Cysteiniphilum litorale gen. nov., sp. nov., isolated

TAXONOMIC DESCRIPTION Liu et al., Int J Syst Evol Microbiol DOI 10.1099/ijsem.0.001917

Cysteiniphilum litorale gen. nov., sp. nov., isolated from coastal seawater Lan Liu,1† Nimaichand Salam,1† Jian-Yu Jiao,1 Shun-Mei E,2,3 Cha Chen,2,3 Bao-Zhu Fang,1 Min Xiao,1 Mo Li,2,3 Wen-Jun Li1,* and Ping-Hua Qu2,3,*

Abstract A taxonomic study was performed on strain SYSU D3-2T, isolated from coastal seawater near the estuary of Pearl River in southern China. The strain was observed to be Gram-reaction-negative, non-motile and non-spore-forming. Cells were found to be of coccobacilli shape. Chemotaxonomic analysis of the plasma membrane revealed ubiquinone-8 as the respiratory quinone, diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminolipid, an unidentified aminophospholipid and an unidentified phospholipid as the polar lipids, and anteiso-C15 : 0, C18 : 0 and anteiso-C17 : 0 as the major fatty acids (>10 % of total fatty acids). Comparison of 16S rRNA gene sequences showed that strain SYSU D3-2T shared maximum similarities with Caedibacter taeniospiralis 51T (92.3 %) and Fangia hongkongensis UST040201-002T (90.6 %), while sharing 85.8–90.0 % similarity with species of the genera Allofrancisella and Francisella. Phylogenetic dendrograms based on the 16S rRNA gene sequences showed that the strain clustered within the family Francisellaceae, but formed a separate lineage closely linked to Caedibacter taeniospiralis 51T and F. hongkongensis UST040201-002T. Based on the findings of the polyphasic taxonomic study, strain SYSU D3-2T is proposed to be recognized as a representative of a novel species of a new genus within the order Thiotrichales, with the name Cysteiniphilum litorale gen. nov., sp. nov. The type strain of the type species is SYSU D3-2T (=NBRC 112441T=DSM 101832T=KCTC 52386T=CGMCC 1.15758T).

As part of a programme to determine the diversity of members of the family Francisellaceae from different ecological niches, seawater samples were collected during September 2014 from the seashore of Wailingding island (113.27 E 23.12 N), near the estuary of Pearl River in southern China. Concentration and pretreatment of the water sample was done as described previously [1, 2]. The pretreated sample was then spread-plated on BCYEa [3] supplemented with 3 g glycine l 1, 1 mg vancomycin l 1, 80 000 IU polymyxin B sulphate l 1 and 80 mg cycloheximide l 1 (BCYEa-GVPC) and BCYEaGVPC with cefepime discs, and the inoculated plates were incubated at 35 C under 5 % CO2 atmosphere for 1 week. Subculturing and purification were done on BCYEa agar medium under similar culture conditions. From among the strains isolated, a Francisella-like, Gram-rection-negative, coccobacillus bacterium with low partial 16S rRNA gene sequence similarities to other recognized strains, designated strain SYSU D3-2T, was selected for detailed characterization using a polyphasic taxonomic approach.

Extraction of genomic DNA and PCR amplification of the 16S rRNA gene were performed according to our previously standardized protocol [4]. The amplified product was purified, cloned and sequenced [5]. The cloned sequences were qualitychecked and assembled using Seqman version 5.0, and vector sequences were removed with the VecScreen program of the NCBI (www.ncbi.nlm.nih.gov/tools/vecscreen/). In the case of resemblance between the clones, a single sequence was chosen for further analysis. The sequence was then compared with available 16S rRNA gene sequences of cultured species from the EzTaxon-e server (http://ezbiocloud.net/) [6] and the NCBI nucleotide database via BLAST search [7]. Multiple alignments with sequences of the most closely related taxa and calculations of levels of sequence similarity were carried out using CLUSTAL X version 1.83 [8]. Phylogenetic dendrograms were generated with three tree-making algorithms, the neighbour-joining [9], maximum-likelihood [10] and maximumparsimony [11] methods, using the software package MEGA version 7.0 [12]. Kimura’s two parameter model [13] was used

Author affiliations: 1State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China; 2Department of Laboratory Medical Science, the Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China; 3Department of Clinical Laboratory, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou 510006, PR China. *Correspondence: Wen-Jun Li, [email protected]; Ping-Hua Qu, [email protected] Keywords: Cysteiniphilum litorale gen. nov., sp. nov.; 16S rRNA gene; polyphasic taxonomy. †These authors contributed equally to this work. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SYSU D3-2T is KX817994. Four supplementary figures are available with the online Supplementary Material. 001917 ã 2017 IUMS

Downloaded from www.microbiologyresearch.org by IP: 113.108.133.53 1 On: Sat, 29 Jul 2017 05:52:50

Liu et al., Int J Syst Evol Microbiol

to calculate evolutionary distance matrices of the neighbourjoining phylogenetic tree. Bootstrap analysis was performed with 1000 replications [14]. Escherichia coli DSM 30083T (X80725) was used as the outgroup. The G+C content of the genomic DNA was determined by using reversed-phase HPLC [15] with E. coli DH5a as the reference strain.

fatty acid methyl ester (FAME) profiles were determined by GC (7890B; Agilent) according to the standard protocol of the Microbial Identification System (Sherlock version 6.2; database: TSBA6; MIDI). Cells for cellular fatty acid analysis were harvested from cultures grown on BCYEa agar for 48 h at 28 C under 5 % CO2 atmosphere.

Cell motility was studied by the development of turbidity throughout a tube of semi-solid medium [16] by using Haemophilus testing medium (HTM). The morphological features of strain SYSU D3-2T were observed by using scanning electron microscopy (JSM-6330F; JEOL) according to the protocols of Liu et al. [17]. Formation of spores was monitored by phase-contrast microscopy on cells cultured on BCYEa agar medium at 28 C for up to 5 days. Gram staining was carried out by the standard Gram’s reaction and was confirmed by the KOH test [18]. Growth characteristics were examined on several media common to clinical laboratories, such as BCYEa agar, Columbia blood agar (Oxoid), cysteine heart agar (brain heart infusion agar supplemented 5 % glucose, 0.04 % cysteine and 9 % heated sheep red blood cells), Haemophilus chocolate 2 agar (BioMerieux), HTM (Oxoid), Kligler’s iron agar (Difco), MacConkey agar (Oxoid) and nutrient agar (Difco); the requirement for Lcysteine was also tested on the above media. Anaerobic growth was examined with an anaerobic bag (BioMerieux). Microaerophilic respiratory type was tested by using an automatic anaerobic or aerobic system (Mart). Growth at different temperatures (5, 10, 14, 18, 23, 28, 32, 37 and 40 C) and NaCl tolerance at various concentrations (0– 10 %, w/v, at intervals of 0.5 %) was determined on BCYEa agar medium. The pH range (pH 4.0–10.0, at intervals of 1.0 pH unit) for growth was tested in BCYEa broth medium by using the buffer system described by Xu et al. [19]. Oxidase activity was determined by the oxidation of tetramethyl-p-phenylenediamine [20]. Catalase activity was analysed by assessing the production of bubbles on addition of a drop of 3 % (v/v) H2O2. Biochemical analyses (activities of b-lactamase and urease, Voges–Proskauer and indole tests, reduction of nitrate, hydrolysis of aesculin, and requirement for X and V factors in growth medium) were performed according to the methods described by Aslanzadeh [21]. Utilization of carbon sources was tested using the GEN III MicroPlate (Biolog) according to the manufacturer’s instructions. Other enzyme activities and biochemical characteristics were determined using API ZYM and API NH kits (bioMerieux) according to the manufacturer’s instructions. Antibiotic susceptibility testing was performed on HTM agar with E-test strips (Oxoid). Susceptibility criteria were interpreted with reference to the minimum inhibitory concentration breakpoint of Francisella tularensis as listed in the CLSI M45-A2 guideline for Potential Bacterial Agents of Bioterrorism.

Comparison of an almost-complete 16S rRNA gene sequence of strain SYSU D3-2T (1489 bp) with the curated bacterial sequence database in the EzTaxon-e server showed that strain SYSU D3-2T shared the highest sequence similarity with Caedibacter taeniospiralis 51T (92.3 %) and Fangia hongkongensis UST040201-002T (90.6 %). The sequence similarities with members of the genera Allofrancisella and Francisella were in the range of 85.8–90.0 %. In the neighbour-joining phylogenetic dendrogram based on 16S rRNA sequences, strain SYSU D3-2T clustered with members of the family Francisellaceae, while forming a separate lineage closely linked to Caedibacter taeniospiralis 51T and Fangia hongkongensis UST040201-002T (Fig. 1). The stability of the phylogenetic relationship was further confirmed from the dendrograms generated with the maximum-likelihood and maximum-parsimony methods (Figs S1 and S2, available in the online Supplementary Material). The DNA G+C content of strain SYSU D3-2T was determined to be 38.1 mol%.

Respiratory quinones were extracted and purified as described by Collins et al. [22] and analysed by HPLC [23]. Polar lipids were extracted as described by Minnikin et al. [24] and identified by two-dimensional TLC [25]. Cellular

Strain SYSU D3-2T was observed to be Gram-reaction-negative, microaerophilic and non-motile. Scanning electron microscopy showed that cells of strain SYSU D3-2T were coccobacilli in shape, measuring 0.4~0.50.6~0.9 µm (Fig. S3). The growth of strain SYSU D3-2T was enhanced by Lcysteine supplementation, and strain SYSU D3-2T after incubation for 2 days grew well on BCYEa agar, CHAB, Haemophilus chocolate 2 agar and HTM agar, weakly on Columbia blood agar, but not on Kligler’s iron agar, MacConkey agar or nutrient agar. Growth was observed at 18 to 37 C (optimum range, 28–32 C), pH 6 to 10 (optimum, pH 7–8) and in the presence of up to 7.5 % NaCl (w/v). The strain was positive for b-lactamase and catalase activities but negative for oxidase, urease, Voges–Proskauer and indole tests, reduction of nitrate, hydrolysis of aesculin, and X and V factor requirement tests. Strain SYSU D3-2T was resistant to cephalosporins, but sensitive to chloramphenicol, ciprofloxacin, doxycycline, gentamicin, levofloxacin and tetracycline. Differential characteristics of strain SYSU D32T and the closely related type strains are listed in Table 1 while the detailed characteristics of the strain are given in the species description. The respiratory quinone detected for strain SYSU D3-2T was ubiquinone-8. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminolipid, an unidentified aminophospholipid and an unidentified phospholipid (Fig. S4). The major fatty acids (>10 %) of strain SYSU D3-2T consisted of two branched-chain, saturated fatty acids: anteiso-C15 : 0 (34.4 %) and anteiso-C17 : 0 (10.7 %), and one evenchain, saturated fatty acid: C18 : 0 (20.4 %) (Table 2).



100* Francisella noatunensis subsp. orientalis Ehime-1T/EU683030 85* Francisella asiatica PQ 1104T/EU597812

0.02

Francisella noatunensis subsp. noatunensis LMG 23800T/DQ295795 52* 100* Francisella piscicida GM2212T/DQ309246 91* Francisella halioticida LMG26062T/AB449247 100* Francisella philomiragia ATCC 25015T/ABYY01000004 Francisella persica ATCC VR-331T/M21292 98*

Francisella hispaniensis FhSp1T/FN252413

T 99* Francisella tularensis subsp. novicida U112 /CP000439 T 93* Francisella tularensis subsp. mediasiatica GIEM 543 /CP000915 T 69* Francisella tularensis subsp. holarctica OSU18 /CP000437 87* Francisella tularensis subsp. tularensis ATCC 6223T/Z21931

100*

100*

100*

Allofrancisella guangzhouensis 08HL01032T/CP010427

89*

Allofrancisella inopinata SYSUYG23T/JN620412 Allofrancisella frigidaquae SYSU 10HL1970T/JN620409 Fangia hongkongensis UST040201-002T/AB176554

100*

Cysteiniphilum litorale SYSU D3-2T/KX817994 100*

Caedibacter taeniospiralis 51T/AY102612

Legionella cardiaca H63T/JF831047

100*

Legionella erythra SE-32A-C8T/Z32638 Ectothiorhodospira variabilis WN22T/AM943121

79

100* Methylomicrobium japanense NIT/D89279 100* Methylomicrobium alcaliphilum 20ZT/FO082060

98*

Methylomicrobium kenyense AMO1T/AJ132384 100*

69*

Methylosarcina lacus LW14T/AZUN01000001 Methylomonas lenta R-45377T/FR798962 Methylobacter psychrophilus Z-0021T/AF152597

76*

Methylobacter marinus A45T/ARVS01000003

72* 100*

Methylobacter whittenburyi ACM 3310T/JQNS01000002 Escherichia coli DSM 30083T/X80725

Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of strain SYSU D3-2T and closely related strains. Bootstrap values (expressed as percentages of 1000 replications) of above 50 % are shown at the branch points. Asterisks denote nodes that were also recovered using the maximum-parsimony and maximum-likelihood methods. The sequence of E. coli DSM 30083T was used as the outgroup. Bar, 0.02 substitutions per nucleotide position.

Strain SYSU D3-2T showed low levels of 16S rRNA gene sequence similarities with known members of the family Francisellaceae. Strain SYSU D3-2T could also be distinguished from the closely related strains Fangia hongkongensis UST040201-002T, Allofrancisella guangzhouensis 08HL010 32T, Allofrancisella frigidaquae SYSU 10HL1970T and Allofrancisella inopinata SYSU YG23T by several physiological characteristics as listed in Table 1 and by the composition of the major fatty acids (Table 2). anteiso-C15 : 0, the major constituent of strains SYSU D3-2T and Fangia hongkongensis UST040201-002T, was either present in negligible amounts or absent in strains belonging to the genus Allofrancisella. In comparison, strains representing the genus Allofrancisella were characterized by larger amounts of C10 : 0 and C18 : 0 3OH, which were absent or detected in lesser quantities in strains SYSU D3-2T and Fangia hongkongensis UST040201002T. Strains SYSU D3-2T and Fangia hongkongensis

UST040201-002T could be distinguished from each other by the quantity of C18 : 0. Comparative studies, however, could not be done with strain Caedibacter taeniospiralis 51T as the strain is an endosymbiont and requires the presence of a host for its growth. Based on the differentiating characteristics (Tables 1 and 2, Fig. 1), strain SYSU D3-2T merits recognition as a member of a novel species of a new genus within the order Thiotrichales for which the name Cysteiniphilum litorale gen. nov., sp. nov. is proposed.

DESCRIPTION OF CYSTEINIPHILUM GEN. NOV. Cysteiniphilum (Cy.s.te.i.ni¢phi.lum. N.L. neut. n. cysteinum cysteine; Gr. masc. n. philos friend, loving; N.L. neut. n. Cysteiniphilum cysteine loving). Gram-reaction-negative and coccobacillus. Cells are catalasepositive, non-motile and non-spore forming. Negative for



Table 1. Differential characteristics of strain SYSU D3-2T from the closely related species within the family Francisellaceae Strains: 1, Cysteiniphilum litorale SYSU D3-2T; 2, Fangia hongkongensis UST040201-002T; 3, Allofrancisella guangzhouensis 08HL01032T; 4, Allofrancisella frigidaquae SYSU 10HL1970T; 5, Allofrancisella inopinata SYSU YG23T. Data were from this study unless indicated otherwise. +, Positive; , negative; (+), weakly positive; ND, not determined. Characteristic

1

2*

3

4

5

18–37

Growth range Temperature ( C) pH (optimum) NaCl (%)

18–37

16–40

18–37

18–37

6–10 (7–8)

4.9–8.8 (4.9–6.8)

ND

ND

ND

0–7.5

0.4–7.5

0–5

0–7.5

0–7.5

+

(+)

(+)

+

+

+

+

Oxidase

(+)

API ZYM Alkaline phosphatase

+

b-Galactosidase

+

N-Acetyl-bglucosaminidase Lipase (C14)

+ +

Leucine arylamidase Trypsin

+ +

API NH Acid from sucrose

ND

Acid from maltose

+

Ornithine decarboxylase Utilization of:

ND

D-Fructose

+

a-D-Glucose

+

N-Acetyl-Dglucosamine Glycerol

+

+ ND

+

Maltose D-Mannose D-Serine

+

+

ND

+

ND

+

ND

Sucrose

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

+

L-Alanine

+

ND

ND

L-Histidine

+

ND

ND

ND

Fatty acid

anteiso-C15 : 0, C18 : 0, anteiso-C17 : 0 38.1

anteiso-C17 : 0, anteisoC15 : 0 37.9†

C18 : 0 3-OH, C10 : 0 30

C18 : 0 3-OH, C16 : 0, C10 : 0 32.6

DNA G+C content (mol%)

C10 : 0, C14 : 0, C16 : 0, C18 : 0 3-OH, C18 : 1!9c 32.5

*Data for strain UST040201-002T were taken from Lau et al. [26]. †Data for DNA G+C content from Sjödin et al. [27].

nitrate reduction and urease activity. Do not require X and V factors for growth. The respiratory quinone is ubiquinone-8. Polar lipid profiles consist of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminolipid, an unidentified aminophospholipid and an unidentified phospholipid. The major fatty acids (>10 %) are anteiso-C15 : 0, C18 : 0 and anteiso-C17 : 0. The type species of the genus Cysteiniphilum is Cysteiniphilum litorale.

DESCRIPTION OF CYSTEINIPHILUM LITORALE SP. NOV. Cysteiniphilum litorale (li.to.ra¢le. L. neut. adj. litorale coastal).

The following characteristics are found for the species in addition to the ones listed for the genus description. Grows well on BCYEa agar, CHAB, Haemophilus chocolate 2 agar and HTM agar, weakly on Columbia blood agar but not on Kligler’s iron agar, MacConkey agar or nutrient agar. Growth occurs at 18–37 C (optimum, 28–32 C), within a pH range of pH 6 to 10 (optimum, pH 7–8) and in the presence of up to 7.5 % NaCl (w/v). Positive for b-lactamase activity, but negative for oxidase, hydrolysis of aesculin, Voges–Proskauer and indole tests. Resistant to cephalosporins, but sensitive to chloramphenicol, ciprofloxacin, doxycycline, gentamicin, levofloxacin and tetracycline. Positive for acidic phosphatase, esterase (C4), esterase lipase (C8), naphthol-AS-BI-phosphohydrolase,



Table 2. Cellular fatty acid compositions of strain SYSU D3-2T and closely related strains T

Taxa: 1, Cysteiniphilum litorale gen. nov., sp. nov. SYSU D3-2 (data from this study); 2, Fangia hongkongensis UST040201-002T (data from Lau et al. [26]); 3, Allofrancisella guangzhouensis 08HL01032T (data from this study); 4, Allofrancisella frigidaquae SYSU 10HL1970T (data from Qu et al. [3]); 5, Allofrancisella inopinata SYSU YG23T (data from Qu et al. [3]). Values are percentages of total fatty acids. –, Not detected; TR, trace (