High quality permanent draft genome sequence of Phaseolibacter flectens ATCC 12775^T, a plant pathogen of French bean pods

Abstract

Phaseolibacter flectens strain ATCC 12775^T (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.

Introduction

Phaseolibacter flectens ATCC 12775^T (= CFBP 3281^T , ICMP 745^T , LMG 2187^T , NCPPB 539^T ), was isolated from infected French bean ( Phaseolus vulgaris ) pods in Queensland, Australia by Johnson (1956) [1]. Johnson, identified strain ATCC 12775^T as Pseudomonas flectens [1], however, 29 years later, De Vos et al. [2] argued, that Ps. flectens Johnson (1956) does not belong to the genus Pseudomonas and thus should be removed from this genus. Anzai et al. [3] demonstrated that Ps. flectens should be included in the cluster of the Enterobacteriaceae family [4]. Recently, Halpern et al. [5], 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. [5]. 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 [4].

Johnson [1], studied a disease which caused blighting and twisting of French bean pods. He isolated strain ATCC 12775^T 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 12775^T , 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 [1]. Johnson [1] 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 [1].

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 12775^T ).

Organism information

Classification and features

Ph. flectens strain ATCC 12775^T share typical characteristics of Enterobacteriaceae members such as: Gram negative, facultative anaerobic, chemoheterotrophic rod, positive for catalase and glucose fermentation and negative for oxidase [5] (Table 1). The phylogenetic tree based on the 16S rRNA also supports the fact that strain ATCC 12775^T is a member of the family Enterobacteriaceae (Fig. 1), as was already suggested by Anzai et al. [3]. Ph. flectens is the type species of the genus Phaseolibacter , which currently comprises only one species [5].

Table 1 Classification and general features of Phaseolibacter flectens strain ATCC 12775^T according to the MIGS recommendations [24], published by the genome standards consortium [25] and the names for life database [26]

Fig. 1

Phylogenetic tree highlighting the position of Phaseolibacter flectens relative to type species within the family Enterobacteriaceae. The sequence alignments were performed by using the CLUSTAL W program and the tree was generated using the neighbor joining method in MEGA 5 software [23]. Bootstrap values (from 1,000 replicates) greater than 40 % are shown at the branch points. The bar indicates a 0.5 % sequence divergence

Cells of Ph. flectens strain ATCC 12775^T 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 [5]. 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 [5].

Fig. 2

Electron micrograph of negatively stained cells of strain Phaseolibacter flectens. Cells are nonflagellated rods when grown on media supplemented with sucrose. However, a flagellum can be seen when the strain is grown on media without the supplementation of sucrose. Bar, 200 nm

Chemotaxonomic data

The major fatty acids are: C_16:0; Summed feature 2 (one or more of C_14:0 3-OH, iso-C_16:1 I and unknown ECL 10.928) and Summed feature 3 (C_16:1 ω7c and/or iso-C_15:0 2-OH) [2]. Minor fatty acids are: unknown 13.957; C_17:0 cyclo; C_18:1 ω7c; C_12:0; C_14:0 2-OH and C_14:0 [5].

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 [8]. The goal of the KMG-I study is to increase the coverage of sequenced reference microbial genomes [9]. The project is registered in the Genomes OnLine Database [10] 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 [11]. A summary of the project information is shown in Table 2.

Table 2 Genome sequencing project information

Growth conditions and genomic DNA preparation

Ph. flectens strain ATCC 12775^T 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 12775^T.

Genome sequencing and assembly

The draft genome of Ph. flectens was generated at the DOE Joint genome Institute (JGI) using the Illumina technology [12]. 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 [13], (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 [14]. 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.

Genome annotation

The assembled sequence was annotated using the JGI prokaryotic annotation pipeline [15] and was further reviewed using the Integrated Microbial Genomes—Expert Review platform [16]. Genes were identified using Prodigal [17]. CRISPR elements were detected using CRT [18] and PILER-CR [19]. The final annotated genome is available from the Integrated Microbial Genome system [20].

Genome properties

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.

Table 3 Genome statistics

Table 4 Number of genes associated with the general COG functional categories

Insights from the genome sequence

Ph. flectens was isolated from pods of diseased French bean plants. The genome of Ph. flectens strain ATCC 12775^T 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 12775^T , 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 [21]. Another feature of bacterial phytopathogenesis is the secretion of pectinolytic enzymes by microorganisms [22]. 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).

Conclusions

In the current study we characterized the genome of Ph. flectens strain ATCC 12775^T , that was isolated from French bean pods in Queensland, Australia [1]. Strain ATCC 12775^T 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 [1]. Genes indicating the potential of strain ATCC 12775^T 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 [21] and pectate lyase that has the potential to degrade the pectic components of the plant cell wall [22].