The Challenge of Regulation in a Minimal Photoautotroph: Non-Coding RNAs in Prochlorococcus

Prochlorococcus, an extremely small cyanobacterium that is very abundant in the world's oceans, has a very streamlined genome. On average, these cells have about 2,000 genes and very few regulatory proteins. The limited capability of regulation is thought to be a result of selection imposed by a relatively stable environment in combination with a very small genome. Furthermore, only ten non-coding RNAs (ncRNAs), which play crucial regulatory roles in all forms of life, have been described in Prochlorococcus. Most strains also lack the RNA chaperone Hfq, raising the question of how important this mode of regulation is for these cells. To explore this question, we examined the transcription of intergenic regions of Prochlorococcus MED4 cells subjected to a number of different stress conditions: changes in light qualities and quantities, phage infection, or phosphorus starvation. Analysis of Affymetrix microarray expression data from intergenic regions revealed 276 novel transcriptional units. Among these were 12 new ncRNAs, 24 antisense RNAs (asRNAs), as well as 113 short mRNAs. Two additional ncRNAs were identified by homology, and all 14 new ncRNAs were independently verified by Northern hybridization and 5′RACE. Unlike its reduced suite of regulatory proteins, the number of ncRNAs relative to genome size in Prochlorococcus is comparable to that found in other bacteria, suggesting that RNA regulators likely play a major role in regulation in this group. Moreover, the ncRNAs are concentrated in previously identified genomic islands, which carry genes of significance to the ecology of this organism, many of which are not of cyanobacterial origin. Expression profiles of some of these ncRNAs suggest involvement in light stress adaptation and/or the response to phage infection consistent with their location in the hypervariable genomic islands

RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli

Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and transformation regulation remain unclear in this new type of plasmid transformation. In this study, we developed our previous plasmid transformation system and induced competence at early stationary phase. Despite of inoculum size, the development of competence was determined by optical cell density. DNase I interruption experiment showed that DNA was taken up exponentially within the initial 2 minutes and most transforming DNA entered E. coli cells within 10 minutes on LB-agar plates. A half-order kinetics between recipient cells and transformants was identified when cell density was high on plates. To determine whether the stationary phase master regulator RpoS plays roles in plasmid transformation, we investigated the effects of inactivating and over-expressing its encoding gene rpoS on plasmid transformation. The inactivation of rpoS systematically reduced transformation frequency, while over-expressing rpoS increased plasmid transformation. Normally, RpoS recognizes promoters by its lysine 173 (K173). We found that the K173E mutation caused RpoS unable to promote plasmid transformation, further confirming a role of RpoS in regulating plasmid transformation. In classical transformation, DNA was transferred across membranes by DNA uptake proteins and integrated by DNA processing proteins. At stationary growth phase, RpoS regulates some genes encoding membrane/periplasmic proteins and DNA processing proteins. We quantified transcription of 22 of them and found that transcription of only 4 genes (osmC, yqjC, ygiW and ugpC) encoding membrane/periplasmic proteins showed significant differential expression when wildtype RpoS and RpoSK173E mutant were expressed. Further investigation showed that inactivation of any one of these genes did not significantly reduce transformation, suggesting that RpoS may regulate plasmid transformation through other/multiple target genes

nocoRNAc: Characterization of non-coding RNAs in prokaryotes

<p>Abstract</p> <p>Background</p> <p>The interest in non-coding RNAs (ncRNAs) constantly rose during the past few years because of the wide spectrum of biological processes in which they are involved. This led to the discovery of numerous ncRNA genes across many species. However, for most organisms the non-coding transcriptome still remains unexplored to a great extent. Various experimental techniques for the identification of ncRNA transcripts are available, but as these methods are costly and time-consuming, there is a need for computational methods that allow the detection of functional RNAs in complete genomes in order to suggest elements for further experiments. Several programs for the genome-wide prediction of functional RNAs have been developed but most of them predict a genomic locus with no indication whether the element is transcribed or not.</p> <p>Results</p> <p>We present <smcaps>NOCO</smcaps>RNAc, a program for the genome-wide prediction of ncRNA transcripts in bacteria. <smcaps>NOCO</smcaps>RNAc incorporates various procedures for the detection of transcriptional features which are then integrated with functional ncRNA loci to determine the transcript coordinates. We applied RNAz and <smcaps>NOCO</smcaps>RNAc to the genome of <it>Streptomyces coelicolor </it>and detected more than 800 putative ncRNA transcripts most of them located antisense to protein-coding regions. Using a custom design microarray we profiled the expression of about 400 of these elements and found more than 300 to be transcribed, 38 of them are predicted novel ncRNA genes in intergenic regions. The expression patterns of many ncRNAs are similarly complex as those of the protein-coding genes, in particular many antisense ncRNAs show a high expression correlation with their protein-coding partner.</p> <p>Conclusions</p> <p>We have developed <smcaps>NOCO</smcaps>RNAc, a framework that facilitates the automated characterization of functional ncRNAs. <smcaps>NOCO</smcaps>RNAc increases the confidence of predicted ncRNA loci, especially if they contain transcribed ncRNAs. <smcaps>NOCO</smcaps>RNAc is not restricted to intergenic regions, but it is applicable to the prediction of ncRNA transcripts in whole microbial genomes. The software as well as a user guide and example data is available at <url>http://www.zbit.uni-tuebingen.de/pas/nocornac.htm</url>.</p