Antrihabitans stalagmiti sp. nov., isolated from a larva cave and a proposal to transfer Rhodococcus cavernicola Lee et al. 2020 to a new genus Spelaeibacter as Spelaeibacter cavernicola gen. nov. comb. nov.

An actinobacterial strain, designated YC3-6T, was isolated from a larva cave in Jeju, Republic of Korea. The novel isolate was found to grow at 10-30 °C, pH 5.0-10.0 and 0-4% (w/v) NaCl. The 16S rRNA gene phylogeny showed that the novel isolate formed a distinct subline within the family Nocardiaceae. Levels of 16S rRNA gene similarity indicated that the close relatives are Rhodococcus cavernicola (98.4% sequence similarity) and "Rhodococcus psychrotolerans" (98.2%) followed by Antrihabitans stalactiti (96.8%). However, the core gene-based phylogeny revealed that the novel isolate formed a tight cluster with A. stalactiti and was separated from R. cavernicola and other members of the family Nocardiaceae. The morphological and chemotaxonomic characteristics of strain YC3-6T are in line with those of the genus Antrihabitans. Strain YC3-6T showed an average nucleotide identity of 75.5% and a digital DDH of 20.3% with A. stalactiti. In addition, the core gene analysis showed that R. cavernicola formed a distinct subline between an Antrihabitans cluster and Aldersonia kunmingensis, and well separated from members of the genus Rhodococcus. The average amino acid identity values of R. cavernicola to closely related neighbours were 69.3-69.4% with members of the genus Antrihabitans and 67.3% with Ald. kunmingensis, while the POCP values ranged from 56.9 to 63.6%. On the basis of results obtained here, strain YC3-6T is concluded to represent a novel species of the genus Antrihabitans, for which the name Antrihabitans stalagmiti sp. nov. (type strain, YC3-6T = KACC 19963T = DSM 107561T) is proposed. Based on overall genome relatedness and chemotaxonomic differences, it is also proposed that R. cavernicola Lee et al. 2020 be transferred to a new genus Spelaeibacter as Spelaeibacter cavernicola gen. nov., comb. nov.

Correlation of the lung microbiota with metabolic profiles in bronchoalveolar lavage fluid in HIV infection.

BACKGROUND: While 16S ribosomal RNA (rRNA) sequencing has been used to characterize the lung's bacterial microbiota in human immunodeficiency virus (HIV)-infected individuals, taxonomic studies provide limited information on bacterial function and impact on the host. Metabolic profiles can provide functional information on host-microbe interactions in the lungs. We investigated the relationship between the respiratory microbiota and metabolic profiles in the bronchoalveolar lavage fluid of HIV-infected and HIV-uninfected outpatients. RESULTS: Targeted sequencing of the 16S rRNA gene was used to analyze the bacterial community structure and liquid chromatography-high-resolution mass spectrometry was used to detect features in bronchoalveolar lavage fluid. Global integration of all metabolic features with microbial species was done using sparse partial least squares regression. Thirty-nine HIV-infected subjects and 20 HIV-uninfected controls without acute respiratory symptoms were enrolled. Twelve mass-to-charge ratio (m/z) features from C18 analysis were significantly different between HIV-infected individuals and controls (false discovery rate (FDR) = 0.2); another 79 features were identified by network analysis. Further metabolite analysis demonstrated that four features were significantly overrepresented in the bronchoalveolar lavage (BAL) fluid of HIV-infected individuals compared to HIV-uninfected, including cystine, two complex carbohydrates, and 3,5-dibromo-L-tyrosine. There were 231 m/z features significantly associated with peripheral blood CD4 cell counts identified using sparse partial least squares regression (sPLS) at a variable importance on projection (VIP) threshold of 2. Twenty-five percent of these 91 m/z features were associated with various microbial species. Bacteria from families Caulobacteraceae, Staphylococcaceae, Nocardioidaceae, and genus Streptococcus were associated with the greatest number of features. Glycerophospholipid and lineolate pathways correlated with these bacteria. CONCLUSIONS: In bronchoalveolar lavage fluid, specific metabolic profiles correlated with bacterial organisms known to play a role in the pathogenesis of pneumonia in HIV-infected individuals. These findings suggest that microbial communities and their interactions with the host may have functional metabolic impact in the lung.

[Recent advance on the genus nocardioides--a review].

The Nocardioides genus was established by Prauser H. in 1976 according to morphological and physiological characteristics, as well as partial chemotaxonomic analyses of 17 nocardio-form actinobacteria isolated from soil, based on which a novel species Nocardioides albus was proposed as the type species. With the development in the technologies of isolation, purification and taxonomy, more and more members of this genus with varied morphological, physiological and biochemical characteristics were increasingly discovered from different sources, while all of them shared the same diagnostic characteristics of the genus. In the past 50 years, some of the members of the genus Nocardioides were ever transferred in or out and then some species description was ever emended. Till date, there were 56 validly described species in this genus. Some members of this genus were used in agriculture and industry. The objective of this review is to summarize the research advances in the genus Nocardioides, such as the changes of the taxonomic position and emendation description of the species as well as the application prospect in industry and agriculture.

Production of a new glycolipid biosurfactant from marine Nocardiopsis lucentensis MSA04 in solid-state cultivation.

Considering the need of potential biosurfactant producers and economic production processes using industrial waste, the present study aims to develop solid-state culture (SSC) of a marine actinobacterium for biosurfactant production. A potential biosurfactant producer Nocardiopsis lucentensis MSA04 was isolated from the marine sponge Dendrilla nigra. Among the substrates screened, wheat bran increased the production significantly (E(24) 25%) followed by oil seed cake and industrial waste such as tannery pretreated sludge, treated molasses (distillery waste) and pretreated molasses. Enhanced biosurfactant production was achieved under SSC conditions using kerosene as carbon source, beef extract as nitrogen source and wheat bran as substrate. The maximum production of biosurfactant by MSA04 occurred at a C/N ratio of 0.5 envisaging that a higher amount of nitrogen source is required by the strain compared to that of the carbon source. The kerosene and beef extract interactively increase the production and a stable production was attained with the influence of both factors independently. A significant interactive influence of secondary control factors such as copper sulfate and inoculum size was validated in response surface methods-based experiments. The surface active compound produced by MSA04 was characterized as glycolipid with a hydrophobic non-polar hydrocarbon chain (nonanoic acid methyl ester) and hydrophilic sugar, 3-acetyl 2,5 dimethyl furan. In conclusion, the strain N. lucentensis MSA04 was a potential source of glycolipid biosurfactant, could be used for the development of bioremediation processes in the marine environment.

Mechanism controlling the extended lag period associated with vinyl chloride starvation in Nocardioides sp. strain JS614.

The extended lag period associated with vinyl chloride (VC) starvation in VC- and ethene-assimilating Nocardioides sp. strain JS614 was examined. The extended lag periods were variable (3-7 days), only associated with growth on VC or ethene, and were observed in VC- or ethene-grown cultures following 24 h carbon starvation and mid-exponential phase cultures grown on non-alkene carbon sources (e.g. acetate). Alkene monooxygenase (AkMO) and epoxyalkane:coenzyme M transferase (EaCoMT) are the initial enzymes of VC and ethene biodegradation in strain JS614. Reverse-transcription PCR confirmed that the AkMO gene etnC was expressed in response to epoxyethane, a metabolic intermediate of ethene biodegradation. Epoxyethane (0.5 mM) eliminated the extended lag period in both starved and mid-exponential phase cultures, suggesting that epoxyethane accumulation activates AkMO expression in strain JS614. AkMO activity in ethene-grown cultures was not detected after 6.7 h of carbon starvation, while 40% of the initial EaCoMT activity remained after 24 h. Acetate eliminated the extended lag period in starved cultures but not in mid-exponential phase cultures suggesting that acetate reactivates extant AkMO in starved VC- or ethene-grown cultures. The imbalance between AkMO and EaCoMT activities during starvation likely contributes to the extended lag period by delaying epoxide accumulation and subsequent AkMO induction.

Physiological and molecular genetic analyses of vinyl chloride and ethene biodegradation in Nocardioides sp. strain JS614.

Nocardioides sp. strain JS614 utilizes vinyl chloride and ethene as carbon and energy sources. JS614 could be influential in natural attenuation and biogeochemical ethene cycling, and useful for bioremediation, biocatalysis and metabolic engineering, but a fundamental understanding of the physiological and genetic basis of vinyl chloride and ethene assimilation in strain JS614 is required. Alkene monooxygenase (AkMO) activity was demonstrated in whole-cell assays and epoxyalkane:coenzyme M transferase (EaCoMT) activity was detected in JS614 cell-free extracts. Pulsed-field gel electrophoresis revealed a 290-kb plasmid (pNoc614) in JS614. Curing experiments and PCR indicated that pNoc614 encodes vinyl chloride/ethene-degradation genes. JS614 vinyl chloride/ethene catabolic genes and flanking DNA (34.8 kb) were retrieved from a fosmid clone. AkMO and EaCoMT genes were found in a putative operon that included CoA transferase, acyl-CoA synthetase, dehydrogenase, and reductase genes. Adjacent to this gene cluster was a divergently transcribed gene cluster that encoded possible coenzyme M biosynthesis enzymes. Reverse transcription-PCR demonstrated the vinyl chloride- and ethene-inducible nature of several genes. Genes encoding possible plasmid conjugation, integration, and partitioning functions were also discovered on the fosmid clone.

Nocardiopsis aegyptia sp. nov., isolated from marine sediment.

An actinomycete, strain SNG49(T), was isolated from marine sediment of Abu Qir Bay, on the western seashore of Alexandria, Egypt. The bacterium was aerobic and Gram-positive. It produced beige to light-yellow aerial mycelium, brown substrate mycelium and straight to flexuous hyphae, but no specific spore chains. 16S rDNA sequence analysis and chemotaxonomic markers were consistent with classification of strain SNG49(T) in the genus Nocardiopsis, i.e. meso-diaminopimelic acid; no diagnostic sugars; phosphatidylcholine, phosphatidylmethylethanolamine, phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol as polar lipids; menaquinones of the MK-10 series from MK-10(H(0)) to MK-10(H(8)); and iso/anteiso-branched and 10-methyl-branched fatty acids, the principal fatty acids being anteiso-17 : 0 and tuberculostearic acid. Nocardiopsis lucentensis and Nocardiopsis alba are the phylogenetic neighbours of strain SNG49(T), respectively showing 98.8 and 98.7 % 16S rRNA gene sequence similarity; however, moderate DNA-DNA reassociation values between these two species and strain SNG49(T) (44 and 60 %, respectively) showed that strain SNG49(T) could be clearly separated from them. These data, together with distinct physiological traits, led to the conclusion that this isolate represents a novel species within the genus Nocardiopsis, for which the name Nocardiopsis aegyptia is proposed. The type strain is SNG49(T) (=DSM 44442(T)=NRRL B-24244(T)).

Nocardioides aquiterrae sp. nov., isolated from groundwater in Korea.

A bacterial strain, GW-9T, which was isolated from groundwater in Korea, was subjected to a polyphasic taxonomic study using phenotypic characterization and phylogenetic and genetic methods. Phylogenetic analysis based on 16S rDNA sequences showed that strain GW-9T forms an evolutionary lineage within the radiation enclosing Nocardioides species and, in particular, a coherent cluster with Nocardioides pyridinolyticus. The cell-wall peptidoglycan type of strain GW-9T was based on LL-diaminopimelic acid as the diamino acid, indicating wall chemotype I. The predominant menaquinone was MK-8(H4). Strain GW-9T had a cellular fatty acid profile containing straight-chain, branched, unsaturated and 10-methyl fatty acids. The major fatty acid was iso-C(16:0). The DNA G+C content of strain GW-9T was 73 mol%. The 16S rDNA of strain GW-9T was 99.2% similar to that of the type strain of Nocardioides pyridinolyticus and 94.9-96.0% similar to sequences of the type strains of other Nocardioides species. Differences in phenotypic characteristics and genetic distinctiveness indicate that strain GW-9T is separate from previously described Nocardioides species. Therefore, on the basis of the data presented, a novel species of the genus Nocardioides, Nocardioides aquiterrae sp. nov., is proposed. The type strain is strain GW-9T (=KCCM 41647T=JCM 11813T).

21-Acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione conversion by Nocardioides simplex VKM Ac-2033D.

The conversion of 21-acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (I) by Nocardioides simplex VKM Ac-2033D was studied purposed selective production of its 1(2)-dehydroanalogues-value precursors in the synthesis of modern glucocorticoids starting from 9alpha-hydroxyandrostenes. 21-Acetoxy-pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (II), pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (III) and pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (IV) were revealed as metabolites, and the structures were confirmed by mass spectrometry and (1)H nuclear magnetic resonance (NMR) spectroscopy. The metabolic pathways of I by N. simplex included 1(2)-dehydrogenation and deacetylation. The sequence of the reactions was shown to depend on the transformation conditions. The presence of both soluble and membrane associated steroid esterases in N. simplex was demonstrated using cell fractionation. Unlike inducible 1(2)-dehydrogenase, steroid esterase was shown to be constitutive. The conditions providing selective accumulation of II from I by whole N. simplex cells were determined.

Aeromicrobium marinum sp. nov., an abundant pelagic bacterium isolated from the German Wadden Sea.

An obligately salt-dependent Gram-positive bacterium, designated strain T2(T), was isolated from surface waters of the German Wadden Sea. The organism exhibited optimum growth at salt concentrations similar to that of sea water. On the basis of phenotypic, chemotaxonomic and phylogenetic differences, it is concluded that strain T2(T) (=DSM 15272(T)=LMG 21768(T)) is the first marine species of the genus Aeromicrobium to be identified, for which the name Aeromicrobium marinum is proposed. It is also the first described marine bacterium within the family Nocardioidaceae. Strain T2(T) is a rod-shaped, aerobic, heterotrophic bacterium containing LL-diaminopimelic acid in the peptidoglycan and MK-9(H(4)) as the major menaquinone. The bacterium is characterized by high proportions of the fatty acids palmitic acid, oleic acid, tuberculostearic acid and hydroxypalmitic acid. DNA-DNA hybridization analysis showed the marine bacterium to display 29.1 % relatedness with Aeromicrobium fastidiosum DSM 10552(T) and 44.4 % relatedness with Aeromicrobium erythreum DSM 8599(T). A. marinum was demonstrated to be an abundant member of the pelagic bacterial community in the German Wadden Sea since it represented about 1 % of the total bacterial population as revealed by dot-blot hybridization and most-probable-number counts.

Molecular detection, isolation, and physiological characterization of functionally dominant phenol-degrading bacteria in activated sludge.

DNA was isolated from phenol-digesting activated sludge, and partial fragments of the 16S ribosomal DNA (rDNA) and the gene encoding the largest subunit of multicomponent phenol hydroxylase (LmPH) were amplified by PCR. An analysis of the amplified fragments by temperature gradient gel electrophoresis (TGGE) demonstrated that two major 16S rDNA bands (bands R2 and R3) and two major LmPH gene bands (bands P2 and P3) appeared after the activated sludge became acclimated to phenol. The nucleotide sequences of these major bands were determined. In parallel, bacteria were isolated from the activated sludge by direct plating or by plating after enrichment either in batch cultures or in a chemostat culture. The bacteria isolated were classified into 27 distinct groups by a repetitive extragenic palindromic sequence PCR analysis. The partial nucleotide sequences of 16S rDNAs and LmPH genes of members of these 27 groups were then determined. A comparison of these nucleotide sequences with the sequences of the major TGGE bands indicated that the major bacterial populations, R2 and R3, possessed major LmPH genes P2 and P3, respectively. The dominant populations could be isolated either by direct plating or by chemostat culture enrichment but not by batch culture enrichment. One of the dominant strains (R3) which contained a novel type of LmPH (P3), was closely related to Valivorax paradoxus, and the result of a kinetic analysis of its phenol-oxygenating activity suggested that this strain was the principal phenol digester in the activated sludge.

Inter- and intraspecific phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rDNA sequences.

The 16S rDNAs from 38 strains of the genus Nocardioides, two Aeromicrobium species and Terrabacter tumescens were directly sequenced and then analysed. The mean nucleotide similarity value between the type strains of validly described Nocardioides species was 96.1 +/- 3.0%. The mean nucleotide similarity value between the type strains of validly described Nocardioides species and the two Aeromicrobium species was 93.7 +/- 1.4% T. tumescens was distantly related to the genera Nocardioides and Aeromicrobium. The mean intraspecific nucleotide similarity value of 16S rDNA sequences from Nocardioides albus was 99.5 +/- 0.5%. The mean intraspecific nucleotide similarity of 16S rDNA sequences from Nocardioides simplex was 100%, except for N. simplex strains ATCC 13260, ATCC 19565 and ATCC 19566, which were shown not to be members of the genus Nocardioides. 'Nocardioides flavus' strains IFO 14396T and IFO 14397, and 'Nocardioides fulvus' JCM 3335T showed a 16S rDNA similarity value of 100% with Nocardioides luteus KCTC 9575T and Nocardioides albus JCM 5854. 'N. fulvus' IFO 14399 shared its highest 16S rDNA similarity with Nocardioides sp. ATCC 39419 at 99%. 'N. fulvus' IFO 14399 and Nocardioides sp. ATCC 39419 formed a phylogenetic lineage distinct from the genera Nocardioides and Aeromicrobium. 'Nocardioides thermolilacinus' strains IFO 14335T and IFO 14336 displayed a close relationship to the genus Streptomyces. From 16S rDNA sequence analyses, it is considered that some strains that have been attributed to the genus Nocardioides should be taxonomically re-evaluated.

Biochemical and molecular characterization of 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. KP7.

1-Hydroxy-2-naphthoate dioxygenase, which cleaves the singly hydroxylated aromatic ring, was purified from phenanthrene-degrading Nocardioides sp. strain KP7. The purified enzyme had a molecular mass of 45 kDa by SDS-polyacrylamide gel electrophoresis and 270 kDa by gel filtration chromatography. The apparent Km and kcat values of this enzyme for 1-hydroxy-2-naphthoate were 10 microM and 114 s-1, respectively. One mole of molecular oxygen was consumed when 1 mol of 1-hydroxy-2-naphthoate was oxidized. This enzyme contained 1 mol of Fe(II)/mol of the subunit and was inactivated by o-phenanthroline. The enzyme that had been inactivated by o-phenanthroline was reactivated by incubating with FeSO4 and ascorbic acid. Thus, Fe(II) was required for the enzyme to exhibit activity. The structural gene for this enzyme was screened from a cosmid library and then sequenced, the length of the 1-hydroxy-2-naphthoate gene being 1161 base pairs. The deduced amino acid sequence of this enzyme was different from those of other ring-cleaving dioxygenases that cleave the doubly hydroxylated aromatic ring.

Reclassification of Nocardioides simplex ATCC 13260, ATCC 19565, and ATCC 19566 as Rhodococcus erythropolis.

Our phylogenetic analysis based on 16S ribosomal DNA (rDNA) sequences and chemotaxonomic analyses showed that Nocardioides simplex ATCC 13260, ATCC 19565, and ATCC 19566 are more closely related to the genus Rhodococcus, especially Rhodococcus erythropolis, than to the genus Nocardioides. N. simplex ATCC 13260 and N. simplex ATCC 19565 and ATCC 19566 exhibited levels of 16S rDNA similarity of 99.4 and 100%, respectively, to R. erythropolis DSM 43066T. Strains ATCC 13260, ATCC 19565, and ATCC 19566 had mesodiaminopimelic acid in their peptidoglycan and MK-8(H2) as their predominant menaquinone. These three strains produced cellular fatty acid patterns similar to those of R. erythropolis strains rather than those of Nocardioides species. Therefore, N. simplex ATCC 13260, ATCC 19565, and ATCC 19566 should be reclassified as strains of R. erythropolis Gray and Thornton 1928.

An acyl-carrier-protein-thioesterase domain from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea. High-level production, purification and characterisation in Escherichia coli.

The C-terminal region of a multifunctional polypeptide from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea is predicted to contain an acyl carrier protein and a thioesterase or acyltransferase activity [Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. & Leadlay, P. F. (1990) Nature 348, 176-178]. Site-directed mutagenesis by means of the polymerase chain reaction was used to construct an efficient pT7-based expression plasmid for this domain. The recently developed technique of electrospray mass spectrometry was used to demonstrate that the purified protein had not been post-translationally modified by attachment of a 4'-phosphopantetheine group. However, treatment with the serine proteinase inhibitor phenylmethylsulphonyl fluoride led to highly selective labelling of the predicted active site of the thioesterase or acyltransferase.

Primary sequence of the glucanase gene from Oerskovia xanthineolytica. Expression and purification of the enzyme from Escherichia coli.

A 2.7-kilobase fragment of DNA from Oerskovia xanthineolytica containing the gene for a beta-1,3-glucanase has been isolated and its complete nucleotide sequence determined. The sequence was found to contain two large open reading frames. Purification of the mature native enzyme and subsequent amino-terminal sequencing defined the glucanase gene in one reading frame which potentially encodes a protein of 548 amino acids. We have expressed this glucanase gene in Escherichia coli under control of the lacUV5 promoter and found the product to be secreted into the periplasm as a mature enzyme of about the same molecular weight as that of the native protein. The recombinant enzyme was purified to near homogeneity by a single step of high performance liquid chromatography. The ability of the recombinant enzyme to digest beta-glucan substrates and to lyse viable yeast cells was found to be indistinguishable from that of the native protein. Deletion of the cysteine-rich carboxyl-terminal 117 amino acids of the enzyme, which also contain two duplicated segments, abolished the lytic activity but did not significantly affect the glucanase function of the protein. The possible involvement of this domain in interaction with the yeast cell wall is discussed.

Pradimicins M, N, O and P, new dihydrobenzo[a]naphthacenequinones produced by blocked mutants of Actinomadura hibisca P157-2.

Four blocked mutants which accumulated new dihydrobenzo[a]naphthacenequinone metabolites, designated pradimicins M, N, O and P, have been isolated from cultures of mutants of Actinomadura hibisca P157-2 resulting from treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The structures of the four compounds were determined by spectral analysis. Pradimicins N, O and P contain D-alanine, while pradimicin M does not. The conformations at C-5 and C-6 of these compounds are different from those of the original pradimicins.

Microbial conversion of anthracyclinones to carminomycins by a blocked mutant of Actinomadura roseoviolacea.

New anthracycline antibiotics, 1-hydroxy-11-deoxycarminomycin II and 11-deoxycarminomycin II were produced by a blocked mutant MuW1 of Actinomadura roseoviolacea from epsilon-pyrromycinone and aklavinone, respectively. We found that the enzyme catalyzing hydroxylation at the C-11 position was not lost but was down regulated in the strain MuW1.

Molecular taxonomic studies on some LL-diaminopimelic acid-containing coryneforms from herbage: description of Nocardioides fastidiosa sp. nov.

Molecular taxonomic studies were performed on two LL-diaminopimelic acid-containing coryneform isolates from herbage. The results indicate that the herbage strains represent a new species of the genus Nocardioides for which the name Nocardioides fastidiosa sp. nov. is proposed. The type strain is NCIB 12713.