Prediction of Transcriptional Terminators in Bacillus subtilis and Related Species

In prokaryotes, genes belonging to the same operon are transcribed in a single mRNA molecule. Transcription starts as the RNA polymerase binds to the promoter and continues until it reaches a transcriptional terminator. Some terminators rely on the presence of the Rho protein, whereas others function independently of Rho. Such Rho-independent terminators consist of an inverted repeat followed by a stretch of thymine residues, allowing us to predict their presence directly from the DNA sequence. Unlike in Escherichia coli, the Rho protein is dispensable in Bacillus subtilis, suggesting a limited role for Rho-dependent termination in this organism and possibly in other Firmicutes. We analyzed 463 experimentally known terminating sequences in B. subtilis and found a decision rule to distinguish Rho-independent transcriptional terminators from non-terminating sequences. The decision rule allowed us to find the boundaries of operons in B. subtilis with a sensitivity and specificity of about 94%. Using the same decision rule, we found an average sensitivity of 94% for 57 bacteria belonging to the Firmicutes phylum, and a considerably lower sensitivity for other bacteria. Our analysis shows that Rho-independent termination is dominant for Firmicutes in general, and that the properties of the transcriptional terminators are conserved. Terminator prediction can be used to reliably predict the operon structure in these organisms, even in the absence of experimentally known operons. Genome-wide predictions of Rho-independent terminators for the 57 Firmicutes are available in the Supporting Information section

Characterization of relationships between transcriptional units and operon structures in Bacillus subtilis and Escherichia coli

BACKGROUND: Operon structures play an important role in transcriptional regulation in prokaryotes. However, there have been fewer studies on complicated operon structures in which the transcriptional units vary with changing environmental conditions. Information about such complicated operons is helpful for predicting and analyzing operon structures, as well as understanding gene functions and transcriptional regulation. RESULTS: We systematically analyzed the experimentally verified transcriptional units (TUs) in Bacillus subtilis and Escherichia coli obtained from ODB and RegulonDB. To understand the relationships between TUs and operons, we defined a new classification system for adjacent gene pairs, divided into three groups according to the level of gene co-regulation: operon pairs (OP) belong to the same TU, sub-operon pairs (SOP) that are at the transcriptional boundaries within an operon, and non-operon pairs (NOP) belonging to different operons. Consequently, we found that the levels of gene co-regulation was correlated to intergenic distances and gene expression levels. Additional analysis revealed that they were also correlated to the levels of conservation across about 200 prokaryotic genomes. Most interestingly, we found that functional associations in SOPs were more observed in the environmental and genetic information processes. CONCLUSION: Complicated operon strucutures were correlated with genome organization and gene expression profiles. Such intricately regulated operons allow functional differences depending on environmental conditions. These regulatory mechanisms are helpful in accommodating the variety of changes that happen around the cell. In addition, such differences may play an important role in the evolution of gene order across genomes

Investigation of the Molecular Basis for Transcriptional Regulation of Tn916 and Macrolide Resistance in Bacillus subtilis

Antibiotic resistance (AR) is one of the most serious threats to modern healthcare today. To understand how resistance spreads, we need to investigate the genetic basis of transferable AR. Conjugative transposons (CTns) have acquired the vast majority of resistance genes we currently know about which makes them one of the major vectors involved in their spread. This study aims to investigate how Tn916 and Tn916-like elements maintain their stability following insertion into a bacterial genome. We identified putative rho-independent terminators upstream of the conjugation genes of Tn2010, Tn5397, Tn6000, Tn6002, Tn6003, Tn6087 and Tn916 and hypothesised that their role is to prevent transcriptional readthrough into the conjugation genes upon integration into a new insertion site. To verify this experimentally, the terminator was cloned in between the tet(M) promoter and a gusA reporter in pHCMC05. We demonstrated the level of β-glucuronidase enzyme activity decreased, confirming termination activity. We have for the first time, identified and verified a group of conserved terminators in the conjugation region of the Tn916-like family of CTns. Further data supports our hypothesis that the terminator efficiency is modulated upon excision and circularisation of Tn916, which is the exact time when Tn916 would require expression of its conjugation genes. A fundamental understanding of the current antibiotic resistance mechanisms employed by bacteria is also essential to minimise the emergence of resistance and to devise effective resistance-control strategies. Another aim of this study is to investigate the molecular mechanism underlying macrolide resistance in Bacillus subtilis. Macrolide-resistant B. subtilis were generated as part of the project and analysis revealed a new genetic mutation to be responsible for the macrolide resistance phenotype. Comparative genome analysis revealed 21 bp and 54 bp duplication in the rplV of these mutants in comparison to the wild type strain. The rplV encodes the large ribosomal subunit protein, L22. Alteration in L22 has led to a predicted alteration in the C-terminal loop of the protein, predicted to change the shape of the exit tunnel within the ribosome. Ectopic expression of the rplV mutants containing the 21 bp and 54 bp duplication in B. subtilis BS34A confers resistance to macrolides. This is the first observation of macrolide resistance due to 54 bp duplication in the B. subtilis rplV gene

RNIE: genome-wide prediction of bacterial intrinsic terminators

Bacterial Rho-independent terminators (RITs) are important genomic landmarks involved in gene regulation and terminating gene expression. In this investigation we present RNIE, a probabilistic approach for predicting RITs. The method is based upon covariance models which have been known for many years to be the most accurate computational tools for predicting homology in structural non-coding RNAs. We show that RNIE has superior performance in model species from a spectrum of bacterial phyla. Further analysis of species where a low number of RITs were predicted revealed a highly conserved structural sequence motif enriched near the genic termini of the pathogenic Actinobacteria, Mycobacterium tuberculosis. This motif, together with classical RITs, account for up to 90% of all the significantly structured regions from the termini of M. tuberculosis genic elements. The software, predictions and alignments described below are available from http://github.com/ppgardne/RNIE

The transcriptionally active regions in the genome of Bacillus subtilis

The majority of all genes have so far been identified and annotated systematically through in silico gene finding. Here we report the finding of 3662 strand-specific transcriptionally active regions (TARs) in the genome of Bacillus subtilis by the use of tiling arrays. We have measured the genome-wide expression during mid-exponential growth on rich (LB) and minimal (M9) medium. The identified TARs account for 77.3% of the genes as they are currently annotated and additionally we find 84 putative non-coding RNAs (ncRNAs) and 127 antisense transcripts. One ncRNA, ncr22, is predicted to act as a translational control on cstA and an antisense transcript was observed opposite the housekeeping sigma factor sigA. Through this work we have discovered a long conserved 3′ untranslated region (UTR) in a group of membrane-associated genes that is predicted to fold into a large and highly stable secondary structure. One of the genes having this tail is efeN, which encodes a target of the twin-arginine translocase (Tat) protein translocation system

A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation

Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA‐K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other Staphylococci, and RsaE is also present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE‐mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C−rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanism

DBTBS: a database of transcriptional regulation in Bacillus subtilis containing upstream intergenic conservation information

DBTBS, first released in 1999, is a reference database on transcriptional regulation in Bacillus subtilis, summarizing the experimentally characterized transcription factors, their recognition sequences and the genes they regulate. Since the previous release, the original content was extended by the addition of the data contained in 569 new publications, the total of which now reaches 947. The number of B. subtilis promoters annotated in the database was more than doubled to 1475. In addition, 463 experimentally validated B. subtilis operons and their terminators have been included. Given the increase in the number of fully sequenced bacterial genomes, we decided to extend the usability of DBTBS in comparative regulatory genomics. We therefore created a new section on the conservation of the upstream regulatory sequences between homologous genes in 40 Gram-positive bacterial species, as well as on the presence of overrepresented hexameric motifs that may have regulatory functions. DBTBS can be accessed at: http://dbtbs.hgc.jp

A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation

Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA‐K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other Staphylococci, and RsaE is also present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE‐mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C−rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanis