Comparative transcriptomics of pathogenic and non-pathogenic Listeria species

Comparative RNA-seq analysis of two related pathogenic and non-pathogenic bacterial strains reveals a hidden layer of divergence in the non-coding genome as well as conserved, widespread regulatory structures called ‘Excludons', which mediate regulation through long non-coding antisense RNAs

Comparative transcriptomics across the prokaryotic tree of life

Whole-transcriptome sequencing studies from recent years revealed an unexpected complexity in transcriptomes of bacteria and archaea, including abundant non-coding RNAs, cis-antisense transcription and regulatory untranslated regions (UTRs). Understanding the functional relevance of the plethora of non-coding RNAs in a given organism is challenging, especially since some of these RNAs were attributed to ‘transcriptional noise’. To allow the search for conserved transcriptomic elements we produced comparative transcriptome maps for multiple species across the microbial tree of life. These transcriptome maps are detailed in annotations, comparable by gene families, and BLAST-searchable by user provided sequences. Our transcriptome collection includes 18 model organisms spanning 10 phyla/subphyla of bacteria and archaea that were sequenced using standardized RNA-seq methods. The utility of the comparative approach, as implemented in our web server, is demonstrated by highlighting genes with exceptionally long 5′UTRs across species, which correspond to many known riboswitches and further suggest novel putative regulatory elements. Our study provides a standardized reference transcriptome to major clinically and environmentally important microbial phyla. The viewer is available at http://exploration.weizmann.ac.il/TCOL, setting a framework for comparative studies of the microbial non-coding genome

Transcriptome dynamics of a broad host-range cyanophage and its hosts

Cyanobacteria are highly abundant in the oceans and are constantly exposed to lytic viruses. The T4-like cyanomyoviruses are abundant in the marine environment and have broad host-ranges relative to other cyanophages. It is currently unknown whether broad host-range phages specifically tailor their infection program for each host, or employ the same program irrespective of the host infected. Also unknown is how different hosts respond to infection by the same phage. Here we used microarray and RNA-seq analyses to investigate the interaction between the Syn9 T4-like cyanophage and three phylogenetically, ecologically and genomically distinct marine Synechococcus strains: WH7803, WH8102 and WH8109. Strikingly, Syn9 led a nearly identical infection and transcriptional program in all three hosts. Different to previous assumptions for T4-like cyanophages, three temporally regulated gene expression classes were observed. Furthermore, a novel regulatory element controlled early-gene transcription, and host-like promoters drove middle gene transcription, different to the regulatory paradigm for T4. Similar results were found for the P-TIM40 phage during infection of Prochlorococcus NATL2A. Moreover, genomic and metagenomic analyses indicate that these regulatory elements are abundant and conserved among T4-like cyanophages. In contrast to the near-identical transcriptional program employed by Syn9, host responses to infection involved host-specific genes primarily located in hypervariable genomic islands, substantiating islands as a major axis of phage-cyanobacteria interactions. Our findings suggest that the ability of broad host-range phages to infect multiple hosts is more likely dependent on the effectiveness of host defense strategies than on differential tailoring of the infection process by the phage

A Global Transcriptional Switch between the Attack and Growth Forms of <i>Bdellovibrio bacteriovorus</i>

<div><p><i>Bdellovibrio bacteriovorus</i> is an obligate predator of bacteria ubiquitously found in the environment. Its life cycle is composed of two essential phases: a free-living, non-replicative, fast swimming attack phase (AP) wherein the predator searches for prey; and a non-motile, actively dividing growth phase (GP) in which it consumes the prey. The molecular regulatory mechanisms governing the switch between AP and GP are largely unknown. We used RNA-seq to generate a single-base-resolution map of the <i>Bdellovibrio</i> transcriptome in AP and GP, revealing a specific "AP" transcriptional program, which is largely mutually exclusive of the GP program. Based on the expression map, most genes in the <i>Bdellovibrio</i> genome are classified as "AP only" or "GP only". We experimentally generated a genome-wide map of 140 AP promoters, controlling the majority of AP-specific genes. This revealed a common sigma-like DNA binding site highly similar to the <i>E. coli</i> flagellar genes regulator sigma28 (FliA). Further analyses suggest that FliA has evolved to become a global AP regulator in <i>Bdellovibrio</i>. Our results also reveal a non-coding RNA that is massively expressed in AP. This ncRNA contains a c-di-GMP riboswitch. We suggest it functions as an intracellular reservoir for c-di-GMP, playing a role in the rapid switch from AP to GP.</p></div

RT-PCR verification of mutually exclusive expression.

<p>Total RNA retrieved from AP or GP <i>B. bacteriovorus</i> HD100 during a synchronous predation of <i>E. coli</i> ML35 (0.5, 1 and 3 hrs post inoculation) was subjected to RT-PCR. Sixteen representative genes predicted by RNA-seq analysis to be AP-specific (left) or GP-specific (right) were amplified. Coli, control genomic DNA of <i>E. coli</i> ML35; AP, cDNA from AP cells; GP0.5, cDNA of GP cells 0.5 hr post inoculation; GP1, cDNA of GP cells 1 hr post inoculation; GP3, cDNA of GP cells 3 hrs post inoculation. DNA, <i>B. bacteriovorus</i> genomic DNA.</p

An AP-specific, massively expressed, riboswitch-containing sRNA.

<p>(A) Genomic organization and expression of merRNA. X-axis, position on the <i>B. bacteriovorus</i> genome; Y-axis, coverage of RNA-seq data. (B) Normalized expression of merRNA and other structural RNAs in attack phase. Expression is presented in RPKM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061850#pone.0061850-Mortazavi1" target="_blank">[21]</a> values, which are normalized for gene size and for the amount of reads in the library. (C) RT-PCR verification of merRNA expression being dominant in attack phase (AP) and reduced with infection of <i>E. coli</i> (30,60, and 180 mins after infection). Ctrl, <i>B. bacteriovorus</i> genomic DNA; ML35, genomic DNA of <i>E. coli</i> ML35.</p

AP-specific small RNAs.

<p>AP-specific small RNAs.</p

RNA-seq data used in this study.

<p>RNA-seq data used in this study.</p

GC-content distribution of attack-phase and growth-phase genes.

<p>GC-content distribution of attack-phase and growth-phase genes.</p