analysis of the structure of one of the few well-studied bacterial ncRNAs, 6S RNA, demonstrated that unlike optimal and consensus structures, suboptimal structures are capable of capturing RNA homology even in divergent bacterial species. A computational procedure for the identification of homologous ncRNAs using suboptimal structures was created. The suggested procedure was applied to strongly divergent bacterial species and was capable of identifying homologous ncRNAs.

Kinetic modelling and meta-analysis of the B. subtilis SigA regulatory network during spore germination and outgrowth

This study describes the meta-analysis and kinetic modelling of gene expression control by sigma factor SigA of Bacillus subtilis during germination and outgrowth based on microarray data from 14 time points. The analysis computationally models the direct interaction among SigA, SigA-controlled sigma factor genes (sigM, sigH, sigD, sigX), and their target genes. Of the >800 known genes in the SigA regulon, as extracted from databases, 311 genes were analysed, and 190 were confirmed by the kinetic model as being controlled by SigA. For the remaining genes, alternative regulators satisfying kinetic constraints were suggested. The kinetic analysis suggested another 214 genes as potential SigA targets. The modelling was able to (i) create a particular SigA-controlled gene expression network that is active under the conditions for which the expression time series was obtained, and where SigA is the dominant regulator, (ii) suggest new potential SigA target genes, and (iii) find other possible regulators of a given gene or suggest a new mechanism of its control by identifying a matching profile of unknown regulator(s). Selected predicted regulatory interactions were experimentally tested, thus validating the model.

Protein profiles of Streptomyces aureofaciens producing tetracyclines: reappraisal of the effect of benzyl thiocyanate.

Cell protein profiles of submerged cultures of Streptomyces aureofaciens cultivated in the absence or presence of 12 μm benzyl thiocyanate (BT) were analyzed by one-dimensional SDS polyacrylamide gel electrophoresis. Substantial increase in the intensity of the 13, 35, 37, 60, and 100 kDa protein bands was observed in cultures treated with BT. Similar increase in the 35, 37, and 60 kDa bands was found in a mutant blocked in the last chlortetracycline biosynthesis step. Effect of BT on the solid medium-grown cultures was also observed, with a more intensive substrate mycelium pigmentation and alteration in the spore size and shape as the most characteristic features. Earlier studies of BT effect involving those on the stimulation of chlortetracycline biosynthesis are summarized and a possible signal-transducing mechanism is discussed from the point of view of adaptation of S. aureofaciens to the uncoupling of oxidative phosphorylation.