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Growth Forms of Bdellovibrio Bacteriovorus A Global Transcriptional Switch between the Attack and Growth Forms of Bdellovibrio bacteriovorus Iris Karunker1., Or Rotem2., Mally Dori-Bachash2, Edouard Jurkevitch2, Rotem Sorek1* 1 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel, 2 Department of Plant Pathology and Microbiology, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel Abstract Bdellovibrio bacteriovorus 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 Bdellovibrio 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 Bdellovibrio 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 E. coli flagellar genes regulator sigma28 (FliA). Further analyses suggest that FliA has evolved to become a global AP regulator in Bdellovibrio. 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. Citation: Karunker I, Rotem O, Dori-Bachash M, Jurkevitch E, Sorek R (2013) A Global Transcriptional Switch between the Attack and Growth Forms of Bdellovibrio bacteriovorus. PLoS ONE 8(4): e61850. doi:10.1371/journal.pone.0061850 Editor: Brian Stevenson, University of Kentucky College of Medicine, United States of America Received December 20, 2012; Accepted March 14, 2013; Published April 16, 2013 Copyright: ß 2013 Karunker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: RS was supported, in part, by the ERC-StG program (grant 260432), the Minerva Foundation, and by a DIP grant from the Deutsche Forschungsgemeinschaft. EJ was supported by the Israel Science Foundation (grant 1290/08). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work Introduction microarrays [7]. This study reported 479 genes up regulated 30 minutes after the shift from AP to GP (early GP genes) and 230 Bdellovibrio bacteriovorus is an obligate predator of gram negative genes that were downregulated (AP genes). In agreement with bacteria ubiquitously found in bulk soil, in the rhizosphere of previous studies that were based on proteomics [8] the AP genes plants, and in freshwater bodies such as rivers and water treatment functions largely included motility, chemotaxis, adhesion and facilities [1]. This organism can prey on a wide range of bacteria, outer membrane genes, which are required for detection, including many pathogens of humans and plants, and has thus attachment, and penetration into the prey cell [9,10,11]. The been suggested as an effective "living antibiotics" [2]. B. bacteriovorus GP genes include those associated with growth and division such displays a dimorphic life cycle, with two main phenotypes: Attack as ribosomal proteins and RNA polymerase components, as well Phase (AP) and Growth Phase (GP). AP cells are small, fast- as various transport and peptidoglycan metabolizing genes swimming, non-replicating "hungry" cells that search for prey, presumably involved in processing and internalization of prey while GP cells are non-motile replicative cells that actively materials [7]. Despite the recent advancement in understanding consume the prey [3]. B. bacteriovorus grows and replicates within the genetic program differentiating between AP and GP gene the periplasm of the prey cells, using their cytoplasmic content as expression [12,13], the molecular regulatory cascade leading to the its source of energy and nutrients [4]. Prior to growth, the prey cell dramatic switch between the AP and GP phenotypes of Bdellovibrio is killed and remodelled into a spherical bdelloplast. Growth is still largely unknown. within the prey cell takes place as an aseptate and polynucleoid Transcriptome-wide cDNA sequencing (RNA-seq) is a modern filament with cell division controlled by the availability of soluble method that is gradually replacing microarrays for bacterial host components [5]. The GP cycle of B. bacteriovorus lasts about transcriptome research [14]. This method allows more accurate 4 hours, with the prey being killed within 30 minutes, and quantification of RNA levels, and provides a complete coverage of bdelloplast exhaustion and division of elongated B. bacteriovorus expressed regions to a single nucleotide resolution [15]. It cells occurring about 3 hours after the initial attachment to the therefore allows a direct interrogation of the transcriptional prey [6]. The cycle is completed by a subsequent rupturing of the landscape of a genome [14]. As opposed to microarrays, RNA- bdelloplast envelope and a release of daughter AP cells. seq allows the detection of previously unannotated genes such as Expression of Bdellovibrio genes during the switch from AP to GP non-coding RNA regulators (ncRNAs). Indeed, RNA-seq studies phases was studied by Lambert et al using oligonucleotide PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e61850 Transcriptional Switch in Bdellovibrio in bacterial and archaeal organisms have revealed a plethora of five volumes of 0.85% NaCl and centrifugation at 20,000 g for functional ncRNAs and antisense RNAs, including the recently 10 min 4uC and stored as a pellet at 280uC. discovered long antisense "excludon" structures in Listeria [16,17]. Attack phase cells were obtained from a 10 mL starter culture Specific RNA-seq protocols also allow genome-wide mapping of filtered through a 0.45 mm filter, to eliminate remaining prey cells. transcription start sites (TSSs), enabling single-base resolution Cells were pelleted by centrifugation at 12000 g at 4uC for 10 min. mapping of promoter sequences for all expressed genes [17,18,19]. To date, no regulatory ncRNA has been reported in B. bacteriovorus, RNA isolation and its promoters are largely unmapped. Pellets were resuspended in 5 mL TRI reagent (Sigma), Here we investigated the organization of gene expression during homogenized 40 sec with a pipette and incubated 5 min at room the attack and growth phases in synchronized cultures of B. temperature (RT). 0.5 mL of 1-bromo-3-chloropropane was bacteriovorus using RNA-seq, mapping expressed genes as well as added, manually mixed for 15 sec, incubated at RT for 10 min their transcription start sites (TSSs). Our data revealed a sharp and centrifuged at 1500 g at 4uC for 30 min. The upper, dichotomy in gene expression between the two phases with, in the transparent phase was then transferred to a clean tube, 2.5 mL attack phase, exceptionally high levels of expression of a non- isopropanol added and manually mixed, incubated at RT for coding RNA containing a cyclic-di GMP riboswitch, and the about 10 min, then centrifuged at 1500 g at 4uC for 30 min. The expansion of gene expression control by a secondary sigma factor. RNA pellet was washed with 5 mL of a 75% ethanol solution prepared with DEPC-treated water, gently mixed, and centrifuged Materials and Methods again at 1500 g at 4uC for 10 min. The ethanol was gently removed, the pellet was dried and resuspended in 200 mL Bacterial strains and growth nuclease-free TE (Sigma). The RNA samples were treated with Bdellovibrio bacteriovorus HD100 was grown in HEPES buffer TurboDNase (Ambion). rRNA depletion was performed using (25 mM HEPES, 2 mM CaCl2.2H2O, 3 mM MgCl2.6H2O, MicrobExpress (Ambion). RNA purity and concentration were pH 7.8), at 30uC and 250 rpm in two-membered cultures with E. determined using a NanoDrop spectrophotometer and Agilent coli MG1655 (ATCC #700926) as prey. Fresh attack phase (AP) Bioanalyzer. cells were obtained from overnight cultures by inoculating 100 mL HEPES buffer with 26109 colony forming units (cfu) per mL of E. 7 21 RNA sequencing coli prey MG1655 and about 10 mL predatory cells. For the whole transcriptome sequencing, cDNA libraries were prepared by the Illumina mRNA-seq protocol according to the Synchronization of predatory cultures manufacturer’s protocol without the polyA isolation stage. Synchronization was obtained as previously described [7,8]. Sequencing was performed on an Illumina GAIIx sequencing Prey grown over night in NB at 37uC was diluted 1:100 and machine. TAP(+) and TAP(-) 5’-end cDNA libraries for TSS cultivated under the same conditions for about 2.5 h, to an optical mapping were prepared as previously described [17]. density (OD570) of 0.6 to 0.8. The cells were then pelleted at 4uC for 10 min at 4500 g, washed three times in HEPES buffer and Read mapping and expression calling resuspended in HEPES to a final volume of about 15 mL at Genome sequences of B. bacteriovorus (Genbank: NC_005363) OD570 ,10. The suspension was then stored at 4uC overnight. A and E. coli (Genbank: NC_000913) were downloaded from NCBI. starter predatory culture was established by adding 5 mL of the Sequencing reads were mapped to the genomes as previously prey suspension to 45 mL HEPES inoculated with 250 to 600 mL described [17,20].
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