Phylogenetic analysis of family Neisseriaceae based on genome sequences and description of Populibacter corticis gen. nov., sp. nov., a member of the family Neisseriaceae, isolated from symptomatic bark of Populus × euramericana canker

Two Gram-stain negative aerobic bacterial strains were isolated from the bark tissue of Populus × euramericana. The novel isolates were investigated using a polyphasic approach including 16S rRNA gene sequencing, genome sequencing, average nucleotide identity (ANI) and both phenotypic and chemotaxonomic assays. The genome core gene sequence and 16S rRNA gene phylogenies suggest that the novel isolates are different from the genera Snodgrassella and Stenoxybacter. Additionally, the ANI, G+C content, main fatty acids and phospholipid profile data supported the distinctiveness of the novel strain from genus Snodgrassella. Therefore, based on the data presented, the strains constitute a novel species of a novel genus within the family Neisseriaceae, for which the name Populibacter corticis gen. nov., sp. nov. is proposed. The type strain is 15-3-5T (= CFCC 13594T = KCTC 42251T).


Introduction
In 2006, poplar bark canker with abundant white sour exudates was observed for the first time in China's Henan and Shandong provinces. Initially diseased plants had stem or branch bark that cracked and exuded a frothy fluid, when the disease progressed, many cankers (50 to 150 by 3 to 8 cm, length by width) rapidly appeared. The bacterium Lonsdalea quercina subsp. populi was demonstrated to be the causal agent of these cankers [1]. P. × euramericana were found to develop bark cancer disease in China. Of four susceptible cultivars, P. × euramericana 'Zhonglin 46' was the most susceptible cultivar, followed bycultivars '74/76' and P. deltoids PLOS  'Zhonghe 1' [1]. A specific canker symptom has been detected in poplar (P.× euramericana) stands in the central part of Hungary since 2007 which was also observed in China. The bark of symptomatic trees is vertically cracked, and a sticky, brown-colored fluid oozes from the canker. Typically, huge numbers of the free-living, bacteriophagous nematode Panagrellus redivivus feed on the oozing fluid and on the creamy mass under the cracked bark. Lonsdalea quercina subsp. populi were isolated and identified in 2013 [2]. During our investigations on the bacterial diversity of the bark tissue of P. × euramericana canker, two isolates, 15-3-5 T and TQ2-3 belonging to the family Neisseriaceae, were isolated from symptomatic bark tissue of a P. × euramericana canker sample collected from Neihuang, Anyang City, and Taiqian, Puyang city, Henan Province, China in August 2010 and September 2015, respectively. To determine the taxonomic and phylogenetic position of this organism, the morphological, physiological, biochemical and chemotaxonomic characteristics, 16S rRNA gene sequences of the two novel isolates were analyzed. The results indicated that the novel isolates should be placed as a novel species of a novel genus in the family Neisseriaceae. Up to now, there are 15 valid published genera in family Neisseriaceae listed on www.bacterio. net. Some species of genus Neisseria are very important pathogen of human and animals, such as Neisseria gonorrhoeae, which is responsible for the sexually transmitted infection gonorrhea [3].

Morphological, physiological and biochemical characterization
Observations of the colony morphology, motility and growth conditions were performed as previously described by Li et al. [4]. Catalase and oxidase activities were measured as recommended by Smibert and Krieg [5]. Gram staining was performed as reported by Schroeter [6]. Anaerobic growth was examined at 30˚C for 1 week in anaerobic jars [7]. Hydrolysis of gelatin was tested according to the methods described by Smibert & Krieg [5]. Carbon source assimilation of two novel isolates and reference strain were tested by API 20NE (bioMérieux). API ZYM and API 50CHB/E (bioMérieux) were used for testing enzyme activities and acid production on two novel isolates. The assays were conducted according to the manufacturer's instructions.
For the cellular fatty acid analysis, two novel strains were grown on tryptic soy agar (TSA, Difco) at 30˚C and harvested in the exponential phase (for 24 h). Strain Snodgrassella alvi wkB2 T was grown on TSA at 30˚C for 24 h under 3-5% CO 2 . Fatty acid methyl esters were extracted and analyzed according to the standard protocol of the Sherlock Microbial Identification System (MIDI, version 6.0) [8]. Chemotaxonomic analyses of polar lipids of strain 15-3-5 T were performed using 100 mg of freeze-dried cell material for two-dimensional thin-layer chromatography (TLC) as described by Minnikin et al. [9]. Menaquinones of strain 15-3-5 T were extracted according to the method of Collins et al. [10], analyzed by high performance liquid chromatography [11,12] and confirmed by liquid chromatography/mass spectrometry (MS).

16S rRNA and acetate kinase gene sequence analysis
The 16S rRNA gene was amplified by PCR using primers 8F/1525R as described previously [13,14]. After multiple sequence alignment using Clustal W, phylogenetic analysis with related reference species based on the maximum-likelihood and neighbor-joining methods was performed using MEGA5.1 by applying the Jukes-Cantor model including the proportion of invariable sites and gamma distribution (partial deletion of gaps/missing data treatment and a site coverage cut-off of 90%). The resulting trees were evaluated by 1000 bootstrap replicates [15,16,17]. For confirming phylogenetic relationships for the novel isolate (15-3-5 T ) and reference species of the family Neisseriaceae, the acetate kinase gene (ack) was also use to construct a maximum-likelihood tree using the amino acid sequence. The phylogenetic analysis was done as description above.

Genome sequence analysis
The novel isolate (15-3-5 T ) was selected for genome sequencing as described by Romero-Hernández [18]. Pyrosequencing was performed using Ion Torrent technology with an Ion 316 v2 chip. The preliminary assembly was obtained using the MIRA software. Protein-coding genes were annotated by performing a BLAST search against the COGs database (http://www.ncbi. nlm.nih.gov/COG/).
Comparative sequence analysis of the genome core gene sequences was then used to discern phylogenetic relationships for the novel species (15-3-5 T ) and reference species of the family Neisseriaceae. The genome core gene sequences were identified and selected by the PanGP [19] software package, and a BLAST Matrix was constructed using a cutoff of 1e-5, 40% identity and 50% coverage. Aligned core genes were connected together with the same direction (MLST-like). The tree was based on alignment of the genome core gene sequences and was constructed using the neighbor-joining method (MEGA version 6.06) [20]. Nodal supports were assessed by 1000 bootstrap replicates.

Average Nucleotide Identity (ANI)
ANI is a similarity measure between two genome sequences that may be used to replace DNA-DNA hybridization [21,22]. In this study, the ANI among strain 15-3-5 T , Snodgrassella alvi wkB2 T and Stenoxybacter acetivorans DSM19021 T was determined using the OrthoANI [23].

Percentage of Conserved Proteins (POCP)
The percentage of conserved proteins (POCP) analysis was performed as described by Qin et al. [24]. In brief, the conserved proteins between a pair of genomes were determined by aligning all of the genome protein sequences of strain 15-3-5 T with all of the protein sequences of reference genome using the BLASTP program. The percentage of conserved proteins between two genomes was calculated as [(C1+C2)/(T1+T2)] Á 100%, where C1 and C2 represent the conserved numbers of proteins in the two genomes being compared and T1 and T2 represent the total numbers of proteins in the two genomes being compared.

Morphological, physiological and biochemical characterization
Colonies are milk white, opaque, circular, concave with entire margins, glossy and approximately 1-2 mm in diameter after 72 h growth at 30˚C on TSA (pH 7). Growth occurs between 10 and 37˚C and pH 5 to 10, with optimal growth at 28-30˚C and pH 6.0-8.0. Growth is present under 0-3% (w/v) salinity. The detailed results were showed in species description and Table 1.
The major fatty acids detected in two isolates were C 16:0 , C 18:1 ω7c, C 16:1 ω7c/C 16:1 ω6c (detailed in S1 Table), and were similar to the ones in the closely related genus Snodgrassella. However, the novel isolate can be distinguished from the related reference strain of genus Snodgrassella by the percentages of the main fatty acids (C 18:1 ω7c, C 16:1 ω7c/C 16:1 ω6c, Table 1).

16S rRNA and ack gene sequence analysis
A 1421-bp sequence, corresponding to nucleotide positions 4-1457, was obtained. The GenBank accession numbers for the 16S rRNA gene sequence of strains 15-3-5 T and TQ2-3 are KT988024 and KU513552, respectively. Based on a comparison of 16S rRNA gene sequences, two novel isolates have identical sequences, and shared the highest sequence similarity with Snodgrassella alvi wkB2 T (94.5%), followed by Neisseria animaloris LMG 23011 T (94.3%), Alysiella crassa IAM 14969 T (94.2%), Uruburuella testudinis 07_OD624 T (94.1%), and Alysiella filiformis ATCC 15532 T (94.0%), Stenoxybacter acetivorans TAM-DN1 T (94.0%), and less than 94% sequence similarity with all of the other species with validly published names (http://eztaxon-e.ezbiocloud.net) [26]. In the maximum-likelihood phylogenetic tree, two novel isolates clustered together with Stenoxybacter acetivorans TAM-DN1 T , Snodgrassella alvi wkB2 T and Alysiella filiformis ATCC 15532 T , forming one large branch, but the novel isolates branch was not related closely to any genus within the family Neisseriaceae (Fig 1). In the maximum-likelihood phylogenetic tree based on ack gene, the novel isolates were clustered together with Snodgrassella alvi wkB2 T and Stenoxybacter acetivorans TAM-DN6b and TAM-DN6b, and the novel isolates branch was close to Snodgrassella alvi wkB2 T branch (S2 Fig).

Genome sequence analysis, ANI and POCP
The genome is 2361536 bp long, and the final assembly identified 131 large contigs. The genome accession number of strain 15-3-5 T is LQXR00000000. The max sequence length is 355320 bp. There are 2399 predicted genes, including 2013 protein-coding genes, 50 RNAs and 2 CRISPR repeats. The G+C content is 47.8%. The G+C content of the novel strain is higher than in Snodgrassella alvi wkB2 T (41.3%).

Discussion
Two novel strains were isolated from poplar tree canker samples which are very complicated and special environment. Only two strains were isolated from more than 100 symptomatic canker bark samples because it is not the preponderant species in the poplar canker environment. Other researches showed that a sequence identity of 94.5% or lower for two 16S rRNA genes is strong evidence for distinct genera [27,28]. In present study, the novel genus shared the highest sequence similarity with Snodgrassella alvi wkB2 T (94.5%). The 16S rRNA gene data suggest that the novel organism should represent a separate genus. The phylogenetic tree based on ack gene also indicated that the novel isolates were obviously distinguished from genera Snodgrassella and Stenoxybacter (S2 Fig). While, recent research by Qin et al. reported that generally, a percentage of conserved proteins (POCP) value of 50% can be proposed as a genus boundary for prokaryotic lineages being analyzed. While the interspecies POCP value ranged from 43% to 97%, and the average interspecies POCP values were 63% [24]. In their research, the interspecies and intergenera POCP analyses were performed only for 12 families/orders, including 97 genera and 235 species. With more and more bacterial genome sequencing, POCP value of 50% as a genus boundary for prokaryotic lineages should be further proved by using more genome data. In this study, the novel isolate showed the POCP identity values with Snodgrassella alvi wkB2 T and Stenoxybacter acetivorans DSM 19021 T are all more than 50% and less than 60% which are located in the boundary of the genera.
In the case of a strain or set of strains shown to be novel taxa, they should be characterized as comprehensively as possible [29]. In present study, the G + C content, major fatty acids and phospholipid profile data also support the distinctiveness of the novel species from genus Snodgrassella. Additionally, the novel species and Snodgrassella alvi can be differentiated from each other by the ability of the strain to utilize capric acid and citrate as unique carbon source to grow aerobicaly and anaerobically. (Table 1). Furthermore, the two novel strains can be differentiated from one another by DNA fingerprinting (ERIC, S3 Fig) and the activities of esterase lipase (C8), valine arylamidase, cystine arylamidase and trypsin. Therefore, two isolates are considered to represent a novel species representing a new genus of the family Neisseriaceae, for which the name Populibacter corticis gen. nov., sp. nov. is proposed Description of Populibacter gen. nov. Description of Populibacter corticis sp. nov.