Polycyclic Aromatic Hydrocarbon Degradation by a New Marine Bacterium, Neptunomonas naphthovorans gen. nov., sp. nov.

Two strains of bacteria were isolated from creosote-contaminated Puget Sound sediment based on their ability to utilize naphthalene as a sole carbon and energy source. When incubated with a polycyclic aromatic hydrocarbon (PAH) compound in artificial seawater, each strain also degraded 2-methylnaphthalene and 1-methylnaphthalene; in addition, one strain, NAG-2N-113, degraded 2,6-dimethylnaphthalene and phenanthrene. Acenaphthene was not degraded when it was used as a sole carbon source but was degraded by both strains when it was incubated with a mixture of seven other PAHs. Degenerate primers and the PCR were used to isolate a portion of a naphthalene dioxygenase iron-sulfur protein (ISP) gene from each of the strains. A phylogenetic analysis of PAH dioxygenase ISP deduced amino acid sequences showed that the genes isolated in this study were distantly related to the genes encoding naphthalene dioxygenases of Pseudomonas and Burkholderia strains. Despite the differences in PAH degradation phenotype between the new strains, the dioxygenase ISP deduced amino acid fragments of these organisms were 97.6% identical. 16S ribosomal DNA-based phylogenetic analysis placed these bacteria in the gamma-3 subgroup of the Proteobacteria, most closely related to members of the genus Oceanospirillum. However, morphologic, physiologic, and genotypic differences between the new strains and the oceanospirilla justify the creation of a novel genus and species, Neptunomonas naphthovorans. The type strain of N. naphthovorans is strain NAG-2N-126.     

Polycyclovorans algicola gen. nov., sp. nov., an Aromatic-Hydrocarbon-Degrading Marine Bacterium Found Associated with Laboratory Cultures of Marine Phytoplankton

A strictly aerobic, halotolerant, rod-shaped bacterium, designated strain TG408, was isolated from a laboratory culture of the marine diatom Skeletonema costatum (CCAP1077/1C) by enrichment with polycyclic aromatic hydrocarbons (PAHs) as the sole carbon source. 16S rRNA gene sequence analysis placed this organism within the order Xanthomonadales of the class Gammaproteobacteria. Its closest relatives included representatives of the Hydrocarboniphaga-Nevskia-Sinobacter clade (<92% sequence similarity) in the family Sinobacteraceae. The strain exhibited a narrow nutritional spectrum, preferring to utilize aliphatic and aromatic hydrocarbon compounds and small organic acids. Notably, it displayed versatility in degrading two- and three-ring PAHs. Moreover, catechol 2,3-dioxygenase activity was detected in lysates, indicating that this strain utilizes the meta-cleavage pathway for aromatic compound degradation. Cells produced surface blebs and contained a single polar flagellum. The predominant isoprenoid quinone of strain TG408 was Q-8, and the dominant fatty acids were C_16:0, C_16:1 ω7c, and C_18:1 ω7c. The G+C content of the isolate''s DNA was 64.3 mol% ± 0.34 mol%. On the basis of distinct phenotypic and genotypic characteristics, strain TG408 represents a novel genus and species in the class Gammaproteobacteria for which the name Polycyclovorans algicola gen. nov., sp. nov., is proposed. Quantitative PCR primers targeting the 16S rRNA gene of this strain were developed and used to show that this organism is found associated with other species of marine phytoplankton. Phytoplankton may be a natural biotope in the ocean where new species of hydrocarbon-degrading bacteria await discovery and which contribute significantly to natural remediation processes.     

Fibrivirga algicola gen. nov., sp. nov., an algicidal bacterium isolated from a freshwater river

An aerobic, gram-stain-negative, pink-colored, non-motile and rod-shaped algicidal bacterium, designated as JA-25^T was isolated from freshwater in Geumgang River, Republic of Korea. Strain JA-25^T grew at 15–30 °C and pH 6–9, and did not require NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JA-25^T belongs to the family ‘Spirosomaceae’ and is most closely related to Fibrella aestuarina BUZ 2^T (93.6%). Strain JA-25^T showed?<?90% sequence similarity to other members of the family ‘Spirosomaceae’. The average nucleotide identity(ANI), in silico DNA-DNA hybridization and average amino acid identity(AAI) values based on the genomic sequences of JA-25^T and F. aestuarina BUZ 2^T were 74.4, 20.5, and 73.6%, respectively. Strain JA-25^T showed an algicidal effect on the marine flagellate alga Heterocapsa triquetra, but no effect on fresh water cyanobacterium (Nostoc). In genome analysis, RIPP-like peptides were detected and predicted to resemble the indolmycin biosynthetic gene cluster, which possibly influence its algicidal effect. Furthermore, a bacteriorhodopsin gene with photoheterotrophic characteristics was detected. The genomic DNA G?+?C content was 52.5 mol%. The major cellular fatty acids were summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:1 ω5c, C16:0 (>?10%). The major respiratory quinone was menaquinone 7 and major polar lipids were phosphatidylethanolamine, two unidentified aminolipids, two phospholipids, and five unidentified lipids. Considering the phylogenetic inference, phenotypic, and chemotaxonomic data, strain JA-25^T should be classified as a novel species in the novel genus Fibrivirga, with the proposed name Fibrivirga algicola sp. nov. The type strain is JA-25^T (=?KCCM 43334^T?=?NBRC 114259^T).

     

Characterization of Sphingomonas sp. Ant 17, an Aromatic Hydrocarbon-Degrading Bacterium Isolated from Antarctic Soil

Sphingomonas sp. strain Ant 17 was isolated from fuel-contaminated soil collected at Scott Base, Ross Island, Antarctica. We anticipated that Ant 17 would be a good model organism for studying cold climate bioremediation, and therefore determined its biodegradation capabilities and tolerance of potentially growth-limiting environmental conditions. Sphingomonas sp. Ant 17 degrades the aromatic fraction of several different crude oils, jet fuel, and diesel fuel at low temperatures and without nutrient amendment. It utilizes or transforms a broad range of pure aromatic substrates, including hydrocarbons, heterocycles, and aromatic acids and alcohols. Ant 17 grows at temperatures of 1 degree C to 35 degrees C and mineralizes radiolabeled phenanthrene over a range of more than 24 degrees C. This psychrotolerant isolate appears to utilize hydrocarbons more efficiently at low temperatures than would be predicted by mesophilic enzyme kinetics. The optimum pH for growth was 6.4 at 22 degrees C, with extended lag phases observed in more alkaline media. However, there was less effect of pH on lag phase at lower temperatures. Ant 17 displayed greater tolerance to UV irradiation and freeze-thaw cycles than the hydrocarbon-degrading isolate Sphingomonas sp. WPO-1, which may reflect adaptation to its Antarctic soil environment. However, it was more sensitive than expected to desiccation and to low concentrations of NaCl and CaCl(2). Ant 17 was phenotypically stable and lacked detectable plasmids, suggesting a chromosomal location for genes encoding aromatic degradation enzymes. Its broad aromatic substrate range and tolerance of low and fluctuating temperature and low nutrients make Sphingomonas sp. Ant 17 a valuable microbe for examining fuel spill bioremediation in cold soils.     

Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24

Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX.     

Numerical and Genetic Analysis of Polycyclic Aromatic Hydrocarbon-Degrading Mycobacteria

Ability to degrade high molecular weight polycyclic aromatic hydrocarbons (PAHs) has been found in diverse species of fast-growing mycobacteria. This study included several PAH-degrading mycobacteria from heavily contaminated sites and an uncontaminated humus soil in the Natural Park, Schwäbische Alb, Germany. The numerical analysis with a total of 131 tests showed that isolates from humus soil and contaminated sites had similar substrate utilization patterns for primary alcohols from ethanol to pentanol, 1,4-butanediol, benzyl alcohol, hexadecane, ethyl acetate, fluoranthene, phenanthrene, and pyrene as the sole carbon and energy (C/E) sources. Significant differences between the two subgroups isolated from humus soil and contaminated sites were observed in the utilization of polyalcoholic sugars, including adonitol, D-arabitol, L-arabitol, erythritol, inositol, rhamnose, sorbitol, and xylitol. Among isolates from humus soil, strain PYR100 showed high similarity in 16S rDNA sequence with M. vanbaalenii strain PYR-1 (=DSM 7251, 100%) and M. austroafricanum ATCC 33464 (99.9%). In addition to the numerical analysis, the 16S–23S intergenic spacer sequence was useful for discriminating between the closely related strains PYR100 and PYR-1 (98% similarity). The patterns of the variable V2 and V3 regions in the ribosomal RNA gene corresponding to Escherichia coli positions 179 to 197 and 1006 to 1023, respectively, were useful for dividing fast-growing and thermosensitive PAH-degrading mycobacteria into ten subgroups consistent with the phylogenetic positions.     

Syntrophomonas wolfei gen. nov. sp. nov., an Anaerobic, Syntrophic, Fatty Acid-Oxidizing Bacterium

An anaerobic, nonphototrophic bacterium that β-oxidizes saturated fatty acids (butyrate through octanoate) to acetate or acetate and propionate using protons as the electron acceptor (H₂ as electron sink product) was isolated in coculture with either a non-fatty acid-degrading, H₂-utilizing Desulfovibrio sp. or methanogens. Three strains of the bacterium were characterized and are described as a new genus and species, Syntrophomonas wolfei. S. wolfei is a gram-negative, slightly helical rod with round ends that possesses between two to eight flagella laterally inserted along the concave side of the cell. It has a multilayered cell wall of the gram-negative type. The presence of muramic acid, inhibition of growth by penicillin, and increased sensitivity of the cells to lysis after treatment with lysozyme indicate that peptidoglycan is present in the cell wall. Cells of S. wolfei contain poly-β-hydroxybutyrate. Isoheptanoate was degraded to acetate, isovalerate, and H₂. Carbohydrates, proteinaceous materials, alcohols, or other tested organic compounds do not support growth. Common electron acceptors are not utilized with butyrate as the electron donor. Growth and degradation of fatty acids occur only in syntrophic association with H₂-using bacteria. The most rapid generation time obtained by cocultures of S. wolfei with Desulfovibrio and Methanospirillum hungatei is 54 and 84 h, respectively. The addition of Casamino Acids but neither Trypticase nor yeast extract stimulated growth and resulted in a slight decrease in the generation time of S. wolfei cocultured with M. hungatei. The addition of H₂ to the medium stopped growth and butyrate degradation by S. wolfei.     

Polycyclic Aromatic Hydrocarbon (PAH) Degradation Coupled to Methanogenesis

Baltimore Harbor (Baltimore, MD) sediments were utilized to initiate anaerobic enrichment cultures with polycyclic aromatic hydrocarbons (PAHs) in the absence of supplementary electron acceptors. Cultures amended with naphthalene and phenanthrene exhibited sustained, transferable degradation of the PAHs. Bromoethanesulfonic acid, a selective inhibitor of methanogenesis, inhibited the degradation of 200 μm naphthalene and phenanthrene; molecular characterization based on 16S rRNA sequences confirmed that methanogenic Archaea were eliminated, thus providing evidence that methanogenesis is involved in the degradation pathway. Revisions requested 16 November 2005; Revisions received 14 December 2005     

Description of four novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus budapestensis sp. nov., Xenorhabdus ehlersii sp. nov., Xenorhabdus innexi sp. nov., and Xenorhabdus szentirmaii sp. nov

The taxonomic affiliation was determined for four Xenorhabdus strains isolated from four Steinernema hosts from different countries. As compared to the five validly described Xenorhabdus species, i.e., X. nematophila, X. japonica, X. beddingii, X. bovienii and X. poinarii, these isolates represented novel species on the basis of 16S rRNA gene sequences and riboprint patterns, as well as by physiological and metabolic properties. They were named Xenorhabdus budapestensis sp. nov., type strain DSM 16342^T, isolated from Steinernema bicornutum; Xenorhabdus ehlersii sp. nov., type strain DSM 16337^T, isolated from Steinernema serratum; Xenorhabdus innexi sp. nov., type strain DSM 16336^T isolated from Steinernema scapterisci; and Xenorhabdus szentirmaii sp. nov., type strain DSM 16338^T, isolated from Steinernema rarum.     

Description of Massilia rubra sp. nov., Massilia aquatica sp. nov., Massilia mucilaginosa sp. nov., Massilia frigida sp. nov., and one Massilia genomospecies isolated from Antarctic streams,lakes and regoliths

Bacteria of the genus Massilia often colonize extreme ecosystems, however, a detailed study of the massilias from the Antarctic environment has not yet been performed. Here, sixty-four Gram-stain-negative, aerobic, motile rods isolated from different environmental samples on James Ross Island (Antarctica) were subjected to a polyphasic taxonomic study. The psychrophilic isolates exhibited slowly growing, moderately slimy colonies revealing bold pink-red pigmentation on R2A agar. The set of strains exhibited the highest 16S rRNA gene sequence similarities (99.5–99.9%) to Massilia violaceinigra B2^T and Massilia atriviolacea SOD^T and formed several phylogenetic groups based on the analysis of gyrB and lepA genes. Phenotypic characteristics allowed four of them to be distinguished from each other and from their closest relatives. Compared to the nearest phylogenetic neighbours the set of six genome-sequenced representatives exhibited considerable phylogenetic distance at the whole-genome level. Bioinformatic analysis of the genomic sequences revealed a high number of putative genes involved in oxidative stress response, heavy-metal resistance, bacteriocin production, the presence of putative genes involved in nitrogen metabolism and auxin biosynthesis. The identification of putative genes encoding aromatic dioxygenases suggests the biotechnology potential of the strains. Based on these results four novel species and one genomospecies of the genus Massilia are described and named Massilia rubra sp. nov. (P3094^T = CCM 8692^T = LMG 31213^T), Massilia aquatica sp. nov. (P3165^T = CCM 8693^T = LMG 31211^T), Massilia mucilaginosa sp. nov. (P5902^T = CCM 8733^T = LMG 31210^T), and Massilia frigida sp. nov. (P5534^T = CCM 8695^T = LMG 31212^T).     

Cloning, Expression, and Characterization of the katG Gene, Encoding Catalase-Peroxidase, from the Polycyclic Aromatic Hydrocarbon-Degrading Bacterium Mycobacterium sp. Strain PYR-1

A 81-kDa protein from Mycobacterium sp. strain PYR-1 was expressed in response to exposure of the strain to the polycyclic aromatic hydrocarbon pyrene and recovered by two-dimensional gel electrophoresis. The N-terminal sequence of the protein indicated that it was similar to catalase-peroxidase. An oligonucleotide probe designed from this sequence was used to screen a genomic library of Mycobacterium sp. strain PYR-1, and a positive clone, containing a part of the gene encoding the 81-kDa protein, was isolated. A gene-walking technique was used to sequence the entire gene, which was identified as katG for catalase-peroxidase. The deduced KatG protein sequence showed significant homology to KatGII of Mycobacterium fortuitum and clustered with catalase-peroxidase proteins from other Mycobacterium species in a phylogenetic tree. The katG gene was expressed in Escherichia coli to produce a protein with catalase-peroxidase activity. Since the induction of this catalase-peroxidase occurred in pyrene-induced cultures and the exposure of these cultures to hydrogen peroxide reduced pyrene metabolism, our data suggest that this enzyme plays a role in polycyclic aromatic hydrocarbon metabolism by strain PYR-1.     

Actinobacillus rossii sp. nov., Actinobacillus seminis sp. nov., nom. rev., Pasteurella bettii sp. nov., Pasteurella lymphangitidis sp. nov., Pasteurella mairi sp. nov., and Pasteurella trehalosi sp. nov

Evidence from numerical taxonomic analysis and DNA-DNA hybridization supports the proposal of new species in the genera Actinobacillus and Pasteurella. The following new species are proposed: Actinobacillus rossii sp. nov., from the vaginas of postparturient sows; Actinobacillus seminis sp. nov., nom. rev., associated with epididymitis of sheep; Pasteurella bettii sp. nov., associated with human Bartholin gland abscess and finger infections; Pasteurella lymphangitidis sp. nov. (the BLG group), which causes bovine lymphangitis; Pasteurella mairi sp. nov., which causes abortion in sows; and Pasteurella trehalosi sp. nov., formerly biovar T of Pasteurella haemolytica, which causes septicemia in older lambs.     

Description of five novel thermophilic species of the genus Thermus: Thermus hydrothermalis sp. nov., Thermus neutrinimicus sp. nov., Thermus thalpophilus sp. nov., Thermus albus sp. nov., and Thermus altitudinis sp. nov., isolated from hot spring sediments

Biological denitrification is a significant process in nitrogen biogeochemical cycle of terrestrial geothermal environments, and Thermus species have been shown to be crucial heterotrophic denitrifier in hydrothermal system. Five Gram-stain negative, aerobic and rod-shaped thermophilic bacterial strains were isolated from hot spring sediments in Tibet, China. Phylogenetic analysis based on 16S rRNA gene and whole genome sequences indicated that these isolates should be assigned to the genus Thermus and were most closely related to Thermus caldifontis YIM 73026^T, and Thermus brockianus YS38^T. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the five strains and the type strains of the genus Thermus were lower than the threshold values (95% and 70%, respectively) recommended for bacterial species, which clearly distinguished the five isolates from other species of the genus Thermus and indicated that they represent independent species. Colonies are circular, convex, non-transparent. Cell growth occurred at 37–80 °C (optimum, 60–65 °C), pH 6.0–8.0 (optimum, pH 7.0) and with 0–2.0% (w/v) NaCl (optimum, 0–0.5%). Denitrification genes (narG, nirK, nirS, and norB genes) detected in their genomes indicated their potential function in nitrogen metabolism. The obtained results combined with those of morphological, physiological, and chemotaxonomic characteristics, including the menaquinones, polar lipids, and cellular fatty acids showed that the isolates are proposed as representing five novel species of the genus Thermus, which are proposed as Thermus hydrothermalis sp. nov. SYSU G00291^T, Thermus neutrinimicus sp. nov. SYSU G00388^T, Thermus thalpophilus sp. nov. SYSU G00506^T, Thermus albus sp. nov. SYSU G00608^T, Thermus altitudinis sp. nov. SYSU G00630^T.     

Methanosarcina acetivorans sp. nov., an Acetotrophic Methane-Producing Bacterium Isolated from Marine Sediments

A new acetotrophic marine methane-producing bacterium that was isolated from the methane-evolving sediments of a marine canyon is described. Exponential phase cultures grown with sodium acetate contained irregularly shaped cocci that aggregated in the early stationary phase and finally differentiated into communal cysts that released individual cocci when ruptured or transferred to fresh medium. The irregularly shaped cocci (1.9 ± 0.2 mm in diameter) were gram negative and occurred singly or in pairs. Cells were nonmotile, but possessed a single fimbria-like structure. Micrographs of thin sections showed a monolayered cell wall approximately 10 nm thick that consisted of protein subunits. The cells in aggregates were separated by visible septation. The communal cysts contained several single cocci encased in a common envelope. An amorphous form of the communal cyst that had incomplete septation and internal membrane-like vesicles was also present in late exponential phase cultures. Sodium acetate, methanol, methylamine, dimethylamine, and trimethylamine were substrates for growth and methanogenesis; H₂-CO₂ (80:20) and sodium formate were not. The optimal growth temperature was 35 to 40°C. The optimal pH range was 6.5 to 7.0. Both NaCl and Mg²⁺ were required for growth, with maximum growth rates at 0.2 M NaCl and 0.05 M MgSO₄. The DNA base composition was 41 ± 1% guanine plus cytosine. Methanosarcina acetivorans is the proposed species. C2A is the type strain (DSM 2834, ATCC 35395).