Isolation of marine xylene-utilizing bacteria and characterization of Halioxenophilus aromaticivorans gen. nov., sp. nov. and its xylene degradation gene cluster

Isolation of marine xylene-utilizing bacteria and characterization of Halioxenophilus... Abstract Seven xylene-utilizing bacterial strains were isolated from seawater collected off the coast of Japan. Analysis of 16S rRNA gene sequences indicated that six isolates were most closely related to the marine bacterial genera Alteromonas, Marinobacter or Aestuariibacter. The sequence of the remaining strain, KU68FT, showed low similarity to the 16S rRNA gene sequences of known bacteria with validly published names, the most similar species being Maricurvus nonylphenolicus strain KU41ET (92.6% identity). On the basis of physiological, chemotaxonomic and phylogenetic data, strain KU68FT is suggested to represent a novel species of a new genus in the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria, for which the name Halioxenophilus aromaticivorans gen. nov., sp. nov. is proposed. The type strain of Halioxenophilus aromaticivorans is KU68FT (=JCM 19134T = KCTC 32387T). PCR and sequence analysis revealed that strain KU68FT possesses an entire set of genes encoding the enzymes for the upper xylene methyl-monooxygenase pathway, xylCMABN, resembling the gene set of the terrestrial Pseudomonas putida strain mt-2. xylene-degrading bacteria, marine bacteria, toluene/xylene methyl-monooxygenase pathway, biodegradation, degrading gene, Halioxenophilus aromaticivorans INTRODUCTION Xylenes are toxic pollutants that are widely distributed in the environment and arise from a variety of industries, gasoline, airplane fuel and natural sources (Fay et al.2007). Therefore, the bioremediation of xylene is of great interest, and numerous xylene-utilizing microorganisms, such as members of the genera Pseudomonas and Rhodococcus, have been isolated, resulting in the reporting of several xylene degradation pathways (Assinder and Williams 1990; Barbieri et al.1993; Jindrová et al.2002; Kim et al.2002; Maruyama et al.2005). The initial steps in these pathways are catalyzed by dioxygenases or monooxygenases that hydroxylate the xylene aromatic ring (ring hydroxylating pathways) and monooxygenases that oxidize the xylene methyl group (methyl monooxygenase pathways). Genetic and/or biochemical data are also available for these pathways (Harayama et al.1986; Kim and Zylstra 1995, 1999; Bertoni et al.1998; Bramucci, Singh and Nagarajan 2002; Kim et al.2004; Maruyama et al.2005). However, these data are derived mainly from terrestrial or freshwater bacteria. While a few studies of marine xylene-utilizing bacteria have been conducted, information on such bacteria from marine environments and their xylene degradation pathways is relatively scarce (Wang et al.2008; Berlendis et al.2010; Jin, Choi and Jeon 2013). Indeed, there are no data on the xylene-degrading genes of marine bacteria. Therefore, additional information concerning marine xylene-degrading bacteria and their degradation pathways is required to facilitate the development of effective bioremediation programs. Here, we report the isolation of novel aerobic xylene-utilizing bacteria. Comparative 16S rRNA gene sequence analysis indicated that one of the isolates, strain KU68FT, forms an independent branch in the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria. Therefore, in this study, we aimed to determine the exact taxonomic position of strain KU68FT and to analyze the sequence properties of the xylene degradation gene cluster present in strain KU68FT. MATERIALS AND METHODS Isolation of xylene-utilizing bacteria from seawater Xylene-utilizing bacteria were isolated from two coastal areas of Japan, Fukui and Tokyo (Ogasawara Islands), according to a previously described procedure with some modifications (Iwaki, Nishimura and Hasegawa 2012). Daigo's IMK-SP medium (Nihon Pharmaceutical) was supplemented with o-, m-, or p-xylene as a vapor. 16S rRNA gene sequencing and phylogenetic analysis Near full-length 16S rRNA genes were amplified by PCR using the bacterial universal primer sets 27f/1492r or 9F/1510R (Table S1, Supporting Information; Lane 1991; Nakagawa and Kawasaki 2001). PCR amplification was carried out using BLEND Taq -plus- (Toyobo, Osaka, Japan) according to the manufacturer's instructions. PCR products were sequenced directly using the primers 27f, 357f, 926f, 342r and 1492r or the primers 9F, 785F, 802R and 1510R (Table S1, Supporting Information; Lane 1991; Nakagawa and Kawasaki 2001). Alignments were generated using the CLUSTALW tool in MEGA version 6 (Tamura et al.2013). Phylogenetic trees were generated using MEGA version 6 as previously described (Iwaki et al.2013). Determination of taxonomic characteristics of strain KU68FT Gram staining was performed using a Favor-G kit (Nissui, Tokyo, Japan), and cells were observed under a light microscope (BX50F4, Olympus, Tokyo, Japan). Cell morphology was examined under a scanning electron microscope (JSM-6320F, JEOL, Tokyo, Japan) at 5 kV by the Hanaichi UltraStructure Research Institute (Okazaki, Japan). Physiological and biochemical characterization was performed as described previously (Iwaki, Fujioka and Hasegawa 2014), except that the cell suspension was adjusted to a 1 McFarland standard for antibiotic susceptibility tests. Contents of DNA G + C, isoprenoid quinones and fatty acids were analyzed at TechnoSuruga Laboratory Co. Ltd (Shizuoka, Japan) as described previously (Iwaki, Fujioka and Hasegawa 2014). PCR detection of genes encoding initial oxygenase To screen for possible genes encoding the initial oxygenase, we used the previously described primer sets XYLA-F/XYLA-R and TODC1-F/TODC1-R (Table S1, Supporting Information; Hendrickx et al.2006). PCR amplification was carried out using BLEND Taq -plus- (Toyobo, Osaka, Japan) according to the manufacturer's instructions. Amplification and sequencing of flanking regions of the partial xylA gene from strain KU68FT The flanking regions of a partial xylA gene from strain KU68FT were obtained by inverse PCR (Ochman, Gerber and Hartl 1988) using the primers invF and invR (Table S1, Supporting Information). The inverse PCR was conducted with step-down cycles using KOD FX Neo DNA polymerase (Toyobo, Osaka, Japan) according to the manufacturer's instructions. The DNA sequence of the inverse-PCR product was determined by direct sequencing and primer walking. DNA sequencing and sequence analysis methods DNA fragments were sequenced with the BigDye Terminator Cycle Sequencing Kit version 3.1 and an ABI PRISM 310 or 3130xl genetic analyzer (Thermo Fisher Scientific, Waltham, MA). DNA sequences were analyzed with GENETYX-Mac software ver. 16 (Genetyx, Tokyo, Japan). Nucleotide and protein sequence similarity searches were done using the BLAST program (Altschul et al.1997) via the National Center for Biotechnology Information server. RESULTS AND DISCUSSION Isolation of xylene-utilizing bacteria and phylogenetic analysis of the 16S rRNA gene sequences of the isolated bacteria Xylene-utilizing bacteria were isolated from seawater collected in both sampled areas, including three strains from Fukui and four from Tokyo (Ogasawara Islands). Among the isolates, two strains, KU67F and KU67G, were isolated from enrichment cultures with o-xylene as the sole carbon source; two strains, KU68F and KU68G, were isolated from enrichment cultures with m-xylene as the sole carbon source; and three strains, KU69F, KU69G1 and KU69G4, were isolated from enrichment cultures with p-xylene as the sole carbon source (Table 1). Table 1. Xylene-degrading isolates from seawater.       16S rRNA gene sequence analysis  Enriched substrate  Source  Strain  Closest species in database (accession number)  Identity (%)  o-xylene  Fukui  KU67F  Marinobacter hydrocarbonoclasticus (X67022)  97.6  o-xylene  Tokyo (Ogasawara islands)  KU67G  Marinobacter shengliensis (KF307780)  99.2  m-xylene  Fukui  KU68F  Maricurvus nonylphenolicus (AB626730)  92.6  m-xylene  Tokyo (Ogasawara islands)  KU68G  Alteromonas macleodii (Y18228)  99.6  p-xylene  Fukui  KU69F  Aestuariibacter aggregatus (FJ847832)  99.1  p-xylene  Tokyo (Ogasawara islands)  KU69G1  Alteromonas macleodii (Y18228)  99.6  p-xylene  Tokyo (Ogasawara islands)  KU69G4  Alteromonas macleodii (Y18228)  97.8        16S rRNA gene sequence analysis  Enriched substrate  Source  Strain  Closest species in database (accession number)  Identity (%)  o-xylene  Fukui  KU67F  Marinobacter hydrocarbonoclasticus (X67022)  97.6  o-xylene  Tokyo (Ogasawara islands)  KU67G  Marinobacter shengliensis (KF307780)  99.2  m-xylene  Fukui  KU68F  Maricurvus nonylphenolicus (AB626730)  92.6  m-xylene  Tokyo (Ogasawara islands)  KU68G  Alteromonas macleodii (Y18228)  99.6  p-xylene  Fukui  KU69F  Aestuariibacter aggregatus (FJ847832)  99.1  p-xylene  Tokyo (Ogasawara islands)  KU69G1  Alteromonas macleodii (Y18228)  99.6  p-xylene  Tokyo (Ogasawara islands)  KU69G4  Alteromonas macleodii (Y18228)  97.8  View Large The isolated bacteria were characterized by analysis of their 16S rRNA gene sequences (Table 1; Fig. S1, Supporting Information). The determined 16S rRNA gene sequences were deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers AB809162 and LC339512 to LC339517. All isolates were phylogenetically affiliated with the class Gammaproteobacteria. Among them, two isolates, KU67F and KU67G, were most closely related to and affiliated with the genus Marinobacter. Two strains of the genus Marinobacter have been previously shown to degrade p-xylene (Berlendis et al.2010), and Marinobacter have been suggested to play an important role in the degradation of several organic compounds in the marine environment (Iwaki, Nishimura and Hasegawa 2012; Iwaki, Takada and Hasegawa 2015). The isolate KU67F was assumed to belong to the same strain as a previously reported phenol degrader, KU17F4, that was isolated from the same seawater sample from Fukui based on its identical 16S rRNA gene sequence (Iwaki, Takada and Hasegawa 2015). Similarly, KU67G and the previously reported phenol degrader KU17G3, which was isolated from the same seawater sample from the Ogasawara Islands, are believed to belong to the same strain. Among the isolates, three strains were most closely related to and affiliated with the genus Alteromonas, and one strain, KU69F, was most closely related to and affiliated with Aestuariibacter, a genus that is closely related to Alteromonas. Strains KU68G and KU69G1 were also assumed to be the same strain, since they exhibited identical 16S rRNA gene sequences. This is the first time that members of the genera Alteromonas and Aestuariibacter have been isolated as xylene degraders. Both culture-dependent and -independent methods have indicated that the members of the genus Alteromonas play key roles in the degradation of polycyclic aromatic hydrocarbons (Jin et al.2012). The remaining isolate, strain KU68FT, shared the highest 16S rRNA gene sequence identity with Maricurvus nonylphenolicus strain KU41ET, a p-n-nonylphenol degrader (Iwaki, Takada and Hasegawa 2012); however, the identity between these strains was only 92.6% (Table 1). Strain KU68FT was also found to be similar to Pseudoteredinibacter isoporae SW-11T (92.5%; Chen et al.2011) and Simiduia aestuariiviva J-MY2 T (92.0%; Park et al.2014) with lower identities. Levels of identity to other described genera were <92.0%, and strain KU68FT formed a distinct lineage within the family Cellvibrionaceae in the order Cellvibrionales: the closest neighbor was Marinagarivorans algicola Z1T (91.6%; Guo et al.2016), as shown in Fig. 1. Therefore, based on the phylogenetic analysis, strain KU68FT should be classified as a novel genus and species in the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria. We therefore selected KU68FT for further study, including the determination of its precise taxonomic position and characterization of the sequences of its xylene-degrading gene cluster. Figure 1. View largeDownload slide Phylogenetic relationships between strain KU68FT and other members of the class Gammaproteobacteria. The tree was constructed using the neighbor-joining algorithm. Numbers at nodes are bootstrap percentages based on 1000 replications; only values >50% are shown. Filled circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum likelihood algorithm. Bar, 0.01 substitutions per nucleotide position. Figure 1. View largeDownload slide Phylogenetic relationships between strain KU68FT and other members of the class Gammaproteobacteria. The tree was constructed using the neighbor-joining algorithm. Numbers at nodes are bootstrap percentages based on 1000 replications; only values >50% are shown. Filled circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum likelihood algorithm. Bar, 0.01 substitutions per nucleotide position. Taxonomic characteristics of strain KU68FT The cells of strain KU68FT are gram-negative and were observed to form aggregations on plates containing Marine Agar 2216 (Fig. S2, Supporting Information). Cells also formed aggregations or flocs in liquid medium. The formation of aggregates may be due to extracellular polymeric substances (Fig. S3, Supporting Information). The strain was capable of utilizing m-xylene and p-xylene but not o-xylene, benzene, toluene, ethylbenzene or cumene as sole carbon and energy sources. Other morphological, phenotypic and chemotaxonomic characteristics are provided in the genus and species descriptions, and those characteristics that differentiate strain KU68FT from phylogenetically related taxa are listed in Tables 2 and 3. Table 2. Differential characteristics of strain KU68FT and closely related genera. Characteristic  1  2  3  4  5  Motility  –  +  +  +  +  NaCl range for growth (%, w/v)  1–4  1–4  2–4  0.5–7.0  0.5–6.0  pH range for growth  6–9.5  7–8  7–8  5.5–8.0  6.5–8.5  Temperature range for growth (°C)  10–30  20–35  10–45  15–40  10–33  Catalase  +  +  +  +  –  Nitrate reduction  –  +  +  +  –  Hydrolysis of:            Starch  –  –  w  +  +  Gelatin  –  +  +  +  +  Enzyme activities (API ZYM tests)            Lipase (C14)  +  +  +  –  –  Cystine arylamidase  W  –  +  –  +  Trypsin  –  –  +  –  –  Chymotrypsin  –  –  +  +  –  Acid phosphatase  +  –  +  +  –  Naphthol-AS-BI-phosphohydrolase  +  +  +  –  +  Carbon utilization of:            D-Arabitol  –  –  +  NR  –  D-Mannose  –  –  +  –  –  L-Rhamnose  +  –  –  NR  –  D-Xylose  –  –  –  +  –  Lactose  +  –  –  NR  –  Cellobiose  +  –  +  +  +  D-Maltose  –  –  –  –  +  Sucrose  +  –  –  +  –  Trehalose  –  +  –  +  –  Glycerol  –  –  +  NR  –  myo-Inositol  +  –  –  NR  –  Acetate  +  +  +  –  NR  Citrate  –  –  –  –  +  Pyruvate  +  +  –  –  NR  Succinate  +  –  +  +  NR  L-Glutamate  +  –  +  –  NR  Susceptibility to:            Ampicillin  +  +  –  +  +  Kanamycin  +  +  +  –  NR  Polymyxin B  +  +  NR  –  +  Tetracycline  +  +  +  +  –  Isoprenoid quinone(s)  Q-8(97.7%) Q-7(1.7%) Q-9(0.6%)  Q-8  Q-9 (79%), Q-8 (21%)  Q-8  Q-8  DNA G + C content (mol%)  51.4  48.6  51.6  54.8  45.1  Characteristic  1  2  3  4  5  Motility  –  +  +  +  +  NaCl range for growth (%, w/v)  1–4  1–4  2–4  0.5–7.0  0.5–6.0  pH range for growth  6–9.5  7–8  7–8  5.5–8.0  6.5–8.5  Temperature range for growth (°C)  10–30  20–35  10–45  15–40  10–33  Catalase  +  +  +  +  –  Nitrate reduction  –  +  +  +  –  Hydrolysis of:            Starch  –  –  w  +  +  Gelatin  –  +  +  +  +  Enzyme activities (API ZYM tests)            Lipase (C14)  +  +  +  –  –  Cystine arylamidase  W  –  +  –  +  Trypsin  –  –  +  –  –  Chymotrypsin  –  –  +  +  –  Acid phosphatase  +  –  +  +  –  Naphthol-AS-BI-phosphohydrolase  +  +  +  –  +  Carbon utilization of:            D-Arabitol  –  –  +  NR  –  D-Mannose  –  –  +  –  –  L-Rhamnose  +  –  –  NR  –  D-Xylose  –  –  –  +  –  Lactose  +  –  –  NR  –  Cellobiose  +  –  +  +  +  D-Maltose  –  –  –  –  +  Sucrose  +  –  –  +  –  Trehalose  –  +  –  +  –  Glycerol  –  –  +  NR  –  myo-Inositol  +  –  –  NR  –  Acetate  +  +  +  –  NR  Citrate  –  –  –  –  +  Pyruvate  +  +  –  –  NR  Succinate  +  –  +  +  NR  L-Glutamate  +  –  +  –  NR  Susceptibility to:            Ampicillin  +  +  –  +  +  Kanamycin  +  +  +  –  NR  Polymyxin B  +  +  NR  –  +  Tetracycline  +  +  +  +  –  Isoprenoid quinone(s)  Q-8(97.7%) Q-7(1.7%) Q-9(0.6%)  Q-8  Q-9 (79%), Q-8 (21%)  Q-8  Q-8  DNA G + C content (mol%)  51.4  48.6  51.6  54.8  45.1  Genera: 1, strain KU68FT; 2, Maricurvus nonylphenolicus KU41ET (data from Iwaki et al.2012); 3, Pseudoteredinibacter isoporae SW-11T (Chen et al.2011); 4, Simiduia aestuariiviva J-MY2 T (Park et al.2014); 5, Marinagarivorans algicola Z1T (Guo et al.2016). +, positive reaction; –, negative reaction; w, weakly positive reaction; NR, not reported. View Large Table 3. Cellular fatty acid compositions (%) of strain KU68FT and closely related genera. Fatty acid  1  2  3  4  5  C9:0  –  1.0  –  –  –  C10:0  –  6.4  3.5  –  5.1  C11:0  –  –  2.5  –  –  C12:0  1.2  –  3.0  –  –  C14:0  1.6  1.3  1.1  2.5  –  C15:0  –  1.8  –  –  –  C16:0  18.1  17.0  10.3  20.7  14.3  C17:0  –  1.8  5.1  4.5  –  C18:0  –  1.6  –  1.5  1.1  C10:0 3-OH  5.9  9.4  3.2  2.7  8.3  C11:0 3-OH  –  –  4.0  –  –  C12:0 3-OH  –  –  2.4  –  –  C12:1 3-OH  –  –  –  2.5  –  C17:1 ω6c  –  –  1.2  1.1  –  C17:1 ω8c  –  5.6  13.4  12.0  –  C17:1 anteiso ω9c  –  –  10.0  –  –  C18:1 ω6c  –  3.0  13.1a  –  49.1  C18:1 ω7c  28.8  19.8    12.3  14.3  Summed Feature 3b  34.5  28.4  22.3c  36.7 c  17.4  Summed Feature 7b  –  –  –  –  1.2  unknown ECL 11.799d  8.5  –  –  –  –  Fatty acid  1  2  3  4  5  C9:0  –  1.0  –  –  –  C10:0  –  6.4  3.5  –  5.1  C11:0  –  –  2.5  –  –  C12:0  1.2  –  3.0  –  –  C14:0  1.6  1.3  1.1  2.5  –  C15:0  –  1.8  –  –  –  C16:0  18.1  17.0  10.3  20.7  14.3  C17:0  –  1.8  5.1  4.5  –  C18:0  –  1.6  –  1.5  1.1  C10:0 3-OH  5.9  9.4  3.2  2.7  8.3  C11:0 3-OH  –  –  4.0  –  –  C12:0 3-OH  –  –  2.4  –  –  C12:1 3-OH  –  –  –  2.5  –  C17:1 ω6c  –  –  1.2  1.1  –  C17:1 ω8c  –  5.6  13.4  12.0  –  C17:1 anteiso ω9c  –  –  10.0  –  –  C18:1 ω6c  –  3.0  13.1a  –  49.1  C18:1 ω7c  28.8  19.8    12.3  14.3  Summed Feature 3b  34.5  28.4  22.3c  36.7 c  17.4  Summed Feature 7b  –  –  –  –  1.2  unknown ECL 11.799d  8.5  –  –  –  –  aC18:1 ω6c and/or C18:1 ω7c; bsummed features are groups of two fatty acids that cannot be separated by GLC using the MIDI system. Summed feature 3 comprises C16:1 ω7c and/or iso-C15:0 2-OH or cC16:1 ω7c and/or C16:1 ω6c; summed feature 7 comprises C19:1 ω6c and/or unknown ECL 18.846; dECL, Equivalent chain-length. Genera: 1, strain KU68FT; 2, Maricurvus nonylphenolicus KU41ET (data from Iwaki et al.2012); 3, Pseudoteredinibacter isoporae SW-11T (Chen et al.2011); 4, Simiduia aestuariiviva J-MY2T (Park et al.2014); 5, Marinagarivorans algicola Z1T (Guo et al.2016). Values are percentages of the total fatty acids; fatty acids that make up <1% of the total are not shown or indicated by ‘–’. View Large As demonstrated by the 16S rRNA gene sequence analysis, strain KU68FT belongs to the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria and forms a lineage distinct from related genera. Furthermore, strain KU68FT can be differentiated from closely related genera by a combination of phenotypic and chemotaxonomic characteristics. Considering the data from the polyphasic study, we suggest that strain KU68FT represents a novel species of a new genus, for which we propose the name Halioxenophilus aromaticivorans gen. nov., sp. nov. Screening for a possible gene encoding initial oxygenase in the first step of xylene degradation in strain KU68FT Terrestrial bacteria degrade xylenes via several pathways that include different initial steps, and these can be distinguished based on the xylene methyl-monooxygenase that catalyzes the hydroxylation of the methyl group or the xylene dioxygenase that catalyzes the hydroxylation of the aromatic ring. m-Xylene is mainly degraded by terrestrial bacteria via the toluene/xylene methyl-monooxygenase pathway (Gibson, Mahadevan and Davey 1974; Jang et al.2005). To characterize the xylene degradation pathway of marine strain KU68FT, we screened for a possible gene encoding an initial oxygenase in the first step of the pathway using PCR. As a result, a xylA gene encoding a toluene/xylene methyl-monooxygenase was detected with the primers XYLA-F/XYLA-R. In contrast, no todC1 gene, encoding the largest subunit of a toluene/xylene dioxygenase, was detected with the primers TODC1-F/TODC1-R. Sequence analysis showed that the deduced amino acid sequence based on the nucleotide sequence of the xylA PCR fragment shared 80.7% identity with that of XylA of Pseudomonas putida strain mt-2 (Suzuki et al.1991). This result suggests that strain KU68FT has a two-component diiron xylene methyl monooxygense system (XylMA) and degrades xylene via the so-called xylene methyl-monooxygenase pathway, which is initiated by the hydroxylation of a methyl group to form methylbenzyl alcohol, followed by the formation of methylbenzealdehyde and methylbenzoate. Isolation and characterization of a possible gene cluster encoding xylene methyl-monooxygenase pathway genes from strain KU68FT To further characterize the xylene degradation pathway of strain KU68FT, we amplified the flanking regions of the partial xylA gene and determined the sequence of a contiguous segment of 9869 bp; this was deposited in the DDBJ under the accession number LC339836. Within this sequence region, seven open reading frames (ORFs) were deduced (Fig. 2). The deduced amino acid sequences of these ORFs were used for BLASTP searches, and five ORFs were assigned to xylCMABNKU68F. The deduced amino acid sequences of xylCMABNKU68F exhibit 77%–87% identity with the corresponding proteins of the well characterized xylene methyl-monooxygenase pathway of Pseudomonas putida strains mt-2 and MT53 (Table S2, Supporting Information). These high identity values are sufficient for determination of the functional identities of the proteins, confirming the presence of a xylene methyl-monooxygenase pathway in strain KU68F and allowing for the assignment of the xyl gene products to the upper part of the xylene methyl-monooxygenase pathway (Fig. 2). The gene order of xylCMABNKU68F is identical to that of terrestrial Pseudomonas spp. A notable difference in the xyl locus between KU68F and terrestrial Pseudomonas involves the fact that two genes with unknown functions in xylene degradation are not present in strain KU68F (Fig. 2): xylU, which is not related to any functionally identified protein in any protein databases, and xylW, which encodes a probable long-chain zinc-dependent alcohol dehydrogenase (Harayama et al.1986; Williams et al.1997). The presence of genes in the xylene methyl-monooxygenase pathway represents a major feature of the species, and the analysis of the xylCMABN genes should aid in species identification. Figure 2. View largeDownload slide Genetic structure of the m-xylene degradation upper pathway gene (xyl) cluster isolated from Halioxenophilus aromaticivorans strain KU68FT and assignment of the xyl gene products to the xylene degradation upper pathway. Genetic organization of the xyl gene clusters in Pseudomonas putida strains mt-2 and MT53 are also shown. Figure 2. View largeDownload slide Genetic structure of the m-xylene degradation upper pathway gene (xyl) cluster isolated from Halioxenophilus aromaticivorans strain KU68FT and assignment of the xyl gene products to the xylene degradation upper pathway. Genetic organization of the xyl gene clusters in Pseudomonas putida strains mt-2 and MT53 are also shown. Detailed sequence features The predicted coding region of xylCKU68F, encoding a methylbenzaldehyde dehydrogenase, is preceded by a putative σ70 –35 sequence, TTGACT, and –10 sequence, TTTGAT (Hertz and Stormo 1996). This coding sequence consists of 1467 nucleotides with appropriately positioned consensus Shine-Dalgarno (SD) sequences, GGAG and GAGG, 2 and 7 bp from the putative ATG start site, respectively. The deduced amino acid sequence of XylCKU68F exhibits 84.0% identity with the sequence of the (methyl)benzaldehyde dehydrogenase, XylC, of P. putida strain mt-2 (Inoue et al.1995). This is a member of the superfamily of NAD(P)+-dependent aldehyde dehydrogenases, which act on a broad variety of aldehyde and semialdehyde substrates by transforming them into carboxylic acids (Hempel, Nicholas and Lindahl 1993). Based on what is generally known about aldehyde dehydrogenases, Cys289 of XylCKU68F is the predicted catalytic amino acid residue, and the GxTxxG sequence at position 233–238 is a putative NAD+ binding site (Yeung et al.2008). Glu255, which is important for the catalytic mechanism of class 2 aldehyde dehydrogenases, is also conserved (Yeung et al.2008). The predicted coding region of xylMKU68F, encoding the hydroxylase component of the xylene methyl-monooxygenase, is 87 bp downstream of xylC in the same direction. This coding sequence consists of 1092 nucleotides with an appropriately positioned consensus SD sequence, AGGAGG, 5 bp from the putative ATG start site. The deduced amino acid sequence of XylMKU68F exhibits 81.3% identity with the sequence of the membrane-integrated hydroxylase component of the xylene methyl-monooxygenase, XylM, of P. putida strain mt-2 (Suzuki et al.1991). Nine histidine residues, which are potential ligands for the diiron atoms, are contained within four conserved motifs, HxxxH, HxxxHH, HxxHH and NYxEHYG, in XylMKU68F. The presence of the histidine residues in these motifs is characteristic of the bacterial integral-membrane hydroxylase family (Shanklin, Whittle and Fox 1994; Ratajczak, Geissdörfer and Hillen 1998; Morikawa 2010). The predicted coding region of xylAKU68F is 298 bp downstream of xylM in the same direction. This coding sequence consists of 1053 nucleotides with an appropriately positioned consensus SD sequence, GGA, 9 bp from the putative ATG start site. The deduced amino acid sequence of the complete XylAKU68F protein is 77.1% identical to that of the NADH:acceptor reductase component of the xylene methyl-monooxygenase, XylA, of P. putida strain mt-2 (Suzuki et al.1991). XylAKU68F possesses consensus sequences characteristic of plant-type iron sulfur proteins for the binding of a [2Fe-2S] cluster (Cys-X4-Cys-X2-Cys-Xn-Cys), which are conserved among various ferredoxin reductases such as the reductases of phthalate dioxygenase (Correll et al.1993) and naphthalene dioxygenase (Simon et al.1993). Consensus sequences involved in a possible FAD-binding domain (RxYS; Dym and Eisenberg 2001) and NAD(P)H-binding domain (GGxGxxP; Correll et al.1992) were also observed in the deduced amino acid sequence of XylAKU68F. The predicted coding region of xylBKU68F, encoding a methylbenzyl alcohol dehydrogenase, is 45 bp downstream of xylA. This coding sequence consists of 1101 nucleotides with an appropriately positioned consensus SD sequence, GGAG, 6 bp from the putative ATG start site. The deduced amino acid sequence of XylBKU68F exhibits 86.3% identity with the sequence of XylB of P. putida strain mt-2, a (methyl)benzyl alcohol dehydrogenase belonging to the zinc-dependent long-chain alcohol dehydrogenase family. Notable sequence features present in XylBKU68F including a catalytic zinc-binding motif (GHExxGxxxxxGxxV), structural zinc-binding motif (CxxCxxCxxxxxxxC) and coenzyme-binding motif (GxGxxG) (Reid and Fewson 1994). An Asp residue at position 218, which is expected to be essential for the binding of NAD+, is also conserved. The predicted coding region of xylNKU68F is 178 bp downstream of xylB. This coding sequence consists of 1395 nucleotides with an appropriately positioned consensus SD sequence, AGG, 5 bp from the putative ATG start site. The deduced amino acid sequence of XylNKU68F exhibits 77.8% identity with XylN from P. putida strain mt-2, an outer membrane protein involved in m-xylene uptake (Kasai, Inoue and Harayama 2001). PSORTb v3.0 (http://www.psort.org/psortb; Yu et al.2010) and PRED-TMBB (http://biophysics.biol.uoa.gr//PRED-TMBB; Bagos et al.2004) predict that XylNKU68F is also an outer membrane protein, like XylN of P. putida strain mt-2. Furthermore, the signal sequence at the N-terminal end of XylNKU68F was predicted using SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP; Petersen et al.2011), with a predicted cleavage site at position 25. The remaining two ORFs, designated orf1 and orf2, encode putative regulators. The predicted orf2 coding region is located upstream of xylC with 665 bp of intervening intergenic sequence, and it is in the opposite orientation from the xylCMABN genes (Fig. 2). A BLAST search revealed that the deduced amino acid sequence of Orf2 shows homology with putative regulators classified in the IclR family. An IclR family helix-turn-helix domain was found in the N-terminal region of the putative Orf2 protein using an InterPro search (Apweiler et al.2000). The predicted orf1 coding region is located 58 bp upstream of orf2 in the opposite orientation from orf2 and the same direction as xylCMABN. A BLAST search revealed that the deduced amino acid sequence of Orf1 shows homology with putative regulators classified in the AraC family, including XylS, which is the toluene/xylene catabolic meta-cleavage pathway operon regulator from P. putida (40.0% identity) (Ramos, Marqués, and Timmis 1997). An AraC family helix-turn-helix domain was found in the C-terminal region of Orf1 according to both Conserved Domain Database (Marchler-Bauer et al.2017) and InterPro searches (Finn et al.2017). The expression of the Pseudomonas xyl upper pathway operon is under the control of a σ54 promoter and the NtrC family regulator XylR. In strain KU68FT, Orf1 and/or Orf2 are probable regulators of the xylCMABN genes, rather than a σ54-dependent NtrC family regulator. This is supported by the presence of a σ70 promoter sequence (TTGACT-N16-TTTGAT) 43 bp upstream of the putative ATG start codon of xylC, which is the first of the xylCMABN genes. A detailed biochemical and genetic study of the xylCMABN operon and its regulation is outside the scope of this study. Description of Halioxenophilus gen. nov. Halioxenophilus (Ha.li.o.xe.no’phi.lus. Gr. adj. halios, of the sea; Gr. adj. xenos, foreign; Gr. masc. n. philos, friend; N.L. masc. n. Halioxenophilus, friend of foreign compounds, referring to the isolation of the type species by enrichment on m-xylene from the sea) Cells are Gram-negative, aerobic pleomorphic rods, and non-motile. Sodium ions are required for their growth. The predominant fatty acids are Summed Feature 3 (C15:0 iso 2-OH and/or C16:1 ω7c), C18:1 ω7c, C16:0, unknown fatty acid (equivalent chain-length 11.799), C10:0 3-OH. The predominant respiratory quinone is Q-8. The type species is Halioxenophilus aromaticivorans. Description of Halioxenophilus aromaticivorans sp. nov. Halioxenophilus aromaticivorans (a.ro.ma.ti.ci.vo’rans. N.L. adj. aromaticus, aromatic, fragrant; L. part. adj. vorans, devouring; N.L. part. adj. aromaticivorans, devouring aromatic (compounds)) The description is identical to that for the genus, with the following additions. Cells are 1.0–1.3 μm in length and 0.4–0.5 μm in width. Colonies are yellow, circular, smooth, pulvinate, 1.0 mm in diameter and with an entire margin after 2 days incubation on Marine Agar 2216, and oxidase- and catalase-positive. Growth occurs at temperatures of 10°C–30°C, at pH 6 from 9.5, and at NaCl concentrations of 1%–4%. The cells are susceptible to ampicillin (10 μg), chloramphenicol (30 μg), gentamicin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), novobiocin (30 μg), penicillin G (10 U), polymyxin B (300 U), rifampicin (5 μg), streptomycin (10 μg) and tetracycline (30 μg), but not to lincomycin (2 μg). In API ZYM system, cells are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase, and weakly positive for cystine arylamidase, but negative for all other enzymes. Hydrolysis of Tween 40, Tween 80 and esculin are positive. In API20NE system, hydrolysis of esculin, β-galactosidase and utilization of d-glucose and caprate are positive, but negative for all other tests. The cells utilize the following compounds as sole carbon and energy sources: m-xylene, p-xylene, d-glucose, l-rhamnose, myo-inositol, cellobiose, lactose, sucrose, acetate, caprate, n-hexanoate, propionate, pyruvate, succinate, l-alanine, l-glutamate, but not the following compounds: o-xylene, benzene, toluene, ethylbenzene, cumene, phenol, l-arabinose, d-arabitol, N-acetyl-glucosamine, d-galactose, d-fructose, d-mannitol, d-mannose, d-ribose, d-sorbitol, d-xylose, glycerol, d-maltose, trehalose, adipate, citrate, gluconate, formate, dl-malate, l-asparagine, l-asparate, l-histidine, l-leucine, l-phenylalanine, l-proline, l-serine, l-threonine. The DNA G + C content is 51.4 mol%. The type strain, KU68FT (=JCM 19134T = KCTC 32387T), was isolated from seawater obtained from the coastal region of Fukui, Japan. SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online. FUNDING This work was financially supported in part by the Kansai University Fund for Supporting Young Scholars (2016). Conflict of interest. None declared. REFERENCES Altschul SF Madden TL Schaffer AAet al.   Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res  1997; 25: 3389– 402. Google Scholar CrossRef Search ADS PubMed  Apweiler R Attwood TK Bairoch Aet al.   InterPro—an integrated documentation resource for protein families, domains and functional sites. Bioinformatics  2000; 16: 1145– 50. Google Scholar CrossRef Search ADS PubMed  Assinder SJ Williams PA. The TOL plasmids: determinants of the catabolism of toluene and the xylenes. Adv Microb Physiol  1990; 31: 1– 69. Google Scholar CrossRef Search ADS PubMed  Bagos PG Liakopoulos TD Spyropoulos ICet al.   PRED-TMBB: a web server for predicting the topology of beta-barrel outer membrane proteins. Nucleic Acids Res  2004; 1: W400– 4. Google Scholar CrossRef Search ADS   Barbieri P Palladino L Di Gennaro Pet al.   Alternative pathways for o-xylene or m-xylene and p-xylene degradation in a Pseu domonas stutzeri stutzeri strain. Biodegradation  1993; 4: 71– 80. Google Scholar CrossRef Search ADS   Berlendis S Cayol J-L Verhé Fet al.   First evidence of aerobic biodegradation of BTEX compounds by pure cultures of Marinobacter. Appl Biochem Biotech  2010; 160: 1992– 9. Google Scholar CrossRef Search ADS   Bertoni G Martino M Galli Eet al.   Analysis of the gene cluster encoding toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1. Appl Environ Microbiol  1998; 64: 3626– 32. Google Scholar PubMed  Bramucci M Singh M Nagarajan V. Biotransformation of p-xylene and 2,6-dimethylnaphthalene by xylene monooxygenase cloned from a Sphingomonas isolate. Appl Microbiol Biot  2002; 59: 679– 84. Google Scholar CrossRef Search ADS   Chen MH Sheu SY Arun ABet al.   Pseudoteredinibacter isoporae gen. nov., sp. nov., a marine bacterium isolated from the reef-building coral Isopora palifera. Int J Syst Evol Microbiol  2011; 61: 1887– 93. Google Scholar CrossRef Search ADS PubMed  Correll CC Batie CJ Ballou DPet al.   Phthalate dioxygenase reductase: a modular structure for electron transfer from pyridine nucleotides to [2Fe-2S]. Science  1992; 258: 1604– 10. Google Scholar CrossRef Search ADS PubMed  Correll CC Ludwig ML Bruns CMet al.   Structural prototypes for an extended family of flavoprotein reductases: comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin. Protein Sci  1993; 2: 2112– 33. Google Scholar CrossRef Search ADS PubMed  Dym O Eisenberg D. Sequence-structure analysis of FAD-containing proteins. Protein Sci  2001; 10: 1712– 28. Google Scholar CrossRef Search ADS PubMed  Fay M Risher JF Wilson JDet al.   Toxicological Profile for Xylene , U.S Department of Health and Human Services, Public Health Service. Atlanta: Agency for Toxic Substance and Disease Registry. 2007 Finn RD Attwood TK Babbitt PCet al.   InterPro in 2017—beyond protein family and domain annotations. Nucleic Acids Res  2017; 45: D190– 9. Google Scholar CrossRef Search ADS PubMed  Gibson DT Mahadevan V Davey JF. Bacterial metabolism of para- and meta-xylene: oxidation of the aromatic ring. J Bacteriol  1974; 119: 930– 6. Google Scholar PubMed  Guo LY Li DQ Sang Jet al.   Marinagarivorans algicola gen. nov., sp. nov., isolated from marine algae. Int J Syst Evol Microbiol  2016; 66: 1593– 9. Google Scholar CrossRef Search ADS PubMed  Harayama S Leppik RA Rekik Met al.   Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product. J Bacteriol  1986; 167: 455– 61. Google Scholar CrossRef Search ADS PubMed  Hempel J Nicholas H Lindahl R. Aldehyde dehydrogenases: widespread structural and functional diversity within a shared framework. Protein Sci  1993; 2: 1890– 900. Google Scholar CrossRef Search ADS PubMed  Hendrickx B Junca H Vosahlova Jet al.   Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site. J Microbiol Methods  2006; 64: 250– 65. Google Scholar CrossRef Search ADS PubMed  Hertz GZ Stormo GD. Escherichia coli promoter sequences: analysis and prediction. Methods Enzymol  1996; 273: 30– 42. Google Scholar CrossRef Search ADS PubMed  Inoue J Shaw JP Rekik Met al.   Overlapping substrate specificities of benzaldehyde dehydrogenase (the xylC gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product) encoded by TOL plasmid pWW0 of Pseu domonas putida. J Bacteriol  1995; 177: 1196– 201. Google Scholar CrossRef Search ADS PubMed  Iwaki H Fujioka M Hasegawa Y. Isolation and characterization of marine nonylphenol-degrading bacteria and description of Pseudomaricurvus alkylphenolicus gen. nov., sp. nov. Curr Microbiol  2014; 68: 167– 73. Google Scholar CrossRef Search ADS PubMed  Iwaki H Nishimura A Hasegawa Y. Isolation and characterization of marine bacteria capable of utilizing phthalate. World J Microb Biot  2012; 28: 1321– 5 Google Scholar CrossRef Search ADS   Iwaki H Takada K Hasegawa Y. Isolation and genetic characterization of phenol-utilizing marine bacteria and their phenol degradation pathway. IJGG  2015; 3: 20– 5. Google Scholar CrossRef Search ADS   Iwaki H Takada K Hasegawa Y. Maricurvus nonylphenolicus gen. nov., sp. nov., a nonylphenol-degrading bacterium isolated from seawater. FEMS Microbiol Lett  2012; 327: 142– 7. Google Scholar CrossRef Search ADS PubMed  Iwaki H Yasukawa N Fujioka Met al.   Isolation and characterization of a marine cyclohexylacetate-degrading bacterium Lutimaribacter litoralis sp. nov., and reclassification of Oceanicola pacificus as Lutimaribacter pacificus comb. nov. Curr Microbiol  2013; 66: 588– 93. Google Scholar CrossRef Search ADS PubMed  Jang JY Kim D Bae HWet al.   Isolation and characterization of a Rhodococcus species strain able to grow on ortho- and para-xylene. J Microbiol  2005; 43: 325– 30. Google Scholar PubMed  Jin HM Choi EJ Jeon CO. Isolation of a BTEX-degrading bacterium, Janibacter sp. SB2, from a sea-tidal flat and optimization of biodegradation conditions. Bioresour Technol  2013; 145: 57– 64. Google Scholar CrossRef Search ADS PubMed  Jin HM Kim JM Lee HJet al.   Alteromonas as a key agent of polycyclic aromatic hydrocarbon biodegradation in crude oil-contaminated coastal sediment. Environ Sci Technol  2012; 46: 7731– 40. Google Scholar CrossRef Search ADS PubMed  Jindrová E Chocová M Demnerová Ket al.   Bacterial aerobic degradation of benzene, toluene, ethylbenzene and xylene. Folia Microbiol  2002; 47: 83– 93. Google Scholar CrossRef Search ADS   Kasai Y Inoue J Harayama S. The TOL plasmid pWW0 xylN gene product from Pseudomonas putida is involved in m-Xylene uptake. J Bacteriol  2001; 183: 6662– 6. Google Scholar CrossRef Search ADS PubMed  Kim D Chae JC Zylstra GJet al.   Identification of a novel dioxygenase involved in metabolism of o-xylene, toluene, and ethylbenzene by Rhodococcus sp. strain DK17. Appl Environ Microbiol  2004; 70: 7086– 92. Google Scholar CrossRef Search ADS PubMed  Kim D Kim YS Kim SKet al.   Monocyclic aromatic hydrocarbon degradation by Rhodococcus sp. strain DK17. Appl Environ Microbiol  2002: 68: 3270– 8. Google Scholar CrossRef Search ADS PubMed  Kim E Zylstra GJ. Molecular and biochemical characterization of two meta-cleavage dioxygenases involved in biphenyl and m-xylene degradation by Beijerinckia sp. strain B1. J Bacteriol  1995; 177: 3095– 103. Google Scholar CrossRef Search ADS PubMed  Kim E Zylstra GJ. Functional analysis of genes involved in biphenyl, naphthalene, phenanthrene, and m-xylene degradation by Sphingomonas yanoikuyae B1. J Ind Microbiol Biotechnol  1999; 23: 294– 302. Google Scholar CrossRef Search ADS PubMed  Lane DJ. 16S/23S rRNA sequencing. In: Stackebrandt E Goodfellow M (eds). Nucleic Acid Techniques in Bacterial Systematics . Chichester, United Kingdom: Wiley, 1991; 115– 75. Marchler-Bauer A Bo Y Han Let al.   CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res  2017; 45: D200– 3. Google Scholar CrossRef Search ADS PubMed  Maruyama T Ishikura M Taki Het al.   Isolation and characterization of o-xylene oxygenase genes from Rhodococcus opacus TKN14. Appl Environ Microbiol  2005; 71: 7705– 15. Google Scholar CrossRef Search ADS PubMed  Morikawa M. Dioxygen activation responsible for oxidation of aliphatic and aromatic hydrocarbon compounds: current state and variants. Appl Microbiol Biot  2010; 87: 1595– 603. Google Scholar CrossRef Search ADS   Nakagawa K Kawasaki H. Determination method of 16S rRNA gene sequence. In: The Society for Antinomycetes (ed.). The Isolation and Characterization of Actinomycetes . Tokyo: Business Center for Academic Societies Japan. 2001; 88– 117 (in Japanese). Ochman H Gerber AS Hartl DL. Genetic applicationsof an inverse polymerase chain reaction. Genetics  1988; 120: 621– 3. Google Scholar PubMed  Park S Kim SI Kang CHet al.   Simiduia aestuariiviva sp. nov., a gammaproteobacterium isolated from a tidal flat sediment. A Van Lee  2014; 106: 927– 34. Google Scholar CrossRef Search ADS   Petersen TN Brunak S von Heijne Get al.   SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods  2011; 8: 785– 6. Google Scholar CrossRef Search ADS PubMed  Ramos JL Marqués S Timmis KN. Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. Annu Rev Microbiol  1997; 51: 341– 73. Google Scholar CrossRef Search ADS PubMed  Ratajczak A Geissdörfer W Hillen W. Alkane hydroxylase from Acinetobacter sp. strain ADP1 is encoded by alkM and belongs to a new family of bacterial integral-membrane hydrocarbon hydroxylases. Appl Environ Microbiol  1998; 64: 1175– 9. Google Scholar PubMed  Reid MF Fewson CA. Molecular characterization of microbial alcohol dehydrogenases. Crit Rev Microbiol  1994; 20: 13– 56. Google Scholar CrossRef Search ADS PubMed  Shanklin J Whittle E Fox BG. Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry  1994; 33: 12787– 94. Google Scholar CrossRef Search ADS PubMed  Simon MJ Osslund TD Saunders Ret al.   Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4. Gene  1993; 127: 31– 7. Google Scholar CrossRef Search ADS PubMed  Suzuki M Hayakawa T Shaw JPet al.   Primary structure of xylene monooxygenase: similarities to and differences from the alkane hydroxylation system. J Bacteriol  1991; 173: 1690– 5. Google Scholar CrossRef Search ADS PubMed  Tamura K Stecher G Peterson Det al.   MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol  2013; 30: 2725– 9. Google Scholar CrossRef Search ADS PubMed  Wang L Qiao N Sun Fet al.   Isolation, gene detection and solvent tolerance of benzene, toluene and xylene degrading bacteria from nearshore surface water and Pacific Ocean sediment. Extremophiles  2008; 12: 335– 42. Google Scholar CrossRef Search ADS PubMed  Williams PA Shaw LM Pitt CWet al.   xyIUW, two genes at the start of the upper pathway operon of TOL plasmid pWWO, appear to play no essential part in determining its catabolic phenotype. Microbiology  1997; 143: 101– 7. Google Scholar CrossRef Search ADS PubMed  Yeung CK Yep A Kenyon GLet al.   Physical, kinetic and spectrophotometric studies of a NAD(P)-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633. Biochim Biophys Acta  2008; 1784: 1248– 55. Google Scholar CrossRef Search ADS PubMed  Yu NY Wagner JR Laird MRet al.   PSORTb 3.0: Improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics  2010; 26: 1608– 15. Google Scholar CrossRef Search ADS PubMed  © FEMS 2018. All rights reserved. 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Isolation of marine xylene-utilizing bacteria and characterization of Halioxenophilus aromaticivorans gen. nov., sp. nov. and its xylene degradation gene cluster

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Abstract

Abstract Seven xylene-utilizing bacterial strains were isolated from seawater collected off the coast of Japan. Analysis of 16S rRNA gene sequences indicated that six isolates were most closely related to the marine bacterial genera Alteromonas, Marinobacter or Aestuariibacter. The sequence of the remaining strain, KU68FT, showed low similarity to the 16S rRNA gene sequences of known bacteria with validly published names, the most similar species being Maricurvus nonylphenolicus strain KU41ET (92.6% identity). On the basis of physiological, chemotaxonomic and phylogenetic data, strain KU68FT is suggested to represent a novel species of a new genus in the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria, for which the name Halioxenophilus aromaticivorans gen. nov., sp. nov. is proposed. The type strain of Halioxenophilus aromaticivorans is KU68FT (=JCM 19134T = KCTC 32387T). PCR and sequence analysis revealed that strain KU68FT possesses an entire set of genes encoding the enzymes for the upper xylene methyl-monooxygenase pathway, xylCMABN, resembling the gene set of the terrestrial Pseudomonas putida strain mt-2. xylene-degrading bacteria, marine bacteria, toluene/xylene methyl-monooxygenase pathway, biodegradation, degrading gene, Halioxenophilus aromaticivorans INTRODUCTION Xylenes are toxic pollutants that are widely distributed in the environment and arise from a variety of industries, gasoline, airplane fuel and natural sources (Fay et al.2007). Therefore, the bioremediation of xylene is of great interest, and numerous xylene-utilizing microorganisms, such as members of the genera Pseudomonas and Rhodococcus, have been isolated, resulting in the reporting of several xylene degradation pathways (Assinder and Williams 1990; Barbieri et al.1993; Jindrová et al.2002; Kim et al.2002; Maruyama et al.2005). The initial steps in these pathways are catalyzed by dioxygenases or monooxygenases that hydroxylate the xylene aromatic ring (ring hydroxylating pathways) and monooxygenases that oxidize the xylene methyl group (methyl monooxygenase pathways). Genetic and/or biochemical data are also available for these pathways (Harayama et al.1986; Kim and Zylstra 1995, 1999; Bertoni et al.1998; Bramucci, Singh and Nagarajan 2002; Kim et al.2004; Maruyama et al.2005). However, these data are derived mainly from terrestrial or freshwater bacteria. While a few studies of marine xylene-utilizing bacteria have been conducted, information on such bacteria from marine environments and their xylene degradation pathways is relatively scarce (Wang et al.2008; Berlendis et al.2010; Jin, Choi and Jeon 2013). Indeed, there are no data on the xylene-degrading genes of marine bacteria. Therefore, additional information concerning marine xylene-degrading bacteria and their degradation pathways is required to facilitate the development of effective bioremediation programs. Here, we report the isolation of novel aerobic xylene-utilizing bacteria. Comparative 16S rRNA gene sequence analysis indicated that one of the isolates, strain KU68FT, forms an independent branch in the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria. Therefore, in this study, we aimed to determine the exact taxonomic position of strain KU68FT and to analyze the sequence properties of the xylene degradation gene cluster present in strain KU68FT. MATERIALS AND METHODS Isolation of xylene-utilizing bacteria from seawater Xylene-utilizing bacteria were isolated from two coastal areas of Japan, Fukui and Tokyo (Ogasawara Islands), according to a previously described procedure with some modifications (Iwaki, Nishimura and Hasegawa 2012). Daigo's IMK-SP medium (Nihon Pharmaceutical) was supplemented with o-, m-, or p-xylene as a vapor. 16S rRNA gene sequencing and phylogenetic analysis Near full-length 16S rRNA genes were amplified by PCR using the bacterial universal primer sets 27f/1492r or 9F/1510R (Table S1, Supporting Information; Lane 1991; Nakagawa and Kawasaki 2001). PCR amplification was carried out using BLEND Taq -plus- (Toyobo, Osaka, Japan) according to the manufacturer's instructions. PCR products were sequenced directly using the primers 27f, 357f, 926f, 342r and 1492r or the primers 9F, 785F, 802R and 1510R (Table S1, Supporting Information; Lane 1991; Nakagawa and Kawasaki 2001). Alignments were generated using the CLUSTALW tool in MEGA version 6 (Tamura et al.2013). Phylogenetic trees were generated using MEGA version 6 as previously described (Iwaki et al.2013). Determination of taxonomic characteristics of strain KU68FT Gram staining was performed using a Favor-G kit (Nissui, Tokyo, Japan), and cells were observed under a light microscope (BX50F4, Olympus, Tokyo, Japan). Cell morphology was examined under a scanning electron microscope (JSM-6320F, JEOL, Tokyo, Japan) at 5 kV by the Hanaichi UltraStructure Research Institute (Okazaki, Japan). Physiological and biochemical characterization was performed as described previously (Iwaki, Fujioka and Hasegawa 2014), except that the cell suspension was adjusted to a 1 McFarland standard for antibiotic susceptibility tests. Contents of DNA G + C, isoprenoid quinones and fatty acids were analyzed at TechnoSuruga Laboratory Co. Ltd (Shizuoka, Japan) as described previously (Iwaki, Fujioka and Hasegawa 2014). PCR detection of genes encoding initial oxygenase To screen for possible genes encoding the initial oxygenase, we used the previously described primer sets XYLA-F/XYLA-R and TODC1-F/TODC1-R (Table S1, Supporting Information; Hendrickx et al.2006). PCR amplification was carried out using BLEND Taq -plus- (Toyobo, Osaka, Japan) according to the manufacturer's instructions. Amplification and sequencing of flanking regions of the partial xylA gene from strain KU68FT The flanking regions of a partial xylA gene from strain KU68FT were obtained by inverse PCR (Ochman, Gerber and Hartl 1988) using the primers invF and invR (Table S1, Supporting Information). The inverse PCR was conducted with step-down cycles using KOD FX Neo DNA polymerase (Toyobo, Osaka, Japan) according to the manufacturer's instructions. The DNA sequence of the inverse-PCR product was determined by direct sequencing and primer walking. DNA sequencing and sequence analysis methods DNA fragments were sequenced with the BigDye Terminator Cycle Sequencing Kit version 3.1 and an ABI PRISM 310 or 3130xl genetic analyzer (Thermo Fisher Scientific, Waltham, MA). DNA sequences were analyzed with GENETYX-Mac software ver. 16 (Genetyx, Tokyo, Japan). Nucleotide and protein sequence similarity searches were done using the BLAST program (Altschul et al.1997) via the National Center for Biotechnology Information server. RESULTS AND DISCUSSION Isolation of xylene-utilizing bacteria and phylogenetic analysis of the 16S rRNA gene sequences of the isolated bacteria Xylene-utilizing bacteria were isolated from seawater collected in both sampled areas, including three strains from Fukui and four from Tokyo (Ogasawara Islands). Among the isolates, two strains, KU67F and KU67G, were isolated from enrichment cultures with o-xylene as the sole carbon source; two strains, KU68F and KU68G, were isolated from enrichment cultures with m-xylene as the sole carbon source; and three strains, KU69F, KU69G1 and KU69G4, were isolated from enrichment cultures with p-xylene as the sole carbon source (Table 1). Table 1. Xylene-degrading isolates from seawater.       16S rRNA gene sequence analysis  Enriched substrate  Source  Strain  Closest species in database (accession number)  Identity (%)  o-xylene  Fukui  KU67F  Marinobacter hydrocarbonoclasticus (X67022)  97.6  o-xylene  Tokyo (Ogasawara islands)  KU67G  Marinobacter shengliensis (KF307780)  99.2  m-xylene  Fukui  KU68F  Maricurvus nonylphenolicus (AB626730)  92.6  m-xylene  Tokyo (Ogasawara islands)  KU68G  Alteromonas macleodii (Y18228)  99.6  p-xylene  Fukui  KU69F  Aestuariibacter aggregatus (FJ847832)  99.1  p-xylene  Tokyo (Ogasawara islands)  KU69G1  Alteromonas macleodii (Y18228)  99.6  p-xylene  Tokyo (Ogasawara islands)  KU69G4  Alteromonas macleodii (Y18228)  97.8        16S rRNA gene sequence analysis  Enriched substrate  Source  Strain  Closest species in database (accession number)  Identity (%)  o-xylene  Fukui  KU67F  Marinobacter hydrocarbonoclasticus (X67022)  97.6  o-xylene  Tokyo (Ogasawara islands)  KU67G  Marinobacter shengliensis (KF307780)  99.2  m-xylene  Fukui  KU68F  Maricurvus nonylphenolicus (AB626730)  92.6  m-xylene  Tokyo (Ogasawara islands)  KU68G  Alteromonas macleodii (Y18228)  99.6  p-xylene  Fukui  KU69F  Aestuariibacter aggregatus (FJ847832)  99.1  p-xylene  Tokyo (Ogasawara islands)  KU69G1  Alteromonas macleodii (Y18228)  99.6  p-xylene  Tokyo (Ogasawara islands)  KU69G4  Alteromonas macleodii (Y18228)  97.8  View Large The isolated bacteria were characterized by analysis of their 16S rRNA gene sequences (Table 1; Fig. S1, Supporting Information). The determined 16S rRNA gene sequences were deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers AB809162 and LC339512 to LC339517. All isolates were phylogenetically affiliated with the class Gammaproteobacteria. Among them, two isolates, KU67F and KU67G, were most closely related to and affiliated with the genus Marinobacter. Two strains of the genus Marinobacter have been previously shown to degrade p-xylene (Berlendis et al.2010), and Marinobacter have been suggested to play an important role in the degradation of several organic compounds in the marine environment (Iwaki, Nishimura and Hasegawa 2012; Iwaki, Takada and Hasegawa 2015). The isolate KU67F was assumed to belong to the same strain as a previously reported phenol degrader, KU17F4, that was isolated from the same seawater sample from Fukui based on its identical 16S rRNA gene sequence (Iwaki, Takada and Hasegawa 2015). Similarly, KU67G and the previously reported phenol degrader KU17G3, which was isolated from the same seawater sample from the Ogasawara Islands, are believed to belong to the same strain. Among the isolates, three strains were most closely related to and affiliated with the genus Alteromonas, and one strain, KU69F, was most closely related to and affiliated with Aestuariibacter, a genus that is closely related to Alteromonas. Strains KU68G and KU69G1 were also assumed to be the same strain, since they exhibited identical 16S rRNA gene sequences. This is the first time that members of the genera Alteromonas and Aestuariibacter have been isolated as xylene degraders. Both culture-dependent and -independent methods have indicated that the members of the genus Alteromonas play key roles in the degradation of polycyclic aromatic hydrocarbons (Jin et al.2012). The remaining isolate, strain KU68FT, shared the highest 16S rRNA gene sequence identity with Maricurvus nonylphenolicus strain KU41ET, a p-n-nonylphenol degrader (Iwaki, Takada and Hasegawa 2012); however, the identity between these strains was only 92.6% (Table 1). Strain KU68FT was also found to be similar to Pseudoteredinibacter isoporae SW-11T (92.5%; Chen et al.2011) and Simiduia aestuariiviva J-MY2 T (92.0%; Park et al.2014) with lower identities. Levels of identity to other described genera were <92.0%, and strain KU68FT formed a distinct lineage within the family Cellvibrionaceae in the order Cellvibrionales: the closest neighbor was Marinagarivorans algicola Z1T (91.6%; Guo et al.2016), as shown in Fig. 1. Therefore, based on the phylogenetic analysis, strain KU68FT should be classified as a novel genus and species in the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria. We therefore selected KU68FT for further study, including the determination of its precise taxonomic position and characterization of the sequences of its xylene-degrading gene cluster. Figure 1. View largeDownload slide Phylogenetic relationships between strain KU68FT and other members of the class Gammaproteobacteria. The tree was constructed using the neighbor-joining algorithm. Numbers at nodes are bootstrap percentages based on 1000 replications; only values >50% are shown. Filled circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum likelihood algorithm. Bar, 0.01 substitutions per nucleotide position. Figure 1. View largeDownload slide Phylogenetic relationships between strain KU68FT and other members of the class Gammaproteobacteria. The tree was constructed using the neighbor-joining algorithm. Numbers at nodes are bootstrap percentages based on 1000 replications; only values >50% are shown. Filled circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum likelihood algorithm. Bar, 0.01 substitutions per nucleotide position. Taxonomic characteristics of strain KU68FT The cells of strain KU68FT are gram-negative and were observed to form aggregations on plates containing Marine Agar 2216 (Fig. S2, Supporting Information). Cells also formed aggregations or flocs in liquid medium. The formation of aggregates may be due to extracellular polymeric substances (Fig. S3, Supporting Information). The strain was capable of utilizing m-xylene and p-xylene but not o-xylene, benzene, toluene, ethylbenzene or cumene as sole carbon and energy sources. Other morphological, phenotypic and chemotaxonomic characteristics are provided in the genus and species descriptions, and those characteristics that differentiate strain KU68FT from phylogenetically related taxa are listed in Tables 2 and 3. Table 2. Differential characteristics of strain KU68FT and closely related genera. Characteristic  1  2  3  4  5  Motility  –  +  +  +  +  NaCl range for growth (%, w/v)  1–4  1–4  2–4  0.5–7.0  0.5–6.0  pH range for growth  6–9.5  7–8  7–8  5.5–8.0  6.5–8.5  Temperature range for growth (°C)  10–30  20–35  10–45  15–40  10–33  Catalase  +  +  +  +  –  Nitrate reduction  –  +  +  +  –  Hydrolysis of:            Starch  –  –  w  +  +  Gelatin  –  +  +  +  +  Enzyme activities (API ZYM tests)            Lipase (C14)  +  +  +  –  –  Cystine arylamidase  W  –  +  –  +  Trypsin  –  –  +  –  –  Chymotrypsin  –  –  +  +  –  Acid phosphatase  +  –  +  +  –  Naphthol-AS-BI-phosphohydrolase  +  +  +  –  +  Carbon utilization of:            D-Arabitol  –  –  +  NR  –  D-Mannose  –  –  +  –  –  L-Rhamnose  +  –  –  NR  –  D-Xylose  –  –  –  +  –  Lactose  +  –  –  NR  –  Cellobiose  +  –  +  +  +  D-Maltose  –  –  –  –  +  Sucrose  +  –  –  +  –  Trehalose  –  +  –  +  –  Glycerol  –  –  +  NR  –  myo-Inositol  +  –  –  NR  –  Acetate  +  +  +  –  NR  Citrate  –  –  –  –  +  Pyruvate  +  +  –  –  NR  Succinate  +  –  +  +  NR  L-Glutamate  +  –  +  –  NR  Susceptibility to:            Ampicillin  +  +  –  +  +  Kanamycin  +  +  +  –  NR  Polymyxin B  +  +  NR  –  +  Tetracycline  +  +  +  +  –  Isoprenoid quinone(s)  Q-8(97.7%) Q-7(1.7%) Q-9(0.6%)  Q-8  Q-9 (79%), Q-8 (21%)  Q-8  Q-8  DNA G + C content (mol%)  51.4  48.6  51.6  54.8  45.1  Characteristic  1  2  3  4  5  Motility  –  +  +  +  +  NaCl range for growth (%, w/v)  1–4  1–4  2–4  0.5–7.0  0.5–6.0  pH range for growth  6–9.5  7–8  7–8  5.5–8.0  6.5–8.5  Temperature range for growth (°C)  10–30  20–35  10–45  15–40  10–33  Catalase  +  +  +  +  –  Nitrate reduction  –  +  +  +  –  Hydrolysis of:            Starch  –  –  w  +  +  Gelatin  –  +  +  +  +  Enzyme activities (API ZYM tests)            Lipase (C14)  +  +  +  –  –  Cystine arylamidase  W  –  +  –  +  Trypsin  –  –  +  –  –  Chymotrypsin  –  –  +  +  –  Acid phosphatase  +  –  +  +  –  Naphthol-AS-BI-phosphohydrolase  +  +  +  –  +  Carbon utilization of:            D-Arabitol  –  –  +  NR  –  D-Mannose  –  –  +  –  –  L-Rhamnose  +  –  –  NR  –  D-Xylose  –  –  –  +  –  Lactose  +  –  –  NR  –  Cellobiose  +  –  +  +  +  D-Maltose  –  –  –  –  +  Sucrose  +  –  –  +  –  Trehalose  –  +  –  +  –  Glycerol  –  –  +  NR  –  myo-Inositol  +  –  –  NR  –  Acetate  +  +  +  –  NR  Citrate  –  –  –  –  +  Pyruvate  +  +  –  –  NR  Succinate  +  –  +  +  NR  L-Glutamate  +  –  +  –  NR  Susceptibility to:            Ampicillin  +  +  –  +  +  Kanamycin  +  +  +  –  NR  Polymyxin B  +  +  NR  –  +  Tetracycline  +  +  +  +  –  Isoprenoid quinone(s)  Q-8(97.7%) Q-7(1.7%) Q-9(0.6%)  Q-8  Q-9 (79%), Q-8 (21%)  Q-8  Q-8  DNA G + C content (mol%)  51.4  48.6  51.6  54.8  45.1  Genera: 1, strain KU68FT; 2, Maricurvus nonylphenolicus KU41ET (data from Iwaki et al.2012); 3, Pseudoteredinibacter isoporae SW-11T (Chen et al.2011); 4, Simiduia aestuariiviva J-MY2 T (Park et al.2014); 5, Marinagarivorans algicola Z1T (Guo et al.2016). +, positive reaction; –, negative reaction; w, weakly positive reaction; NR, not reported. View Large Table 3. Cellular fatty acid compositions (%) of strain KU68FT and closely related genera. Fatty acid  1  2  3  4  5  C9:0  –  1.0  –  –  –  C10:0  –  6.4  3.5  –  5.1  C11:0  –  –  2.5  –  –  C12:0  1.2  –  3.0  –  –  C14:0  1.6  1.3  1.1  2.5  –  C15:0  –  1.8  –  –  –  C16:0  18.1  17.0  10.3  20.7  14.3  C17:0  –  1.8  5.1  4.5  –  C18:0  –  1.6  –  1.5  1.1  C10:0 3-OH  5.9  9.4  3.2  2.7  8.3  C11:0 3-OH  –  –  4.0  –  –  C12:0 3-OH  –  –  2.4  –  –  C12:1 3-OH  –  –  –  2.5  –  C17:1 ω6c  –  –  1.2  1.1  –  C17:1 ω8c  –  5.6  13.4  12.0  –  C17:1 anteiso ω9c  –  –  10.0  –  –  C18:1 ω6c  –  3.0  13.1a  –  49.1  C18:1 ω7c  28.8  19.8    12.3  14.3  Summed Feature 3b  34.5  28.4  22.3c  36.7 c  17.4  Summed Feature 7b  –  –  –  –  1.2  unknown ECL 11.799d  8.5  –  –  –  –  Fatty acid  1  2  3  4  5  C9:0  –  1.0  –  –  –  C10:0  –  6.4  3.5  –  5.1  C11:0  –  –  2.5  –  –  C12:0  1.2  –  3.0  –  –  C14:0  1.6  1.3  1.1  2.5  –  C15:0  –  1.8  –  –  –  C16:0  18.1  17.0  10.3  20.7  14.3  C17:0  –  1.8  5.1  4.5  –  C18:0  –  1.6  –  1.5  1.1  C10:0 3-OH  5.9  9.4  3.2  2.7  8.3  C11:0 3-OH  –  –  4.0  –  –  C12:0 3-OH  –  –  2.4  –  –  C12:1 3-OH  –  –  –  2.5  –  C17:1 ω6c  –  –  1.2  1.1  –  C17:1 ω8c  –  5.6  13.4  12.0  –  C17:1 anteiso ω9c  –  –  10.0  –  –  C18:1 ω6c  –  3.0  13.1a  –  49.1  C18:1 ω7c  28.8  19.8    12.3  14.3  Summed Feature 3b  34.5  28.4  22.3c  36.7 c  17.4  Summed Feature 7b  –  –  –  –  1.2  unknown ECL 11.799d  8.5  –  –  –  –  aC18:1 ω6c and/or C18:1 ω7c; bsummed features are groups of two fatty acids that cannot be separated by GLC using the MIDI system. Summed feature 3 comprises C16:1 ω7c and/or iso-C15:0 2-OH or cC16:1 ω7c and/or C16:1 ω6c; summed feature 7 comprises C19:1 ω6c and/or unknown ECL 18.846; dECL, Equivalent chain-length. Genera: 1, strain KU68FT; 2, Maricurvus nonylphenolicus KU41ET (data from Iwaki et al.2012); 3, Pseudoteredinibacter isoporae SW-11T (Chen et al.2011); 4, Simiduia aestuariiviva J-MY2T (Park et al.2014); 5, Marinagarivorans algicola Z1T (Guo et al.2016). Values are percentages of the total fatty acids; fatty acids that make up <1% of the total are not shown or indicated by ‘–’. View Large As demonstrated by the 16S rRNA gene sequence analysis, strain KU68FT belongs to the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria and forms a lineage distinct from related genera. Furthermore, strain KU68FT can be differentiated from closely related genera by a combination of phenotypic and chemotaxonomic characteristics. Considering the data from the polyphasic study, we suggest that strain KU68FT represents a novel species of a new genus, for which we propose the name Halioxenophilus aromaticivorans gen. nov., sp. nov. Screening for a possible gene encoding initial oxygenase in the first step of xylene degradation in strain KU68FT Terrestrial bacteria degrade xylenes via several pathways that include different initial steps, and these can be distinguished based on the xylene methyl-monooxygenase that catalyzes the hydroxylation of the methyl group or the xylene dioxygenase that catalyzes the hydroxylation of the aromatic ring. m-Xylene is mainly degraded by terrestrial bacteria via the toluene/xylene methyl-monooxygenase pathway (Gibson, Mahadevan and Davey 1974; Jang et al.2005). To characterize the xylene degradation pathway of marine strain KU68FT, we screened for a possible gene encoding an initial oxygenase in the first step of the pathway using PCR. As a result, a xylA gene encoding a toluene/xylene methyl-monooxygenase was detected with the primers XYLA-F/XYLA-R. In contrast, no todC1 gene, encoding the largest subunit of a toluene/xylene dioxygenase, was detected with the primers TODC1-F/TODC1-R. Sequence analysis showed that the deduced amino acid sequence based on the nucleotide sequence of the xylA PCR fragment shared 80.7% identity with that of XylA of Pseudomonas putida strain mt-2 (Suzuki et al.1991). This result suggests that strain KU68FT has a two-component diiron xylene methyl monooxygense system (XylMA) and degrades xylene via the so-called xylene methyl-monooxygenase pathway, which is initiated by the hydroxylation of a methyl group to form methylbenzyl alcohol, followed by the formation of methylbenzealdehyde and methylbenzoate. Isolation and characterization of a possible gene cluster encoding xylene methyl-monooxygenase pathway genes from strain KU68FT To further characterize the xylene degradation pathway of strain KU68FT, we amplified the flanking regions of the partial xylA gene and determined the sequence of a contiguous segment of 9869 bp; this was deposited in the DDBJ under the accession number LC339836. Within this sequence region, seven open reading frames (ORFs) were deduced (Fig. 2). The deduced amino acid sequences of these ORFs were used for BLASTP searches, and five ORFs were assigned to xylCMABNKU68F. The deduced amino acid sequences of xylCMABNKU68F exhibit 77%–87% identity with the corresponding proteins of the well characterized xylene methyl-monooxygenase pathway of Pseudomonas putida strains mt-2 and MT53 (Table S2, Supporting Information). These high identity values are sufficient for determination of the functional identities of the proteins, confirming the presence of a xylene methyl-monooxygenase pathway in strain KU68F and allowing for the assignment of the xyl gene products to the upper part of the xylene methyl-monooxygenase pathway (Fig. 2). The gene order of xylCMABNKU68F is identical to that of terrestrial Pseudomonas spp. A notable difference in the xyl locus between KU68F and terrestrial Pseudomonas involves the fact that two genes with unknown functions in xylene degradation are not present in strain KU68F (Fig. 2): xylU, which is not related to any functionally identified protein in any protein databases, and xylW, which encodes a probable long-chain zinc-dependent alcohol dehydrogenase (Harayama et al.1986; Williams et al.1997). The presence of genes in the xylene methyl-monooxygenase pathway represents a major feature of the species, and the analysis of the xylCMABN genes should aid in species identification. Figure 2. View largeDownload slide Genetic structure of the m-xylene degradation upper pathway gene (xyl) cluster isolated from Halioxenophilus aromaticivorans strain KU68FT and assignment of the xyl gene products to the xylene degradation upper pathway. Genetic organization of the xyl gene clusters in Pseudomonas putida strains mt-2 and MT53 are also shown. Figure 2. View largeDownload slide Genetic structure of the m-xylene degradation upper pathway gene (xyl) cluster isolated from Halioxenophilus aromaticivorans strain KU68FT and assignment of the xyl gene products to the xylene degradation upper pathway. Genetic organization of the xyl gene clusters in Pseudomonas putida strains mt-2 and MT53 are also shown. Detailed sequence features The predicted coding region of xylCKU68F, encoding a methylbenzaldehyde dehydrogenase, is preceded by a putative σ70 –35 sequence, TTGACT, and –10 sequence, TTTGAT (Hertz and Stormo 1996). This coding sequence consists of 1467 nucleotides with appropriately positioned consensus Shine-Dalgarno (SD) sequences, GGAG and GAGG, 2 and 7 bp from the putative ATG start site, respectively. The deduced amino acid sequence of XylCKU68F exhibits 84.0% identity with the sequence of the (methyl)benzaldehyde dehydrogenase, XylC, of P. putida strain mt-2 (Inoue et al.1995). This is a member of the superfamily of NAD(P)+-dependent aldehyde dehydrogenases, which act on a broad variety of aldehyde and semialdehyde substrates by transforming them into carboxylic acids (Hempel, Nicholas and Lindahl 1993). Based on what is generally known about aldehyde dehydrogenases, Cys289 of XylCKU68F is the predicted catalytic amino acid residue, and the GxTxxG sequence at position 233–238 is a putative NAD+ binding site (Yeung et al.2008). Glu255, which is important for the catalytic mechanism of class 2 aldehyde dehydrogenases, is also conserved (Yeung et al.2008). The predicted coding region of xylMKU68F, encoding the hydroxylase component of the xylene methyl-monooxygenase, is 87 bp downstream of xylC in the same direction. This coding sequence consists of 1092 nucleotides with an appropriately positioned consensus SD sequence, AGGAGG, 5 bp from the putative ATG start site. The deduced amino acid sequence of XylMKU68F exhibits 81.3% identity with the sequence of the membrane-integrated hydroxylase component of the xylene methyl-monooxygenase, XylM, of P. putida strain mt-2 (Suzuki et al.1991). Nine histidine residues, which are potential ligands for the diiron atoms, are contained within four conserved motifs, HxxxH, HxxxHH, HxxHH and NYxEHYG, in XylMKU68F. The presence of the histidine residues in these motifs is characteristic of the bacterial integral-membrane hydroxylase family (Shanklin, Whittle and Fox 1994; Ratajczak, Geissdörfer and Hillen 1998; Morikawa 2010). The predicted coding region of xylAKU68F is 298 bp downstream of xylM in the same direction. This coding sequence consists of 1053 nucleotides with an appropriately positioned consensus SD sequence, GGA, 9 bp from the putative ATG start site. The deduced amino acid sequence of the complete XylAKU68F protein is 77.1% identical to that of the NADH:acceptor reductase component of the xylene methyl-monooxygenase, XylA, of P. putida strain mt-2 (Suzuki et al.1991). XylAKU68F possesses consensus sequences characteristic of plant-type iron sulfur proteins for the binding of a [2Fe-2S] cluster (Cys-X4-Cys-X2-Cys-Xn-Cys), which are conserved among various ferredoxin reductases such as the reductases of phthalate dioxygenase (Correll et al.1993) and naphthalene dioxygenase (Simon et al.1993). Consensus sequences involved in a possible FAD-binding domain (RxYS; Dym and Eisenberg 2001) and NAD(P)H-binding domain (GGxGxxP; Correll et al.1992) were also observed in the deduced amino acid sequence of XylAKU68F. The predicted coding region of xylBKU68F, encoding a methylbenzyl alcohol dehydrogenase, is 45 bp downstream of xylA. This coding sequence consists of 1101 nucleotides with an appropriately positioned consensus SD sequence, GGAG, 6 bp from the putative ATG start site. The deduced amino acid sequence of XylBKU68F exhibits 86.3% identity with the sequence of XylB of P. putida strain mt-2, a (methyl)benzyl alcohol dehydrogenase belonging to the zinc-dependent long-chain alcohol dehydrogenase family. Notable sequence features present in XylBKU68F including a catalytic zinc-binding motif (GHExxGxxxxxGxxV), structural zinc-binding motif (CxxCxxCxxxxxxxC) and coenzyme-binding motif (GxGxxG) (Reid and Fewson 1994). An Asp residue at position 218, which is expected to be essential for the binding of NAD+, is also conserved. The predicted coding region of xylNKU68F is 178 bp downstream of xylB. This coding sequence consists of 1395 nucleotides with an appropriately positioned consensus SD sequence, AGG, 5 bp from the putative ATG start site. The deduced amino acid sequence of XylNKU68F exhibits 77.8% identity with XylN from P. putida strain mt-2, an outer membrane protein involved in m-xylene uptake (Kasai, Inoue and Harayama 2001). PSORTb v3.0 (http://www.psort.org/psortb; Yu et al.2010) and PRED-TMBB (http://biophysics.biol.uoa.gr//PRED-TMBB; Bagos et al.2004) predict that XylNKU68F is also an outer membrane protein, like XylN of P. putida strain mt-2. Furthermore, the signal sequence at the N-terminal end of XylNKU68F was predicted using SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP; Petersen et al.2011), with a predicted cleavage site at position 25. The remaining two ORFs, designated orf1 and orf2, encode putative regulators. The predicted orf2 coding region is located upstream of xylC with 665 bp of intervening intergenic sequence, and it is in the opposite orientation from the xylCMABN genes (Fig. 2). A BLAST search revealed that the deduced amino acid sequence of Orf2 shows homology with putative regulators classified in the IclR family. An IclR family helix-turn-helix domain was found in the N-terminal region of the putative Orf2 protein using an InterPro search (Apweiler et al.2000). The predicted orf1 coding region is located 58 bp upstream of orf2 in the opposite orientation from orf2 and the same direction as xylCMABN. A BLAST search revealed that the deduced amino acid sequence of Orf1 shows homology with putative regulators classified in the AraC family, including XylS, which is the toluene/xylene catabolic meta-cleavage pathway operon regulator from P. putida (40.0% identity) (Ramos, Marqués, and Timmis 1997). An AraC family helix-turn-helix domain was found in the C-terminal region of Orf1 according to both Conserved Domain Database (Marchler-Bauer et al.2017) and InterPro searches (Finn et al.2017). The expression of the Pseudomonas xyl upper pathway operon is under the control of a σ54 promoter and the NtrC family regulator XylR. In strain KU68FT, Orf1 and/or Orf2 are probable regulators of the xylCMABN genes, rather than a σ54-dependent NtrC family regulator. This is supported by the presence of a σ70 promoter sequence (TTGACT-N16-TTTGAT) 43 bp upstream of the putative ATG start codon of xylC, which is the first of the xylCMABN genes. A detailed biochemical and genetic study of the xylCMABN operon and its regulation is outside the scope of this study. Description of Halioxenophilus gen. nov. Halioxenophilus (Ha.li.o.xe.no’phi.lus. Gr. adj. halios, of the sea; Gr. adj. xenos, foreign; Gr. masc. n. philos, friend; N.L. masc. n. Halioxenophilus, friend of foreign compounds, referring to the isolation of the type species by enrichment on m-xylene from the sea) Cells are Gram-negative, aerobic pleomorphic rods, and non-motile. Sodium ions are required for their growth. The predominant fatty acids are Summed Feature 3 (C15:0 iso 2-OH and/or C16:1 ω7c), C18:1 ω7c, C16:0, unknown fatty acid (equivalent chain-length 11.799), C10:0 3-OH. The predominant respiratory quinone is Q-8. The type species is Halioxenophilus aromaticivorans. Description of Halioxenophilus aromaticivorans sp. nov. Halioxenophilus aromaticivorans (a.ro.ma.ti.ci.vo’rans. N.L. adj. aromaticus, aromatic, fragrant; L. part. adj. vorans, devouring; N.L. part. adj. aromaticivorans, devouring aromatic (compounds)) The description is identical to that for the genus, with the following additions. Cells are 1.0–1.3 μm in length and 0.4–0.5 μm in width. Colonies are yellow, circular, smooth, pulvinate, 1.0 mm in diameter and with an entire margin after 2 days incubation on Marine Agar 2216, and oxidase- and catalase-positive. Growth occurs at temperatures of 10°C–30°C, at pH 6 from 9.5, and at NaCl concentrations of 1%–4%. The cells are susceptible to ampicillin (10 μg), chloramphenicol (30 μg), gentamicin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), novobiocin (30 μg), penicillin G (10 U), polymyxin B (300 U), rifampicin (5 μg), streptomycin (10 μg) and tetracycline (30 μg), but not to lincomycin (2 μg). In API ZYM system, cells are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase, and weakly positive for cystine arylamidase, but negative for all other enzymes. Hydrolysis of Tween 40, Tween 80 and esculin are positive. In API20NE system, hydrolysis of esculin, β-galactosidase and utilization of d-glucose and caprate are positive, but negative for all other tests. The cells utilize the following compounds as sole carbon and energy sources: m-xylene, p-xylene, d-glucose, l-rhamnose, myo-inositol, cellobiose, lactose, sucrose, acetate, caprate, n-hexanoate, propionate, pyruvate, succinate, l-alanine, l-glutamate, but not the following compounds: o-xylene, benzene, toluene, ethylbenzene, cumene, phenol, l-arabinose, d-arabitol, N-acetyl-glucosamine, d-galactose, d-fructose, d-mannitol, d-mannose, d-ribose, d-sorbitol, d-xylose, glycerol, d-maltose, trehalose, adipate, citrate, gluconate, formate, dl-malate, l-asparagine, l-asparate, l-histidine, l-leucine, l-phenylalanine, l-proline, l-serine, l-threonine. The DNA G + C content is 51.4 mol%. The type strain, KU68FT (=JCM 19134T = KCTC 32387T), was isolated from seawater obtained from the coastal region of Fukui, Japan. SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online. FUNDING This work was financially supported in part by the Kansai University Fund for Supporting Young Scholars (2016). Conflict of interest. None declared. REFERENCES Altschul SF Madden TL Schaffer AAet al.   Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res  1997; 25: 3389– 402. Google Scholar CrossRef Search ADS PubMed  Apweiler R Attwood TK Bairoch Aet al.   InterPro—an integrated documentation resource for protein families, domains and functional sites. Bioinformatics  2000; 16: 1145– 50. Google Scholar CrossRef Search ADS PubMed  Assinder SJ Williams PA. The TOL plasmids: determinants of the catabolism of toluene and the xylenes. Adv Microb Physiol  1990; 31: 1– 69. Google Scholar CrossRef Search ADS PubMed  Bagos PG Liakopoulos TD Spyropoulos ICet al.   PRED-TMBB: a web server for predicting the topology of beta-barrel outer membrane proteins. Nucleic Acids Res  2004; 1: W400– 4. Google Scholar CrossRef Search ADS   Barbieri P Palladino L Di Gennaro Pet al.   Alternative pathways for o-xylene or m-xylene and p-xylene degradation in a Pseu domonas stutzeri stutzeri strain. Biodegradation  1993; 4: 71– 80. Google Scholar CrossRef Search ADS   Berlendis S Cayol J-L Verhé Fet al.   First evidence of aerobic biodegradation of BTEX compounds by pure cultures of Marinobacter. Appl Biochem Biotech  2010; 160: 1992– 9. Google Scholar CrossRef Search ADS   Bertoni G Martino M Galli Eet al.   Analysis of the gene cluster encoding toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1. Appl Environ Microbiol  1998; 64: 3626– 32. Google Scholar PubMed  Bramucci M Singh M Nagarajan V. Biotransformation of p-xylene and 2,6-dimethylnaphthalene by xylene monooxygenase cloned from a Sphingomonas isolate. Appl Microbiol Biot  2002; 59: 679– 84. Google Scholar CrossRef Search ADS   Chen MH Sheu SY Arun ABet al.   Pseudoteredinibacter isoporae gen. nov., sp. nov., a marine bacterium isolated from the reef-building coral Isopora palifera. Int J Syst Evol Microbiol  2011; 61: 1887– 93. Google Scholar CrossRef Search ADS PubMed  Correll CC Batie CJ Ballou DPet al.   Phthalate dioxygenase reductase: a modular structure for electron transfer from pyridine nucleotides to [2Fe-2S]. Science  1992; 258: 1604– 10. Google Scholar CrossRef Search ADS PubMed  Correll CC Ludwig ML Bruns CMet al.   Structural prototypes for an extended family of flavoprotein reductases: comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin. Protein Sci  1993; 2: 2112– 33. Google Scholar CrossRef Search ADS PubMed  Dym O Eisenberg D. Sequence-structure analysis of FAD-containing proteins. Protein Sci  2001; 10: 1712– 28. Google Scholar CrossRef Search ADS PubMed  Fay M Risher JF Wilson JDet al.   Toxicological Profile for Xylene , U.S Department of Health and Human Services, Public Health Service. Atlanta: Agency for Toxic Substance and Disease Registry. 2007 Finn RD Attwood TK Babbitt PCet al.   InterPro in 2017—beyond protein family and domain annotations. Nucleic Acids Res  2017; 45: D190– 9. Google Scholar CrossRef Search ADS PubMed  Gibson DT Mahadevan V Davey JF. Bacterial metabolism of para- and meta-xylene: oxidation of the aromatic ring. J Bacteriol  1974; 119: 930– 6. Google Scholar PubMed  Guo LY Li DQ Sang Jet al.   Marinagarivorans algicola gen. nov., sp. nov., isolated from marine algae. Int J Syst Evol Microbiol  2016; 66: 1593– 9. Google Scholar CrossRef Search ADS PubMed  Harayama S Leppik RA Rekik Met al.   Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product. J Bacteriol  1986; 167: 455– 61. Google Scholar CrossRef Search ADS PubMed  Hempel J Nicholas H Lindahl R. Aldehyde dehydrogenases: widespread structural and functional diversity within a shared framework. Protein Sci  1993; 2: 1890– 900. Google Scholar CrossRef Search ADS PubMed  Hendrickx B Junca H Vosahlova Jet al.   Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site. J Microbiol Methods  2006; 64: 250– 65. Google Scholar CrossRef Search ADS PubMed  Hertz GZ Stormo GD. Escherichia coli promoter sequences: analysis and prediction. Methods Enzymol  1996; 273: 30– 42. Google Scholar CrossRef Search ADS PubMed  Inoue J Shaw JP Rekik Met al.   Overlapping substrate specificities of benzaldehyde dehydrogenase (the xylC gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product) encoded by TOL plasmid pWW0 of Pseu domonas putida. J Bacteriol  1995; 177: 1196– 201. Google Scholar CrossRef Search ADS PubMed  Iwaki H Fujioka M Hasegawa Y. Isolation and characterization of marine nonylphenol-degrading bacteria and description of Pseudomaricurvus alkylphenolicus gen. nov., sp. nov. Curr Microbiol  2014; 68: 167– 73. Google Scholar CrossRef Search ADS PubMed  Iwaki H Nishimura A Hasegawa Y. Isolation and characterization of marine bacteria capable of utilizing phthalate. World J Microb Biot  2012; 28: 1321– 5 Google Scholar CrossRef Search ADS   Iwaki H Takada K Hasegawa Y. Isolation and genetic characterization of phenol-utilizing marine bacteria and their phenol degradation pathway. IJGG  2015; 3: 20– 5. Google Scholar CrossRef Search ADS   Iwaki H Takada K Hasegawa Y. Maricurvus nonylphenolicus gen. nov., sp. nov., a nonylphenol-degrading bacterium isolated from seawater. FEMS Microbiol Lett  2012; 327: 142– 7. Google Scholar CrossRef Search ADS PubMed  Iwaki H Yasukawa N Fujioka Met al.   Isolation and characterization of a marine cyclohexylacetate-degrading bacterium Lutimaribacter litoralis sp. nov., and reclassification of Oceanicola pacificus as Lutimaribacter pacificus comb. nov. Curr Microbiol  2013; 66: 588– 93. Google Scholar CrossRef Search ADS PubMed  Jang JY Kim D Bae HWet al.   Isolation and characterization of a Rhodococcus species strain able to grow on ortho- and para-xylene. J Microbiol  2005; 43: 325– 30. Google Scholar PubMed  Jin HM Choi EJ Jeon CO. Isolation of a BTEX-degrading bacterium, Janibacter sp. SB2, from a sea-tidal flat and optimization of biodegradation conditions. Bioresour Technol  2013; 145: 57– 64. Google Scholar CrossRef Search ADS PubMed  Jin HM Kim JM Lee HJet al.   Alteromonas as a key agent of polycyclic aromatic hydrocarbon biodegradation in crude oil-contaminated coastal sediment. Environ Sci Technol  2012; 46: 7731– 40. Google Scholar CrossRef Search ADS PubMed  Jindrová E Chocová M Demnerová Ket al.   Bacterial aerobic degradation of benzene, toluene, ethylbenzene and xylene. Folia Microbiol  2002; 47: 83– 93. Google Scholar CrossRef Search ADS   Kasai Y Inoue J Harayama S. The TOL plasmid pWW0 xylN gene product from Pseudomonas putida is involved in m-Xylene uptake. J Bacteriol  2001; 183: 6662– 6. Google Scholar CrossRef Search ADS PubMed  Kim D Chae JC Zylstra GJet al.   Identification of a novel dioxygenase involved in metabolism of o-xylene, toluene, and ethylbenzene by Rhodococcus sp. strain DK17. Appl Environ Microbiol  2004; 70: 7086– 92. Google Scholar CrossRef Search ADS PubMed  Kim D Kim YS Kim SKet al.   Monocyclic aromatic hydrocarbon degradation by Rhodococcus sp. strain DK17. Appl Environ Microbiol  2002: 68: 3270– 8. Google Scholar CrossRef Search ADS PubMed  Kim E Zylstra GJ. Molecular and biochemical characterization of two meta-cleavage dioxygenases involved in biphenyl and m-xylene degradation by Beijerinckia sp. strain B1. J Bacteriol  1995; 177: 3095– 103. Google Scholar CrossRef Search ADS PubMed  Kim E Zylstra GJ. Functional analysis of genes involved in biphenyl, naphthalene, phenanthrene, and m-xylene degradation by Sphingomonas yanoikuyae B1. J Ind Microbiol Biotechnol  1999; 23: 294– 302. Google Scholar CrossRef Search ADS PubMed  Lane DJ. 16S/23S rRNA sequencing. In: Stackebrandt E Goodfellow M (eds). Nucleic Acid Techniques in Bacterial Systematics . Chichester, United Kingdom: Wiley, 1991; 115– 75. Marchler-Bauer A Bo Y Han Let al.   CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res  2017; 45: D200– 3. Google Scholar CrossRef Search ADS PubMed  Maruyama T Ishikura M Taki Het al.   Isolation and characterization of o-xylene oxygenase genes from Rhodococcus opacus TKN14. Appl Environ Microbiol  2005; 71: 7705– 15. Google Scholar CrossRef Search ADS PubMed  Morikawa M. Dioxygen activation responsible for oxidation of aliphatic and aromatic hydrocarbon compounds: current state and variants. Appl Microbiol Biot  2010; 87: 1595– 603. Google Scholar CrossRef Search ADS   Nakagawa K Kawasaki H. Determination method of 16S rRNA gene sequence. In: The Society for Antinomycetes (ed.). The Isolation and Characterization of Actinomycetes . Tokyo: Business Center for Academic Societies Japan. 2001; 88– 117 (in Japanese). Ochman H Gerber AS Hartl DL. Genetic applicationsof an inverse polymerase chain reaction. Genetics  1988; 120: 621– 3. Google Scholar PubMed  Park S Kim SI Kang CHet al.   Simiduia aestuariiviva sp. nov., a gammaproteobacterium isolated from a tidal flat sediment. A Van Lee  2014; 106: 927– 34. Google Scholar CrossRef Search ADS   Petersen TN Brunak S von Heijne Get al.   SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods  2011; 8: 785– 6. Google Scholar CrossRef Search ADS PubMed  Ramos JL Marqués S Timmis KN. Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. Annu Rev Microbiol  1997; 51: 341– 73. Google Scholar CrossRef Search ADS PubMed  Ratajczak A Geissdörfer W Hillen W. Alkane hydroxylase from Acinetobacter sp. strain ADP1 is encoded by alkM and belongs to a new family of bacterial integral-membrane hydrocarbon hydroxylases. Appl Environ Microbiol  1998; 64: 1175– 9. Google Scholar PubMed  Reid MF Fewson CA. Molecular characterization of microbial alcohol dehydrogenases. Crit Rev Microbiol  1994; 20: 13– 56. Google Scholar CrossRef Search ADS PubMed  Shanklin J Whittle E Fox BG. Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry  1994; 33: 12787– 94. Google Scholar CrossRef Search ADS PubMed  Simon MJ Osslund TD Saunders Ret al.   Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4. Gene  1993; 127: 31– 7. Google Scholar CrossRef Search ADS PubMed  Suzuki M Hayakawa T Shaw JPet al.   Primary structure of xylene monooxygenase: similarities to and differences from the alkane hydroxylation system. J Bacteriol  1991; 173: 1690– 5. Google Scholar CrossRef Search ADS PubMed  Tamura K Stecher G Peterson Det al.   MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol  2013; 30: 2725– 9. Google Scholar CrossRef Search ADS PubMed  Wang L Qiao N Sun Fet al.   Isolation, gene detection and solvent tolerance of benzene, toluene and xylene degrading bacteria from nearshore surface water and Pacific Ocean sediment. Extremophiles  2008; 12: 335– 42. Google Scholar CrossRef Search ADS PubMed  Williams PA Shaw LM Pitt CWet al.   xyIUW, two genes at the start of the upper pathway operon of TOL plasmid pWWO, appear to play no essential part in determining its catabolic phenotype. Microbiology  1997; 143: 101– 7. Google Scholar CrossRef Search ADS PubMed  Yeung CK Yep A Kenyon GLet al.   Physical, kinetic and spectrophotometric studies of a NAD(P)-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633. Biochim Biophys Acta  2008; 1784: 1248– 55. Google Scholar CrossRef Search ADS PubMed  Yu NY Wagner JR Laird MRet al.   PSORTb 3.0: Improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics  2010; 26: 1608– 15. Google Scholar CrossRef Search ADS PubMed  © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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