Björn Voss

Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42.

Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.

An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803

There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ∼64% of all TSS give rise to antisense or ncRNAs in a genome that is to 87% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5′ annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research.

Pure Multiple RNA Secondary Structure Alignments: A Progressive Profile Approach

In functional, noncoding RNA, structure is often essential to function. While the full 3D structure is very difficult to determine, the 2D structure of an RNA molecule gives good clues to its 3D structure, and for molecules of moderate length, it can be predicted with good reliability. Structure comparison is, in analogy to sequence comparison, the essential technique to infer related function. We provide a method for computing multiple alignments of RNA secondary structures under the tree alignment model, which is suitable to cluster RNA molecules purely on the structural level, i.e., sequence similarity is not required. We give a systematic generalization of the profile alignment method from strings to trees and forests. We introduce a tree profile representation of RNA secondary structure alignments which allows reasonable scoring in structure comparison. Besides the technical aspects, an RNA profile is a useful data structure to represent multiple structures of RNA sequences. Moreover, we propose a visualization of RNA consensus structures that is enriched by the full sequence information.

Beyond Mfold: recent advances in RNA bioinformatics.

Abstract Computational analysis of RNA secondary structure is a classical field of biosequence analysis, which has recently gained momentum due to the manyfold regulatory functions of RNA that have become apparent. We present five recent computational approaches that address the problems of synoptic folding space analysis, pseudoknot prediction, structure alignment, comparative structure prediction, and miRNA target prediction. All these programs are in current use and are available via the Bielefeld Bioinformatics Server at http://bibiserv.techfak.uni-bielefeld.de.

Small RNA-mediated control of the Agrobacterium tumefaciens GABA binding protein

Wounded plants activate a complex defence programme in response to Agrobacterium tumefaciens. They synthesize the non‐proteinogenic amino acid γ‐aminobutyric acid (GABA), which stimulates degradation of the quorum sensing signal N‐(3‐oxo‐octanoyl) homoserine lactone. GABA is transported into A. tumefaciens via an ABC transporter dependent on the periplasmic binding protein Atu2422. We demonstrate that expression of atu2422 and two other ABC transporter genes is downregulated by the conserved small RNA (sRNA) AbcR1 (for ABC regulator). AbcR1 is encoded in tandem with another sRNA, which is similar in sequence and structure. Both sRNAs accumulate during stationary phase but only the absence of AbcR1 resulted in significant accumulation of Atu2422 and increased GABA import. AbcR1 inhibits initiation of atu2422 translation by masking its Shine–Dalgarno sequence and thereby reduces stability of the atu2422 transcript. It is the first described bacterial sRNA that controls uptake of a plant‐generated signalling molecule. Given that similar sRNAs and ABC transporter genes are present in various Rhizobiaceae and in Brucella, it is likely that such sRNA‐mediated control impacts a number of host–microbe interactions.

Expression of small RNAs in Rhizobiales and protection of a small RNA and its degradation products by Hfq in Sinorhizobium meliloti

Regulatory RNA plays a pivotal role in the regulation of bacterial gene expression. Here, five small RNAs were studied in Sinorhizobium meliloti - SmrC15, SmrC16, Sra33, 6S and the signal recognition particle (SRP) RNA, which are conserved among at least seven different Rhizobium and Sinorhizobium species. The amount of SmrC16 decreased in stationary phase, while the other RNAs were up-regulated. The smallest changes, maximally 2-fold, were observed for 6S RNA. In the distantly related Bradyrhizobium japonicum, the amount of 6S RNA was not increased in stationary phase, suggesting some functional divergence in the roles of this molecule in Rhizobiales in comparison to Escherichia coli. Different decay rates were observed for SmrC15 and SmrC16 of S. meliloti upon rifampicin treatment, revealing posttranscriptional regulation during growth. The use of a Deltahfq mutant showed that Hfq protects full-length SmrC16 from degradation and stabilises its specific degradation products.

Heterocyst-specific transcription of NsiR1, a non-coding RNA encoded in a tandem array of direct repeats in cyanobacteria.

In response to nitrogen deficiency, some cyanobacteria develop heterocysts, a terminally differentiated cell type, specialized for the fixation of atmospheric nitrogen. In Nostocales, this differentiation process is controlled by two major regulators, NtcA and HetR, but additional unknown factors are likely to be involved as well. In the context of a genome-wide search for potential non-coding RNAs, we identified an array of 12 tandem repeats that is transcribed in large amounts when cells enter conditions that trigger cell differentiation and switch to nitrogen fixation. The main accumulating transcript, which we suggest designating nitrogen stress-induced RNA 1 (NsiR1), has properties similar to regulatory non-coding RNAs. In Anabaena sp. PCC 7120, it is about 60 nt in length, has a very distinct predicted secondary structure, and is expressed very early and transiently after nitrogen step-down. Moreover, its expression requires HetR and NtcA and is restricted to cells that are differentiating into heterocysts, clearly placing NsiR1 within the regulon that controls the switch to nitrogen fixation and heterocyst formation. The genomic arrangement of NsiR1, located upstream of hetF, a gene whose product is involved in heterocyst formation, is conserved in all five Nostocales whose genomes are completely sequenced. Additionally, we detected NsiR1 expression in 19 different heterocyst-forming cyanobacteria. Our data suggest that every repeat is a complete transcriptional unit furnished with a cell-type-specific promoter and a Rho-independent terminator, which gives rise to a very high NsiR1 transcript level. NsiR1 is the first known bacterial non-coding RNA that is specifically upregulated in response to nitrogen step-down.

Small RNAs of the Bradyrhizobium/Rhodopseudomonas lineage and their analysis.

Small RNAs (sRNAs) play a pivotal role in bacterial gene regulation. However, the sRNAs of the vast majority of bacteria with sequenced genomes still remain unknown since sRNA genes are usually difficult to recognize and thus not annotated. Here, expression of seven sRNAs (BjrC2a, BjrC2b, BjrC2c, BjrC68, BjrC80, BjrC174 and BjrC1505) predicted by genome comparison of Bradyrhizobium and Rhodopseudomonas members, was verified by RNA gel blot hybridization, microarray and deep sequencing analyses of RNA from the soybean symbiont Bradyrhizobium japonicum USDA 110. BjrC2a, BjrC2b and BjrC2c belong to the RNA family RF00519, while the other sRNAs are novel. For some of the sRNAs we observed expression differences between free-living bacteria and bacteroids in root nodules. The amount of BjrC1505 was decreased in nodules. By contrast, the amount of BjrC2a, BjrC68, BjrC80, BjrC174 and the previously described 6S RNA was increased in nodules, and accumulation of truncated forms of these sRNAs was observed. Comparative genomics and deep sequencing suggest that BjrC2a is an antisense RNA regulating the expression of inositol-monophosphatase. The analyzed sRNAs show a different degree of conservation in Rhizobiales, and expression of homologs of BjrC2, BjrC68, BjrC1505, and 6S RNA was confirmed in the free-living purple bacterium Rhodopseudomonas palustris 5D.

Non-coding RNAs in marine Synechococcus and their regulation under environmentally relevant stress conditions

Regulatory small RNAs (sRNAs) have crucial roles in the adaptive responses of bacteria to changes in the environment. Thus far, potential regulatory RNAs have been studied mainly in marine picocyanobacteria in genetically intractable Prochlorococcus, rendering their molecular analysis difficult. Synechococcus sp. WH7803 is a model cyanobacterium, representative of the picocyanobacteria from the mesotrophic areas of the ocean. Similar to the closely related Prochlorococcus it possesses a relatively streamlined genome and a small number of genes, but is genetically tractable. Here, a comparative genome analysis was performed for this and four additional marine Synechococcus to identify the suite of possible sRNAs and other RNA elements. Based on the prediction and on complementary microarray profiling, we have identified several known as well as 32 novel sRNAs. Some sRNAs overlap adjacent coding regions, for instance for the central photosynthetic gene psbA. Several of these novel sRNAs responded specifically to environmentally relevant stress conditions. Among them are six sRNAs changing their accumulation level under cold stress, six responding to high light and two to iron limitation. Target predictions suggested genes encoding components of the light-harvesting apparatus as targets of sRNAs originating from genomic islands and that one of the iron-regulated sRNAs might be a functional homolog of RyhB. These data suggest that marine Synechococcus mount adaptive responses to these different stresses involving regulatory sRNAs.

Small RNAs of the halophilic archaeon Haloferax volcanii

In recent years, sRNAs (small non-coding RNAs) have been found to be abundant in eukaryotes and bacteria and have been recognized as a novel class of gene expression regulators. In contrast, much less is known about sRNAs in archaea, except for snoRNAs (small nucleolar RNAs) that are involved in the modification of bases in stable RNAs. Therefore bioinformatic and experimental RNomics approaches were undertaken to search for the presence of sRNAs in the model archaeon Haloferax volcanii, resulting in more than 150 putative sRNA genes being identified. Northern blot analyses were used to study (differential) expression of sRNA genes. Several chromosomal deletion mutants of sRNA genes were generated and compared with the wild-type. It turned out that two sRNAs are essential for growth at low salt concentrations and high temperatures respectively, and one is involved in the regulation of carbon metabolism. Taken together, it could be shown that sRNAs are as abundant in H. volcanii as they are in well-studied bacterial species and that they fulfil important biological roles under specific conditions.