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.

A Novel Trehalose Synthase for the Production of Trehalose and Trehalulose.

A novel putative trehalose synthase gene (treM) was identified from an extreme temperature thermal spring. The gene was expressed in Escherichia coli followed by purification of the protein (TreM). TreM exhibited the pH optima of 7.0 for trehalose and trehalulose production, although it was functional and stable in the pH range of 5.0 to 8.0. Temperature activity profiling revealed that TreM can catalyze trehalose biosynthesis in a wide range of temperatures, from 5°C to 80°C. The optimum activity for trehalose and trehalulose biosynthesis was observed at 45°C and 50°C, respectively. A catalytic reaction performed at the low temperature of 5°C yielded trehalose with significantly reduced by-product (glucose) production in the reaction. TreM displayed remarkable thermal stability at optimum temperatures, with only about 20% loss in the activity after heat (50°C) exposure for 24 h. The maximum bioconversion yield of 74% trehalose (at 5°C) and 90% trehalulose (at 50°C) was obtained from 100 mM maltose and 70 mM sucrose, respectively. TreM was demonstrated to catalyze trehalulose biosynthesis utilizing the low-cost feedstock jaggery, cane molasses, muscovado, and table sugar. IMPORTANCE Trehalose is a rare sugar of high importance in biological research, with its property to stabilize cell membrane and proteins and protect the organism from drought. It is instrumental in the cryopreservation of human cells, e.g., sperm and blood stem cells. It is also very useful in the food industry, especially in the preparation of frozen food products. Trehalose synthase is a glycosyl hydrolase 13 (GH13) family enzyme that has been reported from about 22 bacterial species so far. Of these enzymes, to date, only two have been demonstrated to catalyze the biosynthesis of both trehalose and trehalulose. We have investigated the metagenomic data of an extreme temperature thermal spring to discover a novel gene that encodes a trehalose synthase (TreM) with higher stability and dual transglycosylation activities of trehalose and trehalulose biosynthesis. This enzyme is capable of catalyzing the transformation of maltose to trehalose and sucrose to trehalulose in a wide pH and temperature range. The present investigation endorses the thermal aquatic habitat as a promising genetic resource for the biocatalysts with high potential in producing high-value rare sugars.

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).

Detection of an alkene monooxygenase in vinyl chloride-oxidizing bacteria with GeneFISH.

Fluorescence in situ hybridization (FISH) can provide information on the morphology, spatial arrangement, and local environment of individual cells enabling the investigation of intact microbial communities. GeneFISH uses polynucleotide probes and enzymatic signal amplification to detect genes that are present in low copy numbers. Previously, this technique has only been applied in a small number of closely related organisms. However, many important functional genes, such as those involved in xenobiotic degradation or pathogenesis, are present in diverse microbial strains. Here, we present a geneFISH method for the detection of the functional gene etnC, which encodes the alpha subunit of an alkene monooxygenase used by aerobic ethene and vinyl chloride oxidizing bacteria (etheneotrophs). The probe concentration was optimized and found to be 100 pg/µl, similar to previous geneFISH reports. Permeabilization was necessary for successful geneFISH labeling of Mycobacteria; sequential treatment with lysozyme and achromopeptidase was the most effective treatment. This method was able to detect etnC in several organisms including Mycobacteria and Nocardioides, demonstrating for the first time that a single geneFISH probe can detect a variety of alleles (>80% sequence similarity) across multiple species. Detection of etnC with geneFISH has practical applications for bioremediation. This method can be readily adapted for other functional genes and has broad applications for investigating microbial communities in natural and engineered systems.

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.

Symbiotic riboflavin degradation by Microbacterium and Nocardioides bacteria.

Unlike its biosynthetic mechanisms and physiological function, current understanding of riboflavin degradation in soil is limited to a few bacteria that decompose it to lumichrome. Here, we isolated six Microbacterium and three Nocardioides strains. These strains utilized riboflavin and lumichrome, respectively, as carbon sources. Among these strains, we identified Microbacterium paraoxydans R16 (R16) and Nocardioides nitrophenolicus L16 (L16), which were isolated form the same enrichment culture. Co-cultured R16 and L16 reconstituted a riboflavin-degrading interspecies consortium, in which the R16 strain degraded riboflavin to lumichrome and ᴅ-ribose. The L16 strain utilized the lumichrome as a carbon source, indicating that R16 is required for L16 to grow in the consortium. Notably, rates of riboflavin degradation and growth were increased in co-cultured, compared with monocultured R16 cells. These results indicated that a beneficial symbiotic interaction between M. paraoxydans R16 and N. nitrophenolicus L16 results in the ability to degrade riboflavin.