Aquella oligotrophica gen. nov. sp. nov.: A new member of the family Neisseriaceae isolated from laboratory tap water

Abstract A bacterial strain designated as P08T was isolated from laboratory tap water during a water quality assessment in University of Malaya, Malaysia. The strain was a Gram‐negative, rod‐shaped, nonmotile, and aerobic bacterium. Complete genome of P08T comprised of a 2,820,660 bp chromosome with a G + C content of 36.43%. Both 16S rRNA phylogeny and phylogenetic tree inferred from the core gene matrix demonstrated that P08T formed a hitherto unknown subline within the family Neisseriaceae. Ortho average nucleotide identity (OrthoANI) values and the percentage of conserved proteins (POCP) calculated from complete genome sequence indicated low relatedness between P08T and its phylogenetic neighbors. Respiratory quinone analysis revealed Q‐8 as the only detectable quinone. The predominant cellular fatty acids were identified as C14:0, iso‐C15:0, and summed feature 3 (C16:1 ω7c/C16:1 ω6c). The polar lipids consisted of uncharacterized aminolipid, phosphatidylglycerol, and phosphatidylethanolamine. All aspects of phenotypic and phylogenetic data suggested that strain P08T represents a novel genus within family Neisseriaceae, for which the name Aquella gen. nov. is proposed. The type species of the genus is Aquella oligotrophica sp. nov., and the type strain is P08T (=LMG 29629T =DSM 100970T).

However, there are also 2 genera with uncertain taxonomic status that have been classified into Neisseriaceae; Adeolu and Gupta (2013) suggested that genus Morococcus (Long, Sly, Pham, & Davis, 1981) should be reclassified to Neisseria species and Prolinoborus (Pot, Willems, Gillis, & De Ley, 1992) is likely wrongly assigned to the order of Neisseriales, based on their phylogenetic analysis.
Other than 16S rRNA gene sequence similarities and phylogenetic analysis, the major distinguishing features among genera for Neisseriaceae are cell morphology, biochemical characteristics, such as oxidase and catalase tests, glucose fermentation, nitrite reduction, and mol% G + C content of the genomic DNA (Garrity et al., 2006). Most strains inhabit indigenously in mucosal membranes of humans and animals, although environmental species were recently included in this family with representatives isolated from anthill, hot spring sediment, and freshwater river (Dong et al., 2015;Garrity et al., 2006;Moß et al., 2013;Sheu et al., 2014). During an assessment on laboratory tap water quality, a bacterial strain designated as P08 T was isolated on R2A agar. Here, we report the characterization of strain P08 T that is the first representative of a newly proposed genus Aquella gen. nov. belonging to family Neisseriaceae.

| Isolation of the strain
Strain P08 T was isolated from a laboratory tap water collected for water quality assessment in University of Malaya (3°07′20.9″N, 101°39′23.7″E) on 22 May 2015. After 3 days of incubation on R2A agar (BD Difco) at 37°C, bacteria isolation and purification were performed. The purified strain was routinely cultivated in R2A liquid medium, unless specified. Cells were preserved in 20% (v/v) glycerol at −80°C. The strain P08 T has been deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and the Belgium Coordinated Collections of Microorganisms (BCCM/LMG, Gent, Belgium).

| DNA extraction, genome sequencing, and functional gene annotation
The genomic DNA of strain P08 T was extracted using the MasterPure TM DNA purification kit (Epicenter, WI, USA) following the manufacturer's protocol. Extracted genomic DNA was sheared and constructed into a template library according to the "Guidelines for Preparing 20 kb SMRTbell™ Templates." Genome sequencing was performed in 1 SMRT cell using the PacBio RS II single-molecule real-time (SMRT) sequencing technology (Pacific Biosciences, CA, USA). The reads were de novo assembled using the hierarchical genome assembly process (HGAP) algorithm version 2 (Chin et al., 2013) into complete genome of P08 T . The assembled genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) version 2.10 (Tatusova et al., 2016), Rapid Annotation using Subsystem Technology (RAST) version 3.0 (Aziz et al., 2008;Overbeek et al., 2014), and IMG ER pipeline (Markowitz et al., 2009).
The genome project and the complete genome sequence were deposited in the Genomes OnLine Database (Liolios et al., 2010) and GenBank. A comparison on the genomes of P08 T and available genomes of type strains in the family Neisseriaceae was performed.

| Phylogenetic analyses
The 16S rRNA gene sequence was mined from the complete genome using RNAmmer 1.2 server (Lagesen et al., 2007). The sequence similarity to other validly described type strains was examined from pairwise sequence comparisons using EzBioCloud database (https://www.ezbiocloud.net) (Yoon et al., 2017). Phylogenetic analysis of 16S rRNA genes of strain P08 T and type members of Neisseriaceae were performed using the software package MEGA version 6.0 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013), with neighbor-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981), and maximum parsimony (Fitch, 1971) algorithms. In each case, bootstrap values were calculated based on 1,000 resamplings (Felsenstein, 1985). Kimura's two-parameter model was used to calculate evolutionary distance matrices of the neighbor-joining method and maximum-likelihood method (Kimura, 1980). Vitreoscilla stercoraria was included in the analysis in place of the type species V. beggiatoides that has no sequence data available.

| Average Nucleotide Identity (ANI), Average Amino acid Identity (AAI), and Percentage of conserved proteins (POCP) analysis
ANI between the complete genome of P08 T and each reference genome was calculated using an online ANI calculation tool on the EzBioCloud web server with the OrthoANI algorithm (Lee, Kim, Park, & Chun, 2015). AAI between of complete genome of P08 T and each reference genome was calculated using an online AAI calculation tool available at the web server http://enve-omics.ce.gatech. edu/(Rodriguez-R & Konstantinidis, 2016). The available genome sequences of the family Neisseriaceae were retrieved from GenBank.
The percentage of conserved proteins (POCP) (Qin et al., 2014) in each pair of genomes was calculated as [(C1+C2)/(T1+T2)]×100%, where C1 and C2 represent the numbers of conserved proteins in the two genomes being compared, respectively, and T1 and T2 represent the total numbers of predicted proteins in the two genomes being compared, respectively. Conserved proteins were defined as having a BLASTP match with an E-value of less than 1e −5 , sequence identity of more than 40% and an alignable region of the query protein sequence of more than 50%, as recommended by Qin et al. (2014).

| Morphological observations and physiological tests
Growth of strain P08 T was tested on chocolate agar (Thermo Scientific), Luria-Bertani agar (Merck), Pseudomonas agar (BD Difco), MacConkey agar (Merck), R2A agar (BD Difco), and trypticase soy agar (Merck), and the ability to grow in this media was recorded after incubation of 3 days at 37 °C. Oxidase and catalase activities were examined with solutions of oxidase reagent (bioMérieux) and 3% (v/v) hydrogen peroxide, respectively. The morphology of bacterial cells was observed using tabletop scanning electron microscopy (Hitachi TM3030, Germany). Cellular motility was tested by the hanging drop method (Beveridge, Lawrence, & Murray, 2007).
Gram staining was performed using the standard Gram reaction and was confirmed by using the KOH lysis test method (Cerny, 1978).
Growth temperature was investigated in R2A medium at 4, 12, 16, 20, 25, 30, 35, 37, 40, and 45 °C, up to 14 days of incubation. Growth at pH 4.0-11.0 (intervals of 1 unit) was determined in R2A medium after incubation for 14 days at 37 °C under speed 220 rpm. For pH adjustment of the basal medium, the following buffers were used as follows: 0.1 M citric acid/0.1 M sodium citrate (pH 4.0-5.0), 0.1 M KH 2 PO 4 /0.1 M NaOH (pH 6.0-8.0), 0.1 M NaHCO 3 /0.1 M Na 2 CO 3 (pH 9.0-10.0), and 0.05 M Na 2 HPO 4 /0.1 M NaOH. Growth on medium added with sodium chloride (NaCl) was determined in R2A medium supplemented with 0, 0.5, 1%-6% (intervals of 1%) (w/v) NaCl after 14 days of incubation at 37 °C. Growth under anaerobic condition was determined by incubating strain P08 T on R2A agar in the Oxoid AnaeroGen system. P08 T was tested on R2A agar supplemented with casein (2% skimmed milk, w/v), starch (0.2% soluble starch, w/v), cellulose (0.5% CM-cellulose, w/v), urease, Tweens 20, 40, 60, and 80 for hydrolysis activities, respectively (Tindall, Sikorski, Smibert, & Krieg, 2007). Hydrolysis of DNA was determined using DNase test agar (BD Difco). The production of clear or opaque halo zones around colonies on agar plates was recorded as a positive result after incubation of 3 weeks, except for hydrolysis of starch, cellulose, urease, and DNA (observed on agar plates incubated for 2 weeks). The color and morphology of colonies were determined on R2A agar incubated for 2 days at 37 °C. Other physiological and biochemical tests were performed using API 20E, API 20NE, and API ZYM strips (bioMérieux) according to the manufacturer's instructions, and results were recorded after 2 days of incubation at 37 °C. Utilization of a variety of carbon sources was tested using GEN III MicroPlates (Biolog), along with other major biochemical and physiological properties. Cells grown for 2 days at 37 °C on R2A were suspended in sterilized inoculating fluid C (Biolog) and adjusted to a specific transmittance (60% T) using a turbidimeter according to the manufacturer's instruction. An aliquot (100 μl) of the cell suspension was transferred to each well, and the plate was immediately incubated for 3 days at 37 °C, before visual reading.

| Antibiotic susceptibility testing
Sensitivity of strain P08 T to antibiotics was tested by the disk dif- and resistant at <10 mm as described by Nokhal and Schlegel (1983).

| Fatty acid analyses
Fatty acid methyl esters are extracted from 40 mg cells scraped from Petri dishes by saponification, methylation, and extraction using minor modifications of the method of Kuykendall, Roy, O'neill, and Devine (1988) and Miller (1982). Fatty acid methyl esters were separated and analyzed by the Identification Service of the DSMZ, Braunschweig, Germany, using the Sherlock Microbial Identification System (MIDI Inc, Newark, USA, version 6.1 with database TSBA6) according to the standard protocol. For this purpose, strain P08 T was grown for 2 days on R2A plates at 37 °C to get cultures of the same physiological age.

| Polar lipid and respiratory quinones analyses
Polar lipid analyses and analyses of respiratory quinones were carried by the Identification Service of the DSMZ, Braunschweig, Germany. Cells were freeze-dried before 100 mg cell material was used for extraction of respiratory lipoquinones using the two-stage method described by Tindall (1990a) and Tindall (1990b). Respiratory quinones were then extracted using methanol:hexane, followed by phase separation into hexane (Tindall, 1990a(Tindall, , 1990b. Separation of respiratory lipoquinones into different classes was conducted by thin layer chromatography (TLC) on silica gel (Macherey-Nagel Art. No. 805 023) using hexane:tertbutylmethylether [9:1 (v/v)] as the solvent. UV-absorbing bands corresponding to different quinone classes were removed from the TLC plate and further analyzed on a LDC Analytical (Thermo Separation Products) HPLC fitted with a reverse phase column (Macherey-Nagel, 2 mm × 125 mm, 3 μm, RP18) using methanol:heptane [9:1 (v/v)] as the eluent. Respiratory lipoquinones were detected at 269 nm. Cells were freeze-dried before 100 mg cell material was used for polar lipids extraction with a modified method using a chloroform:methanol:0.3% aqueous NaCl mixture 1:2:0.8 (Bligh & Dyer, 1959). The mixture was stirred overnight, and cell debris was centrifuged for pellet. Polar lipids were obtained from the chloroform phase by adjusting the chloroform:methanol:0.3% aqueous NaCl mixture to a ratio of 1:1:0.9 (v/v/v). Recovered polar lipids were separated by a two-dimensional silica gel TLC (Macherey-Nagel Art. No. 818 135), first in chloroform:methanol:water (65:25:4, v/v/v) and the second in chloroform:methanol:acetic acid:water (80:12:15:4, v/v/v/v). Total lipid material was detected using molybdatophosphoric acid while specific functional groups detected using spray reagents specific for defined functional groups (Tindall et al., 2007).  (Table S1). According to Ludwig et al. (1998), 16S rRNA gene sequence similarities of lesser than 95% between two bacteria are generally affiliated as a novel genus. Therefore, the low 16S rRNA gene sequence similarity to other validly described type strains highly supported that strain P08 T belongs to a novel genus. Figure 1 shows the positions of the type species of genera within the family Neisseriaceae in a neighbor-joining (NJ) phylogenetic tree based on 16S rRNA gene sequences. Strain P08 T formed a branch clearly separated from the remaining genera from Neisseriaceae. As indicated in Figure 1, strain P08 T appeared to be more closely related to

| Phylogenetic analyses
Neisseriaceae, compared to the type species of genus Prolinoborus, which have a questionable taxonomic status in Neisseriaceae (Adeolu & Gupta, 2013). The NJ phylogenetic tree was congruent with the maximum-likelihood (ML) ( Figure S1) and maximum parsimony (MP) ( Figure S2) phylogenetic trees, all constructed based on 16S rRNA gene sequences. For core genes analysis, we have identified 146 orthologous protein clusters (Table S2). The ML phylogenetic tree based on concatenated core gene (Figure 2) showed that all genera in the Neisseriaceae formed distinct phylogenetic lineages. Strain P08 T was in a cluster distantly related to Snodgrassella and was clearly separated in the phylogenetic tree based on concatenated core gene from other genera of Neisseriaceae. This association was supported by a bootstrap value of 100%. Likewise, the concatenated core gene phylogenetic tree was congruent with the 16S rRNA gene phylogenetic analyses in which strain P08 T represents a novel bacterial taxon in the family Neisseriaceae.

| Genomic features and functional gene annotation
A total of 1,985,842,173 reads with a mean read length of 11,876 bp were generated from whole genome sequencing. The reads were de novo assembled using the hierarchical genome assembly process (HGAP) algorithm version 2 (Chin et al., 2013) to generate the complete genome of P08 T , which consists of 1 polished contig with an average genome coverage of 472.61-fold. The genome of P08 T is 2,820,660 bp in length, consists of a circular chromosome, with G + C contents of 36.43 mol%. The assembled and annotated genome of P08 T described in this paper has been deposited in GenBank (accession number: CP024847.1) and JGI portal (GOLD ID: Gp0293937; IMG Taxon ID: 2,770,939,448). Out of a total of 2,625 genes in the genome, 2,564 protein-coding gene, 12 rRNAs, and 46 tRNAs were predicted from the chromosome by PGAP analysis (Table 1 and  (Table 1). Majority of the genes were categorized into classes responsible for central metabolism of the P08 T including translation, ribosomal structure and biogenesis and amino acid transport and metabolism. There are also a number of genes with unknown functions. Figure S3 represents the gene's distribution into different clusters of orthologous groups (COGs) functional categories.

| Genome comparisons with type strains of type species in the family Neisseriaceae
Genome of P08 T was compared to genome of other members in the family Neisseriaceae. With the exception to Crenobacter luteus, Vitreoscilla beggiatoides, and Uruburuella suis, publicly available genomes of all type strains of type species in the family were included.
Genome of C. luteus strain CN10 was selected as the type strain of the type species C. luteus has no available genome information while the genome of V. stercoraria DSM 513 T was incorporated into analysis as the type species V. beggiatoides has no available culture.
Uruburuella is the only genus currently without genomic data. Most of the genomes currently available were draft genomes, and complete genomes obtained were only limited to strains P08 T , Simonsiella muelleri ATCC 29453 T , and Snodgrassella alvi wkB2 T (Table S3). While an accurate comparison on the size would require the complete genomes of the other type strains, it is noteworthy that the complete genome size of P08 T is 2.82 Mb, which is among the largest and only smaller than the genome size of Amantichitinum ursilacus IGB-41 T ,

| Average Nucleotide Identity (ANI), Average Amino acid Identity (AAI), and Percentage of Conserved Proteins (POCP) analysis
Genome-to-genome similarity between strain P08 T and close taxa was performed using OrthoANI algorithm (Lee et al., 2015). OrthoANI values between strain P08 T and phylogenetically related genera in Neisseriaceae ranged from 63.0% to 65.5% (Table 2), which were significantly below the proposed boundary of 95%-96% for defining a novel species (Richter & Rosselló-Móra, 2009). Despite ANI is not suitable for genus delimitation (Qin et al., 2014), this analysis briefly suggested that strain P08 T is not the species or the   (Table S4) (Konstantinidis & Tiedje, 2005). POCP has been applied to support the designation of P08 T as a novel genus, using a suggested threshold value of 50% (Qin et al., 2014). POCP using the cutoff threshold for genus circumscription highly supported that strain P08 T represented a novel genus in family Neisseriaceae, as strain P08 T had an extremely low POCP compared to other representative members of family Neisseriaceae. The intergenera POCP analyses between P08 T and strains from 13 genera of family Neisseriaceae had values ranged from 24% to 31%, which clearly indicated that strain P08 T does not belong to any validly described genera from Neisseriaceae (Table 2).

| D ISCUSS I ON
The 16S rRNA gene sequence similarity of strain P08 T with other validly described strains is lower than the proposed cutoff (95%) value suggested by Ludwig et al. (1998) for a novel bacterial genus.
EzBioCloud similarity-based search in Table S1 shows that strain P08 T has highest 16S rRNA gene sequence similarity with most of the type strains of species belonging to the genus Neisseria. This suggested that even though strain P08 T does not belong to any validly described genera, it is highly related to family Neisseriaceae. The divergent branching pattern between strain P08 T and the cluster of genera of the family Neisseriaceae was highly reproducible with solid bootstrap recovery using the neighbor-joining algorithm (Figure 1).
This was further confirmed in phylogenetic tree constructed using concatenated core genes (Figure 2). In the phylogenetic tree based on concatenated core genes, 146 orthologous protein clusters were concatenated and subjected to ML analysis. Strain P08 T is distantly related to other members of family Neisseriaceae. Interestingly, even though Adeolu and Gupta (2013) proposed that Moroccocus should be reclassified into genus Neisseria, phylogenetic tree based on concatenated core genes showed that Moroccocus is rather closely related to Bergeriella. However, these 3 taxa clustered into the same TA B L E 3 Cellular fatty acid compositions of Aquella oligotrophica gen. nov. sp. nov. strain P08 T

Fatty acid Composition
Straight-chain Note. Strain P08 T was grown on R2A agar at 37 °C for 2 days. Values are in percentages of the total fatty acids; fatty acids that make up <1% of the total are not listed or are indicated by a dash. a Summed features represent groups of two or three fatty acids that cannot be separated by GLC with the MIDI system. Summed feature 3 comprises C 16:1 ω7c and/or C 16:1 ω6c; summed feature 8 comprises C 18:1 ω7c and/or C 18:1 ω6c.
F I G U R E 4 Thin layer chromatography image of polar lipid analysis of P08 T ; AL, aminolipid; PG, phosphatidylglycerol; PE, phosphatidylethanolamine clade and showed that they are more related to each other than to other members from family Neisseriaceae.
We reported in this study the complete genome sequence of P08 T that is important for pairwise comparison with closely related species in terms of ANI and AAI analyses. The level of genome-togenome similarity between strain P08 T and the closest phylogenetic neighbors was significantly below the interspecies level of sequence similarity ( Table 2). The genus delimitation as determined by POCP between strain P08 T and Neisseria gonorrhoeae DSM 9188 T , Amantichitinum ursilacus IGB-41 T , and Crenobacter luteus CN10 was of values 26%, 23%, and 29%, respectively (Table 2). This is far below the proposed threshold for genus delimitation of 50% (Qin et al., 2014), which indicates that strain P08 T does not belong to any of these mentioned genera. In fact, the relatedness among all these genome sequences is extremely low, which also suggests that strain P08 T represents a novel genus.
Physiological and biochemical characteristics such as absence of catalase and oxidase, rod-shaped, absence of motility, inability to reduce nitrate to nitrite, low DNA G + C content, and cellular fatty acid profile clearly distinguished strain P08 T from other members of the family Neisseriaceae (Table 3 & 4). The ability to grow in up to 43°C is also useful in differentiating strain P08 T from the genera Amantichitinum and Rivicola (Table 4). Based on these morphological, physiological, chemotaxonomic, phylogenetic, and phylogenomic properties, strain P08 T is considered to represent a novel species within a new genus, for which the name Aquella oligotrophica gen.

| Description of Aquella gen. nov
Aquella gen. nov. (A.qu.el'la. L. fem. dim. n. aquella, water, referring to the source of isolation of the novel organism).
Cells are Gram-negative, aerobic, nonmotile, rod-shaped. Cells are oxidase and catalase negative. Q-8 is the only quinone type.
The G + C content of the DNA of the type strain of the type species is 36.43 mol%. Based on 16S rRNA sequence analyses, P08 T belongs to the Betaproteobacteria. The type species is Aquella oligotrophica P08 T .
Exhibits the following properties in addition to those given in the genus description. Cell sizes range from 0.7-1.4 μm in length and 0.3-0.5 μm in width ( Figure 5). Cells occur singly, in pairs, in short chains or in irregular clusters. On R2A agar, colonies (<1 mm in diameter) are milky white, circular, and convex with entire margin. Growth occurs at 12-43 °C (optimal growth at 35-40 °C) and at pH 5.0-8.0 (optimal growth at pH 5.0-7.0).
Growth occurs in the presence of NaCl up to 1% (w/v). Negative F I G U R E 5 Scanning electron micrographs of strain P08 T The type strain Aquella oligotrophica P08 T (=LMG 29629 T