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High quality permanent draft genome sequence of Phaseolibacter flectens ATCC 12775T, a plant pathogen of French bean pods
Standards in Genomic Sciencesvolume 11, Article number: 4 (2016)
Phaseolibacter flectens strain ATCC 12775T (Halpern et al., Int J Syst Evol Microbiol 63:268–273, 2013) is a Gram-negative, rod shaped, motile, aerobic, chemoorganotroph bacterium. Ph. flectens is as a plant-pathogenic bacterium on pods of French bean and was first identified by Johnson (1956) as Pseudomonas flectens. After its phylogenetic position was reexamined, Pseudomonas flectens was transferred to the family Enterobacteriaceae as Phaseolibacter flectens gen. nov., comb. nov. Here we describe the features of this organism, together with the draft genome sequence and annotation. The DNA GC content is 44.34 mol%. The chromosome length is 2,748,442 bp. It encodes 2,437 proteins and 89 RNA genes. Ph. flectens genome is part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes study.
Phaseolibacter flectens ATCC 12775T (= CFBP 3281T , ICMP 745T , LMG 2187T , NCPPB 539T ), was isolated from infected French bean ( Phaseolus vulgaris ) pods in Queensland, Australia by Johnson (1956) . Johnson, identified strain ATCC 12775T as Pseudomonas flectens , however, 29 years later, De Vos et al.  argued, that Ps. flectens Johnson (1956) does not belong to the genus Pseudomonas and thus should be removed from this genus. Anzai et al.  demonstrated that Ps. flectens should be included in the cluster of the Enterobacteriaceae family . Recently, Halpern et al. , reclassified the species Ps. flectens Johnson 1956 as the type species of a novel genus Phaseolibacter in the family Enterobacteriaceae , as Phaseolibacter flectens gen. nov., comb. nov. . Currently, the Enterobacteriaceae family comprises more than 60 different genera. Species belonging to this family exist in diverse environments such as water, terrestrial habitats, human, animals, insects or plants .
Johnson , studied a disease which caused blighting and twisting of French bean pods. He isolated strain ATCC 12775T along with other strains that he identified as the same species from the diseased plants and proved that by inoculating healthy bean pods with pure culture of strain ATCC 12775T , the pods became twisted. The fact that the infection with Ph. flectens was confined to the pods, suggested that the introduction of the bacteria to the crop, took place after the flowering . Johnson  demonstrated in experiments that were carried out in the laboratory and in a glasshouse, that bean thrips (Taeniothrips nigricornis), which are tiny, slender insects that feed on pollen and floral tissue, transmitted this plant pathogenic bacterium between the crop plants .
Here we describe a summary classification and a set of the features of the plant pathogenic bacterium Ph. flectens , together with the permanent draft genome sequence description and annotation of the type strain (ATCC 12775T ).
Classification and features
Ph. flectens strain ATCC 12775T share typical characteristics of Enterobacteriaceae members such as: Gram negative, facultative anaerobic, chemoheterotrophic rod, positive for catalase and glucose fermentation and negative for oxidase  (Table 1). The phylogenetic tree based on the 16S rRNA also supports the fact that strain ATCC 12775T is a member of the family Enterobacteriaceae (Fig. 1), as was already suggested by Anzai et al. . Ph. flectens is the type species of the genus Phaseolibacter , which currently comprises only one species .
Cells of Ph. flectens strain ATCC 12775T are motile rods by means of one or two flagella, measuring 0.5–0.8 μm in width and 1.2–2.3 μm in length (Fig. 2). When cells are grown on LB or R2A agar media for 48 h, colonies are 1 mm diameter, however, when cells are grown on the same media supplemented with sucrose, colonies are 3–5 mm diameter, produce huge amount of mucus, smooth, foggy and grayish white colored and motility is not observed. Growth is observed under anaerobic conditions . Grows at 4–44 °C (optimum, 28–30 °C), with 0–60 % sucrose (optimum, 10–25 % sucrose) (Table 1). Growth is observed on MacConkey agar. D-glucose, sucrose, D-melibiose, glycerol, D-fructose are fermented; acetoin is produced; H2S and indole are not produced; gelatin and urea are not hydrolyzed; citrate is not utilized; nitrate is reduced to nitrogen. L-arabinose, D-manitol, inositol, sorbitol, rhamnose, and amygdalin are not fermented. Tryptophane deaminase activity is present; ß-galactosidase, arginine dihydrolase, lysine and ornithine decarboxylases activities are absent .
The major fatty acids are: C16:0; Summed feature 2 (one or more of C14:0 3-OH, iso-C16:1 I and unknown ECL 10.928) and Summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH) . Minor fatty acids are: unknown 13.957; C17:0 cyclo; C18:1 ω7c; C12:0; C14:0 2-OH and C14:0 .
Genome sequencing information
Genome project history
This organism was selected for sequencing based on its phylogenetic position [6, 7] and is part of the study Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes project . The goal of the KMG-I study is to increase the coverage of sequenced reference microbial genomes . The project is registered in the Genomes OnLine Database  and the permanent draft genome sequence is deposited in GenBank. Draft sequencing and assembly were performed at the DOE Joint Genome Institute (jgi.doe.gov) using state of the art sequencing technology . A summary of the project information is shown in Table 2.
Growth conditions and genomic DNA preparation
Ph. flectens strain ATCC 12775T was grown in the appropriate medium as recommended on the web pages of the collection (Nutrient agar or broth). The purity of the culture was determined by growth on general maintenance media. Cells were harvested by centrifugation and genomic DNA was extracted from lysozyme-treated cells using cetyltrimethyl ammonium bromide and phenol-chloroform. The purity, quality and size of the bulk genomic DNA preparation was assessed according to DOE-JGI guidelines. Amplification and partial sequencing of the 16S rRNA gene confirmed the identity of strain 12775T.
Genome sequencing and assembly
The draft genome of Ph. flectens was generated at the DOE Joint genome Institute (JGI) using the Illumina technology . An Illumina std. shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 18,689,832 reads totaling 2,803.5 Mb. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website (jgi.doe.gov). All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts (Mingkun L, Copeland A, Han J. DUK, unpublished, 2011). Following steps were then performed for assembly: (1). filtered Illumina reads were assembled using Velvet , (2). 1–3 kb simulated paired end reads were created from Velvet contigs using wgsim (https://github.com/lh3/wgsim), (3). Illumina reads were assembled with simulated read pairs using Allpaths–LG . Parameters for assembly steps were: (1). Velvet (velveth: 63 –shortPaired and velvetg: −very clean yes –exportFiltered yes –min contig lgth 500 –scaffolding no –cov cutoff 10) (2). wgsim (−e 0 –1 100 –2 100 –r 0 –R 0 –X 0) (3). Allpaths–LG (PrepareAllpathsInputs: PHRED 64 = 1 PLOIDY = 1 FRAG COVERAGE = 125 JUMP COVERAGE = 25 LONG JUMP COV = 50, RunAllpathsLG: THREADS = 8 RUN = std shredpairs TARGETS = standard VAPI WARN ONLY = True OVERWRITE = True). The final draft assembly contained 29 contigs in 26 scaffolds, totalling 2.7 Mb in size. The final assembly was based on 1,500.0 MB of Illumina data.
The assembled sequence was annotated using the JGI prokaryotic annotation pipeline  and was further reviewed using the Integrated Microbial Genomes—Expert Review platform . Genes were identified using Prodigal . CRISPR elements were detected using CRT  and PILER-CR . The final annotated genome is available from the Integrated Microbial Genome system .
The assembly of the draft genome sequence consists of 26 scaffolds amounting to 2,748,442 bp, and the G + C content is 44.34 % (Table 3, Additional file 1: Table S1). Of the 2,526 genes predicted, 2,437 were protein-coding genes, and 89 RNAs. The majority of the protein-coding genes (81.2 %) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.
Insights from the genome sequence
Ph. flectens was isolated from pods of diseased French bean plants. The genome of Ph. flectens strain ATCC 12775T reveals the presence of virulence associated genes which demonstrate that indeed, this species has the potential to attack plant tissues. Salmonella-Shigella invasin protein C (IpaC SipC) gene is present in the genome of Ph. flectens and represents a family of proteins associated with bacterial type III secretion systems, which are injection machines for virulence factors into host cell cytoplasm. A heat labile enterotoxin alpha chain that belongs to the ADP-ribosylation superfamily, is also present in the Ph. flectens genome. Five genes in the genome of Ph. flectens encode the virulence factor hemolysin which has a lytic activity on eukaryotic cells. These genes are: hemolysin activation/secretion protein (two copies); hemolysin-coregulated protein; phospholipase/lecithinase/hemolysin; hemolysins and related proteins containing CBS domains and putative hemolysin. Two copies of a gene encoding filamentous hemagglutinin family N-terminal domain are encoded in the genome of strain ATCC 12775T , representing another virulence potential of this bacterium. Filamentous hemagglutinin-like adhesins are virulence factors in both plant and animal pathogens and have a role in the attachment, aggregation and cell killing . Another feature of bacterial phytopathogenesis is the secretion of pectinolytic enzymes by microorganisms . Pectate lyase (two copies) is found in the genome, demonstrating the potential of this species to degrade the pectic components of the plant cell wall.
The potential of Ph. flectens to produce pili is evident from the presence of seven pili genes: prepilin-type N-terminal cleavage/methylation domain; P pilus assembly protein, pilin FimA (eight copies); P pilus assembly protein, chaperone PapC (two copies); P pilus assembly protein, chaperone PapD (three copies); P pilus assembly/Cpx signaling pathway, periplasmic inhibitor/zinc-resistance associated protein; Type II secretory pathway, ATPase PulE/Tfp pilus assembly pathway, ATPase PilB and CblD like pilus biogenesis initiator (two copies).
The presence of the gene for S-ribosylhomocysteine lyase LuxS indicates that Ph. flectens produces quorum-sensing autoinducer 2 (AI-2).
In the current study we characterized the genome of Ph. flectens strain ATCC 12775T , that was isolated from French bean pods in Queensland, Australia . Strain ATCC 12775T is a plant pathogen that cause pod twist disease in French bean plants. The bacteria cause the destruction of immature bean pods, immediately after the flowering stage. The blighted pods wither and drop to the ground or remain hanging and become twisted. Bean thrips (Taeniothrips nigricornis), are the ones that probably transmit this plant pathogenic bacterium between the crop plants . Genes indicating the potential of strain ATCC 12775T to cause plant disease were found in the bacterial genome. Among them were: injection machine for virulence factors into host cell cytoplasm (invasin protein C (IpaC_SipC)); heat labile enterotoxin; phospholipase/lecithinase/hemolysin which is capable of destroying the Eukaryotic cell membrane; filamentous hemagglutinin-like adhesins which have a role in the attachment, aggregation and host cell killing  and pectate lyase that has the potential to degrade the pectic components of the plant cell wall .
One thousand microbial genomes
Genomic encyclopedia of Bacteria and Archaea
Minimum information about a genome sequence
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This project has been supported by the Community Sequencing Program of the U.S. Department of Energy’s Joint Genome Institute. The sequencing, assembly and automated genome analysis work at the DOE-JGI was supported by the Office of Science of the U.S. Department of Energy under contract no. DE-AC02-05CH11231. AL was supported in part by St. Petersburg State University grant (No 18.104.22.1685). This work was also supported in part by a grant from the Israel Science Foundation (ISF, grant no. 1094/12) (Prof. Ido Izhaki and Prof. Malka Halpern, PIs) and in part by a grant from the German Research Foundation (DFG, the Deutsche Forschungsgemeinschaft, GZ: HO 930/5-1) (Prof. Malka Halpern).
The authors declare that they have no competing interests.
YGA, II and MH characterized strain ATCC 12775T and transferred it from the genus Pseudomonas to Phaseolibacter gen. nov; YGA, II, MH, AL and NCK drafted the manuscript. AC, TBKR, MH, MP, MG, VM, TW and NCK sequenced, assembled and annotated the genome. All authors read and approved the final manuscript.
Scaffolds and contigs of Genomic DNA for Phaseolibacter flectens ATCC 12775T (Topology; linear, Read depth; 1.00). (DOCX 24 kb)