Nocardioides limicola sp. nov., an alkaliphilic alkane degrading bacterium isolated from oilfield alkali-saline soil.

A Gram-stain-positive, rod-shaped, non-spore-forming, alkane degrading bacterium, designated DJM-14T, was isolated from oilfield alkali-saline soil in Heilongjiang, Northeast China. On the basis of 16 S rRNA gene sequencing, strain DJM-14T was shown to belong to the genus Nocardioides, and related most closely to Nocardioides terrigena KCTC 19,217T (95.53% 16 S rRNA gene sequence similarity). Strain DJM-14T was observed to grow at 25-35 °C, pH 7.0-11.0, in the presence of 0-6.0% (w/v) NaCl. The predominant respiratory quinone was MK-8 (H4) and LL-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan. The major fatty acids were identified as iso-C16:0 and C18:1 ω9c. It contained diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the polar lipids. The genome (3,722,608 bp), composed of 24 contigs, had a G + C content of 69.6 mol%. Out of the 3667 predicted genes, 3618 were protein-coding genes, and 49 were ncRNAs. Digital DNA-DNA hybridization (dDDH) estimation and average nucleotide identity (ANI) of strain DJM-14T against genomes of the type strains of related species in the same family ranged between 18.7% and 20.0%; 68.8% and 73.6%, respectively. According to phenotypic, genotypic and phylogenetic data, strain DJM-14T represents a novel species in the genus Nocardioides, for which the name Nocardioides limicola sp. nov. is proposed and the type strain is DJM-14T (= CGMCC 4.7593T, =JCM 33,692T). In addition, novel strains were able to grow with n-alkane (C24-C36) as the sole carbon source. Multiple copies of alkane 1-monooxygenase (alkB) gene, as well as alcohol dehydrogenase gene and aldehyde dehydrogenase gene involved in the alkane assimilation were annotated in the genome of type strain DJM-14T.

Description and genomic characterization of Nocardioides bruguierae sp. nov., isolated from Bruguiera gymnorhiza.

Strains BSK12Z-3T and BSK12Z-4, two Gram-stain-positive, aerobic, non-spore-forming strains, were isolated from Shankou Mangrove Nature Reserve, Guangxi Zhuang Autonomous Region, China. The diagnostic diamino acid in the cell-wall peptidoglycan of strain BSK12Z-3T was LL-diaminopimelic acid and MK-8(H4) was the predominant menaquinone. The polar lipids comprised diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phospholipid (PL). The major fatty acids was iso-C16:0. Phylogenetic analysis based on 16S rRNA gene sequences suggested that the two strains fell within the genus Nocardioides, appearing most closely related to Nocardioides ginkgobilobae KCTC 39594T (97.5-97.6 % sequence similarity) and Nocardioides marinus DSM 18248T (97.4-97.6 %). Genome-based phylogenetic analysis confirmed that strains BSK12Z-3T and BSK12Z-4 formed a distinct phylogenetic cluster within the genus Nocardioides. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of strains BSK12Z-3T, BSK12Z-4 with their most related species N. marinus DSM18248T were within the ranges of 77.2-77.3 % and 21.3-21.4 %, respectively, clearly indicated that strains BSK12Z-3T, BSK12Z-4 represented novel species. Strains BSK12Z-3T and BSK12Z-4 exhibited 99.9 % 16S rRNA gene sequence similarity. The ANI and dDDH values between the two strains were 97.8 % and 81.1 %, respectively, suggesting that they belong to the same species. However, DNA fingerprinting discriminated that they were not from one clonal origin. Based on phylogenomic and phylogenetic analyses coupled with phenotypic and chemotaxonomic characterizatons, strains BSK12Z-3T and BSK12Z-4 could be classified as a novel species of the genus Nocardioides, for which the name Nocardioides bruguierae sp. nov., is proposed. The type strain is BSK12Z-3T (=CGMCC 4.7709T = JCM 34554T).

Description of Nocardioides jiangsuensis sp. nov., and Proposal for Reclassification of the Genus Marmoricola as Nocardioides.

A Gram-staining-positive, non-motile, aerobic, spherical actinobacterium, designated WL0053T, was isolated from the coastal sediment of Nantong City, Jiangsu Province, China. The 16S rRNA gene sequence of strain WL0053T exhibited the highest similarities to Nocardioides mesophilus MSL-22T (98.0%), N. massiliensis GD12T (97.8%), Marmoricola bigeumensis MSL-05T (97.6%), and N. jensenii DSM 20641T (97.3%). The polyphasic taxonomic approach was used for the identification of strain WL0053T. This strain formed white, round, and smooth colonies and grew in the presence of 0-18% (w/v) NaCl (optimum, 0-4.0%), at pH 6.0-9.0 (optimum, pH 7.0) and at 20-37 °C (optimum, 28 °C). The main cellular fatty acids comprised of C17:1 ω8c, C18:1 ω9c, and iso-C16:0. The genomic DNA G + C content was 71.9%. The predominant quinone was MK-8(H4), and the major polar lipid consisted of phosphatidylcholine, glycolipid, phosphatidylethanolamine, and two unidentified phospholipids. Phylogenetic trees of 16S rRNA gene and bac120 gene set indicted that strain WL0053T was closely related to the species N. iriomotensis and N. mesophilus, while these two species clustered in a separate clade together with M. caldifontis YIM 730233T in the bac120 tree. Combined with the analysis of average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH), it can be considered that the strain WL0053T is a new member of the genus Nocardioides and is proposed to be named as Nocardioides jiangsuensis sp. nov.. The type strain is WL0053T (=MCCC 1K05897T=JCM 34671T=GDMCC 4.192T). Furthermore, based on the fact that the genera Nocardioides and Marmoricola both appeared polyphyletic with no significant difference on phenotypic and chemotaxonomic traits, we proposed to reclassify the genus Marmoricola as Nocardioides.

Cotinine Hydroxylase CotA Initiates Biodegradation of Wastewater Micropollutant Cotinine in Nocardioides sp. Strain JQ2195.

Cotinine is a stable toxic contaminant, produced as a by-product of smoking. It is of emerging concern due to its global distribution in aquatic environments. Microorganisms have the potential to degrade cotinine; however, the genetic mechanisms of this process are unknown. Nocardioides sp. strain JQ2195 is a pure-culture strain that has been reported to degrade cotinine at micropollutant concentrations. This strain utilizes cotinine as its sole carbon and nitrogen source. In this study, a 50-kb gene cluster (designated cot), involved in cotinine degradation, was predicted based on genomic and transcriptomic analyses. A novel three-component cotinine hydroxylase gene (designated cotA1A2A3), which initiated cotinine catabolism, was identified and characterized. CotA from Shinella sp. strain HZN7 was heterologously expressed and purified and was shown to convert cotinine into 6-hydroxycotinine. H218O-labeling and electrospray ionization-mass spectrometry (ESI-MS) analysis confirmed that the hydroxyl group incorporated into 6-hydroxycotinine was derived from water. This study provides new molecular insights into the microbial metabolism of heterocyclic chemical pollutants. IMPORTANCE In the human body, cotinine is the major metabolite of nicotine, and 10 to 15% of generated cotinine is excreted in urine. Cotinine is a structural analogue of nicotine and is much more stable than nicotine. Increased tobacco consumption has led to high environmental concentrations of cotinine, which may have detrimental effects on aquatic ecosystems and human health. Nocardioides sp. strain JQ2195 is a unique cotinine-degrading bacterium. However, the underlying genetic and biochemical foundations of cotinine degradation are still unknown. In this study, a 50-kb gene cluster (designated cot) was identified by genomic and transcriptomic analyses as being involved in the degradation of cotinine. A novel three-component cotinine hydroxylase gene (designated cotA1A2A3) catalyzed cotinine to 6-hydroxy-cotinine. This study provides new molecular insights into the microbial degradation and enzymatic transformation of cotinine.

Description of Nocardioides piscis sp. nov., Sphingomonas piscis sp. nov. and Sphingomonas sinipercae sp. nov., isolated from the intestine of fish species Odontobutis interrupta (Korean spotted sleeper) and Siniperca scherzeri (leopard mandarin fish).

A polyphasic taxonomic approach was used to characterize three novel bacterial strains, designated as HDW12AT, HDW-15BT, and HDW15CT, isolated from the intestine of fish species Odontobutis interrupta or Siniperca scherzeri. All isolates were obligate aerobic, non-motile bacteria, and grew optimally at 30°C. Phylogenetic analysis based on 16S rRNA sequences revealed that strain HDW12AT was a member of the genus Nocardioides, and closely related to Nocardioides allogilvus CFH 30205T (98.9% sequence identities). Furthermore, strains HDW15BT and HDW15CT were members of the genus Sphingomonas, and closely related to Sphingomonas lutea JS5T and Sphingomonas sediminicola Dae 20T (97.1% and 97.9% sequence identities), respectively. Strain HDW12AT contained MK-8 (H4), and strains HDW15BT and HDW15CT contained Q-10 as the respiratory quinone. Major polar lipid components of strain HDW12AT were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylinositol, and those of strains HDW15BT and HDW15CT were sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. The G + C content of strains HDW12AT, HDW15BT, and HDW15CT were 69.7, 63.3, and 65.5%, respectively. The results of phylogenetic, phenotypic, chemotaxonomic, and genotypic analyses suggest that strain HDW12AT represents a novel species within the genus Nocardioides, and strains HDW15BT and HDW15CT represent two novel species within the genus Sphingomonas. We propose the names Nocardioides piscis for strain HDW12AT (= KACC 21336T = KCTC 49321T = JCM 33670T), Sphingomonas piscis for strain HDW15BT (= KACC 21341T = KCTC 72588T = JCM 33738T), and Sphingomonas sinipercae for strain HDW15CT (= KACC 21342T = KCTC 72589T = JCM 33739T).

Different genome-wide transcriptome responses of Nocardioides simplex VKM Ac-2033D to phytosterol and cortisone 21-acetate.

BACKGROUND: Bacterial degradation/transformation of steroids is widely investigated to create biotechnologically relevant strains for industrial application. The strain of Nocardioides simplex VKM Ac-2033D is well known mainly for its superior 3-ketosteroid Δ1-dehydrogenase activity towards various 3-oxosteroids and other important reactions of sterol degradation. However, its biocatalytic capacities and the molecular fundamentals of its activity towards natural sterols and synthetic steroids were not fully understood. In this study, a comparative investigation of the genome-wide transcriptome profiling of the N. simplex VKM Ac-2033D grown on phytosterol, or in the presence of cortisone 21-acetate was performed with RNA-seq. RESULTS: Although the gene patterns induced by phytosterol generally resemble the gene sets involved in phytosterol degradation pathways in mycolic acid rich actinobacteria such as Mycolicibacterium, Mycobacterium and Rhodococcus species, the differences in gene organization and previously unreported genes with high expression level were revealed. Transcription of the genes related to KstR- and KstR2-regulons was mainly enhanced in response to phytosterol, and the role in steroid catabolism is predicted for some dozens of the genes in N. simplex. New transcription factors binding motifs and new candidate transcription regulators of steroid catabolism were predicted in N. simplex. Unlike phytosterol, cortisone 21-acetate does not provide induction of the genes with predicted KstR and KstR2 sites. Superior 3-ketosteroid-Δ1-dehydrogenase activity of N. simplex VKM Ac-2033D is due to the kstDs redundancy in the genome, with the highest expression level of the gene KR76_27125 orthologous to kstD2, in response to cortisone 21-acetate. The substrate spectrum of N. simplex 3-ketosteroid-Δ1-dehydrogenase was expanded in this study with progesterone and its 17α-hydroxylated and 11α,17α-dihydroxylated derivatives, that effectively were 1(2)-dehydrogenated in vivo by the whole cells of the N. simplex VKM Ac-2033D. CONCLUSION: The results contribute to the knowledge of biocatalytic features and diversity of steroid modification capabilities of actinobacteria, defining targets for further bioengineering manipulations with the purpose of expansion of their biotechnological applications.