Minjeong Park

Pseudoxanthomonas sacheonensis sp. nov., isolated from BTEX-contaminated soil in Korea, transfer of Stenotrophomonas dokdonensis Yoon et al. 2006 to the genus Pseudoxanthomonas as Pseudoxanthomonas dokdonensis comb. nov. and emended description of the genus Pseudoxanthomonas

A Gram-negative, strictly aerobic, rod-shaped bacterium, designated strain BD-c54(T), was isolated from BTEX (benzene, toluene, ethylbenzene and xylenes)-contaminated soil in Sacheon, Korea. Growth of strain BD-c54(T) was observed at 15-35 degrees C (optimum 25-30 degrees C) and pH 6.0-9.5 (optimum pH 7.0-8.0). The predominant fatty acids were iso-C(15:0), iso-C(17:1)omega9c, iso-C(11:0) 3-OH, iso-C(16:0), iso-C(11:0) and iso-C(17:0). The strain contained large amounts of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol and a small amount of an unknown amino-group-containing polar lipid as polar lipids. The major quinone was ubiquinone-8 (Q-8) and the G+C content of the genomic DNA was 67.5 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain BD-c54(T) formed a tight phylogenetic lineage with Pseudoxanthomonas yeongjuensis GR12-1(T) within the genus Pseudoxanthomonas and was most closely related to P. yeongjuensis GR12-1(T) and [Stenotrophomonas] dokdonensis DS-16(T), with 98.3 and 96.6% 16S rRNA gene sequence similarity, respectively. The DNA-DNA relatedness between strain BD-c54(T) and P. yeongjuensis GR12-1(T) was 24.5%. On the basis of chemotaxonomic data and molecular properties, strain BD-c54(T) represents a novel species within the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas sacheonensis sp. nov. is proposed. The type strain is BD-c54(T) (=KCTC 22080(T) =DSM 19373(T)). In addition, the transfer of Stenotrophomonas dokdonensis to Pseudoxanthomonas as Pseudoxanthomonas dokdonensis comb. nov. and an emended description of the genus Pseudoxanthomonas are proposed.

Rheinheimera soli sp. nov., a gammaproteobacterium isolated from soil in Korea.

A Gram-negative, non-spore-forming bacterium, designated strain BD-d46(T), was isolated from a playground soil sample in Jinju, South Korea. Cells were straight or curved rods and showed catalase- and oxidase-positive reactions. Growth of strain BD-d46(T) was observed between 15 and 35 degrees C (optimum 25-30 degrees C) and between pH 6.5 and 8.0 (optimum pH 7.0-7.5). The predominant fatty acids were summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH), C(12 : 0) 3-OH and C(16 : 0). Strain BD-d46(T) contained phosphatidylethanolamine and phosphatidylglycerol as major polar lipids. Isoprenoid quinones were Q-8 (75 %) and MK-7 (25 %). The G+C content of the genomic DNA was 49.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BD-d46(T) formed a distinct lineage with Rheinheimera chironomi K19414(T) within the genus Rheinheimera. Levels of 16S rRNA gene sequence similarity between strain BD-d46(T) and the type strains of recognized Rheinheimera species ranged from 94.4 to 96.9 %. On the basis of chemotaxonomic data and molecular properties, strain BD-d46(T) is considered to represent a novel species of the genus Rheinheimera, for which the name Rheinheimera soli sp. nov. is proposed. The type strain is BD-d46(T) (=KCTC 22077(T) =DSM 19413(T)).

Molecular and biochemical characterization of 3-hydroxybenzoate 6-hydroxylase from Polaromonas naphthalenivorans CJ2.

ABSTRACT Prior research revealed that Polaromonas naphthalenivorans CJ2 carries and expresses genes encoding the gentisate metabolic pathway for naphthalene. These metabolic genes are split into two clusters, comprising nagRAaGHAbAcAdBFCQEDJI′-orf1-tnpA and nagR2-orf2I″KL (C. O. Jeon, M. Park, H. Ro, W. Park, and E. L. Madsen, Appl. Environ. Microbiol. 72:1086-1095, 2006). BLAST homology searches of sequences in GenBank indicated that the orf2 gene from the small cluster likely encoded a salicylate 5-hydroxylase, presumed to catalyze the conversion of salicylate into gentisate. Here, we report physiological and genetic evidence that orf2 does not encode salicylate 5-hydroxylase. Instead, we have found that orf2 encodes 3-hydroxybenzoate 6-hydroxylase, the enzyme which catalyzes the NADH-dependent conversion of 3-hydroxybenzoate into gentisate. Accordingly, we have renamed orf2 nagX. After expression in Escherichia coli, the NagX enzyme had an approximate molecular mass of 43 kDa, as estimated by gel filtration, and was probably a monomeric protein. The enzyme was able to convert 3-hydroxybenzoate into gentisate without salicylate 5-hydroxylase activity. Like other 3-hydroxybenzoate 6-hydroxylases, NagX utilized both NADH and NADPH as electron donors and exhibited a yellowish color, indicative of a bound flavin adenine dinucleotide. An engineered mutant of P. naphthalenivorans CJ2 defective in nagX failed to grow on 3-hydroxybenzoate but grew normally on naphthalene. These results indicate that the previously described small catabolic cluster in strain CJ2 may be multifunctional and is essential for the degradation of 3-hydroxybenzoate. Because nagX and an adjacent MarR-type regulatory gene are both closely related to homologues in Azoarcus species, this study raises questions about horizontal gene transfer events that contribute to operon evolution.

Caenimonas koreensis gen. nov., sp. nov., isolated from activated sludge.

A Gram-negative, rod-shaped bacterium, designated strain EMB320T, was isolated from activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor. The isolate was strictly aerobic and non-motile. Growth was observed between 10 and 35 degrees C (optimum 30 degrees C) and between pH 6.0 and 9.0 (optimum pH 7.0-8.0). The predominant cellular fatty acids of strain EMB320T were C16 : 0, C18 : 1omega7c and summed feature 3 (C16 : 1omega7c and/or iso-C15 : 0 2-OH). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Strain EMB320T contained ubiquinone-8 (Q-8) as the major respiratory quinone system and 2-hydroxyputrescine and putrescine as the major polyamines, which suggests that it belongs to the Betaproteobacteria. The G+C content of the genomic DNA was 62.7 mol%. Comparative 16S rRNA gene sequence analysis showed that strain EMB320T formed a phyletic lineage distinct from other genera within the family Comamonadaceae. On the basis of chemotaxonomic data and molecular properties, strain EMB320T represents a novel genus and species within the family Comamonadaceae, for which the name Caenimonas koreensis sp. nov. is proposed. The type strain of Caenimonas koreensis is EMB320T (=KCTC 12616T =DSM 17982T).

Gentisate 1,2-dioxygenase, in the third naphthalene catabolic gene cluster of Polaromonas naphthalenivorans CJ2, has a role in naphthalene degradation

Polaromonas naphthalenivorans strain CJ2 metabolizes naphthalene via the gentisate pathway and has recently been shown to carry a third copy of gentisate 1,2-dioxygenase (GDO), encoded by nagI3, within a previously uncharacterized naphthalene catabolic gene cluster. The role of this cluster (especially nagI3) in naphthalene metabolism of strain CJ2 was investigated by documenting patterns in regulation, transcription and enzyme activity. Transcriptional analysis of wild-type cells showed the third cluster to be polycistronic and that nagI3 was expressed at a relatively high level. Individual knockout mutants of all three nagI genes were constructed and their influence on both GDO activity and cell growth was evaluated. Of the three knockout strains, CJ2ΔnagI3 showed severely diminished GDO activity and grew slowest on aromatic substrates. These observations are consistent with the hypothesis that nagI3 may prevent toxic intracellular levels of gentisate from accumulating in CJ2 cells. All three nagI genes from strain CJ2 were cloned into Escherichia coli: the nagI2 and nagI3 genes were successfully overexpressed. The subunit mass of the GDOs were ~36-39 kDa, and their structures were deduced to be dimeric. The K(m) values of NagI2 and NagI3 were 31 and 10 µM, respectively, indicating that the higher affinity of NagI3 for gentisate may protect the wild-type cells from gentisate toxicity. These results provide clues for explaining why the third gene cluster, particularly the nagI3 gene, is important in strain CJ2. The organization of genes in the third gene cluster matched that of clusters in Polaromonas sp. JS666 and Leptothrix cholodnii SP-6. While horizontal gene transfer (HGT) is one hypothesis for explaining this genetic motif, gene duplication within the ancestral lineage is equally valid. The HGT hypothesis was discounted by noting that the nagI3 allele of strain CJ2 did not share high sequence identity with its homologues in Polaromonas sp. JS666 and L. cholodnii SP-6.