Uncovering the potential of novel micromonosporae isolated from an extreme hyper-arid Atacama Desert soil.

The taxonomic status, biotechnological and ecological potential of several Micromonospora strains isolated from an extreme hyper arid Atacama Desert soil were determined. Initially, a polyphasic study was undertaken to clarify the taxonomic status of five micromonosporae, strains LB4, LB19, LB32T, LB39T and LB41, isolated from an extreme hyper-arid soil collected from one of the driest regions of the Atacama Desert. All of the isolates were found to have chemotaxonomic, cultural and morphological properties consistent with their classification in the genus Micromonospora. Isolates LB32T and LB39T were distinguished from their nearest phylogenetic neighbours and proposed as new species, namely as Micromonospora arida sp. nov. and Micromonospora inaquosa sp. nov., respectively. Eluted methanol extracts of all of the isolates showed activity against a panel of bacterial and fungal indicator strains, notably against multi-drug resistant Klebsiella pneumoniae ATCC 700603 while isolates LB4 and LB41 showed pronounced anti-tumour activity against HepG2 cells. Draft genomes generated for the isolates revealed a rich source of novel biosynthetic gene clusters, some of which were unique to individual strains thereby opening up the prospect of selecting especially gifted micromonosporae for natural product discovery. Key stress-related genes detected in the genomes of all of the isolates provided an insight into how micromonosporae adapt to the harsh environmental conditions that prevail in extreme hyper-arid Atacama Desert soils.

Micromonospora vulcania sp. nov., isolated from volcanic sediment.

A novel actinobacterial strain, designated strain NEAU-JM2(T), was isolated from volcanic sediment collected from Longwan, Jilin province, north China and characterized using a polyphasic approach. The strain was found to have morphological and chemotaxonomic characteristics typical of the members of the genus Micromonospora. Phylogenetic analysis of the16S rRNA gene sequence also indicated that strain NEAU-JM2(T) should be classified in the genus Micromonospora and showed that close relatives are Micromonospora maoerensis NEAU-MES19(T) (99.5 %) and Micromonospora matsumotoense JCM 9104(T) (98.8 %). However, phylogenetic analysis based on the gyrB gene sequence showed that the isolate forms a separate subclade away from the close relatives in the neighbour-joining tree and also recovered with the maximum-likelihood algorithm. The low level of DNA-DNA relatedness allowed the isolate to be differentiated from M. maoerensis NEAU-MES19(T) and M. matsumotoense JCM 9104(T). Furthermore, strain NEAU-JM2(T) could also be distinguished from its close phylogenetic relatives by cultural and physiological characteristics. Therefore, it is proposed that strain NEAU-JM2(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora vulcania sp. nov. is proposed. The type strain is NEAU-JM2(T) (=CGMCC 4.7144(T) = DSM 46711(T)).

Micromonospora taraxaci sp. nov., a novel endophytic actinomycete isolated from dandelion root (Taraxacum mongolicum Hand.-Mazz.).

A novel actinomycete, designated strain NEAU-P5(T), was isolated from dandelion root (Taraxacum mongolicum Hand.-Mazz.). Strain NEAU-P5(T) showed closest 16S rRNA gene sequence similarity to Micromonospora chokoriensis 2-19/6(T) (99.5%), and phylogenetically clustered with Micromonospora violae NEAU-zh8(T) (99.3%), M. saelicesensis Lupac 09(T) (99.0%), M. lupini Lupac 14N(T) (98.8%), M. zeae NEAU-gq9(T) (98.4%), M. jinlongensis NEAU-GRX11(T) (98.3%) and M. zamorensis CR38(T) (97.9%). Phylogenetic analysis based on the gyrB gene sequence also indicated that the isolate clustered with the above type strains except M. violae NEAU-zh8(T). The cell-wall peptidoglycan consisted of meso-diaminopimelic acid and glycine. The major menaquinones were MK-9(H8), MK-9(H6) and MK-10(H2). The phospholipid profile contained diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The major fatty acids were C(16:0), iso-C(15:0) and C(17:0). Furthermore, some physiological and biochemical properties and low DNA-DNA relatedness values enabled the strain to be differentiated from members of closely related species. Therefore, it is proposed that strain NEAU-P5(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora taraxaci sp. nov. is proposed. The type strain is NEAU-P5(T) (=CGMCC 4.7098(T) = DSM 45885(T)).

Micromonospora jinlongensis sp. nov., isolated from muddy soil in China and emended description of the genus Micromonospora.

A novel actinomycete, designated strain NEAU-GRX11(T), was isolated from muddy soil collected from a stream of Jinlong Mountain in Harbin, north China. The organism was found to have morphological and chemotaxonomic characteristics typical of the genus Micromonospora. The 16S rRNA gene sequence of strain NEAU-GRX11(T) showed highest similarity to Micromonospora zamorensis CR38(T) (99.2 %), Micromonospora saelicesensis Lupac 09(T) (99.0 %), Micromonospora chokoriensis 2-19/6(T) (98.7 %), Micromonospora coxensis 2-30-b/28(T) (98.5 %), Micromonospora aurantiaca ATCC 27029(T) (98.4 %) and Micromonospora lupini lupac 14N(T) (98.3 %). Phylogenetic analysis based on the 16S rRNA gene and gyrB gene demonstrated that strain NEAU-GRX11(T) was a member of the genus Micromonospora and supported the closest phylogenetic relationship to M. zamorensis CR38(T), M. saelicesensis Lupac 09(T), M. chokoriensis 2-19/6(T) and M. lupini lupac 14N(T). A combination of DNA-DNA hybridization and some phenotypic characteristics indicated that the novel strain could be readily distinguished from these closest phylogenetic relatives. Therefore, it is proposed that NEAU-GRX11(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora jinlongensis sp. nov. is proposed. The type strain is NEAU-GRX11(T) (=CGMCC 4.7103(T)=DSM 45876(T)).

A novel taxonomic marker that discriminates between morphologically complex actinomycetes.

In the era when large whole genome bacterial datasets are generated routinely, rapid and accurate molecular systematics is becoming increasingly important. However, 16S ribosomal RNA sequencing does not always offer sufficient resolution to discriminate between closely related genera. The SsgA-like proteins are developmental regulatory proteins in sporulating actinomycetes, whereby SsgB actively recruits FtsZ during sporulation-specific cell division. Here, we present a novel method to classify actinomycetes, based on the extraordinary way the SsgA and SsgB proteins are conserved. The almost complete conservation of the SsgB amino acid (aa) sequence between members of the same genus and its high divergence between even closely related genera provides high-quality data for the classification of morphologically complex actinomycetes. Our analysis validates Kitasatospora as a sister genus to Streptomyces in the family Streptomycetaceae and suggests that Micromonospora, Salinispora and Verrucosispora may represent different clades of the same genus. It is also apparent that the aa sequence of SsgA is an accurate determinant for the ability of streptomycetes to produce submerged spores, dividing the phylogenetic tree of streptomycetes into liquid-culture sporulation and no liquid-culture sporulation branches. A new phylogenetic tree of industrially relevant actinomycetes is presented and compared with that based on 16S rRNA sequences.

Micromonospora spongicola sp. nov., an actinomycete isolated from a marine sponge in the Gulf of Thailand.

An actinomycete strain, S3-1(T), was isolated from marine sponge sample collected from the Gulf of Thailand. The strain is aerobic, Gram-positive and produced single spores at the tip of the substrate mycelium. Strain S3-1(T) contained meso-diaminopimelic acid in the peptidoglycan, whole-cell sugars were arabinose, galactose, glucose, rhamnose, ribose and xylose. The polar lipid profile of strain S3-1(T) consisted of phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, phosphoglycolipid and unknown polar lipids. Morphological and chemotaxonomic characteristics of the strain were identified as a member of the genus Micromonospora. Phylogenetic analysis based on 16S rRNA gene sequence analysis of the strain showed similarity to Micromonospora nigra DSM 43818(T) (98.8%), Micromonospora yangpuensis FXJ6.011(T) (98.7%) and Micromonospora narathiwatensis BTG4-1(T) (98.6%). The DNA G+C content was 72.7 mol%. The phenotypic characteristics and DNA-DNA relatedness values supported that the classification of this strain as a novel species in the genus Micromonospora, for which the name Micromonospora spongicola sp. nov. (type strain S3-1(T) =BCC 45595(T)=NBRC 108779(T)) is proposed.

Bioprospecting Micromonospora from Kaziranga National Park of India and their anti-infective potential.

Large number of strains was isolated from soils of Kaziranga National Park of North-East India using selective isolation procedure. They were assigned to the genus Micromonospora on the basis of their typical colonial and pigmentation features. The taxonomic identities of the isolates were confirmed on the basis of their molecular characters (16SrDNA). A total of one hundred Micromonospora strains were isolated during the present investigation. The diagnostic cell wall sugar and amino acids were determined from these Micromonospora strains. After preliminary screening most of the isolates exhibited excellent anti-infective activity against human bacterial pathogens Staphylococcus aureas, Bacillus subtilis, Proteus vulgaris, Echerichia coli, Pseudomonas aeroginosa and fungal pathogens Aspergillus niger, Fusarium oxysporum and Candida albicans. Among these isolates one strain designated as HK-10 showed promising activity against human pathogens S. aureas, B. subtilis, P. vulgaris and P. aeroginosa.

Micromonospora yangpuensis sp. nov., isolated from a sponge.

An actinomycete, strain FXJ6.011(T), was isolated from a cup-shaped sponge collected at Dachan reef, Yangpu in the South China Sea. The strain had morphological characteristics of members of the family Micromonosporaceae. Phylogenetic analysis of the 16S rRNA gene sequence of strain FXJ6.011(T) indicated the highest similarity (98.7 %) to Micromonospora auratinigra JCM 12357(T), Micromonospora chaiyaphumensis JCM 12873(T) and Micromonospora echinofusca JCM 3327(T). Analysis of the gyrB gene sequence also showed that strain FXJ6.011(T) should be assigned to the genus Micromonospora. It contained DD-diaminopimelic acid as the major cell-wall diamino acid and MK-10(H(2)) as predominant menaquinone. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidyl mannosides and phosphatidylinositol dimannoside. The major cellular fatty acids were iso-C(16 : 0), C(17 : 1)ω8c and C(16 : 0). Physiological and biochemical data and low DNA-DNA relatedness values enabled the strain to be differentiated from members of closely related species. Based on phenotypic and genotypic data, strain FXJ6.011(T) represents a novel Micromonospora species, for which the name Micromonospora yangpuensis sp. nov. is proposed; the type strain is FXJ6.011(T) ( = CGMCC 4.5736(T) = NBRC 107727(T)).

Micromonospora pisi sp. nov., isolated from root nodules of Pisum sativum.

A novel actinomycete, designated strain GUI 15(T), isolated from the root nodules of a Pisum sativum plant was characterized taxonomically by using a polyphasic approach. The 16S rRNA gene sequence of strain GUI 15(T) showed highest similarity to Micromonospora pattaloongensis TJ2-2(T) (98.7 %) and Polymorphospora rubra TT 97-42(T) (98.5 %). Phylogenetic analysis based on the gyrase B gene also supported the close relationship of these three strains, but indicated that strain GUI 15(T) should be assigned to the genus Micromonospora. Chemotaxonomic results confirmed the position of the isolate in the genus Micromonospora, but revealed differences at the species level. The novel strain could be distinguished from recognized Micromonospora species by using a combination of physiological and biochemical tests. Based on these observations, strain GUI 15(T) is considered to represent a novel species of the genus Micromonospora, for which the name Micromonospora pisi sp. nov. is proposed. The type strain is GUI 15(T) (=DSM 45175(T)=LMG 24546(T)).

Micromonospora rifamycinica sp. nov., a novel actinomycete from mangrove sediment.

An actinomycete strain, AM105(T), that produces rifamycin, was isolated from mangrove sediment samples collected from the South China Sea. The strain showed closest 16S rRNA gene sequence similarity to Micromonospora matsumotoense (98.0%). Chemotaxonomic characteristics of the isolate coincided with members of the genus Micromonospora. The value of DNA-DNA relatedness to M. matsumotoense (53.6%) and phenotypic differences from phylogenetically related Micromonospora species indicated that this isolate belongs to a novel species, for which the name Micromonospora rifamycinica sp. nov. is proposed. The type strain is AM105(T) (=CGMCC 4.2495(T)=DSM 44983(T)).

Micromonospora coriariae sp. nov., isolated from root nodules of Coriaria myrtifolia.

An actinomycete strain, NAR01(T), was isolated from root nodules of a Coriaria plant. The 16S rRNA gene sequence of strain NAR01(T) showed most similarity to the type strains of Micromonospora endolithica (98.94 %) and Micromonospora chersina (98.4 %). The chemotaxonomic results obtained confirmed the taxonomic position of the isolate within the genus Micromonospora, and revealed differences at the species level. Physiological and biochemical tests showed that strain NAR01(T) could be clearly distinguished from its closest phylogenetic neighbours, while DNA-DNA hybridization results indicated that the isolate represents a novel species. On the basis of these results, strain NAR01(T) (=DSM 44875(T)=LMG 23557(T)) is proposed as the type strain of the novel species Micromonospora coriariae sp. nov.

[Antagonistic activity of actinomycetes of Lake Baikal].

It was demonstrated that actinomycetes of Lake Baikal are strong antagonists of other microorganisms. Representatives of the genera Streptomyces and Micromonospora inhibit the growth of bacteria isolated from the lake, as well as of antibiotic-resistant microorganisms causing various human diseases. Baikal actinomycetes display a wide range of antagonistic activity and are potential producers of new biologically active substances.

Differentiation and protease production in Micromonospora echinospora (ATCC 15837).

Micromonospora echinospora differentiates in both submerged and surface cultures producing abundant dark spores after a period of vegetative mycelial growth. In submerged batch cultures, under either carbon or nitrogen limiting conditions, protease activity was found to coincide with sporulation indicating a relationship between proteolytic activity and differentiation in this organism. Further evidence for this link was provided from surface grown cultures wherein sporulation was inhibited by the serine protease inhibitors TLCK and TPCK. The association between proteolysis and differentiation apparent in this organism correlates with evidence of a similar phenomenon observed in the streptomycetes, suggesting that this may be a common response associated with differentiation in filamentous actinomycetes.

Micromonospora siamensis sp. nov., isolated from Thai peat swamp forest.

Morphological and chemotaxonomic characterization of actinomycete strain TT2-4T isolated from peat swamp forest soil in Pattaloong Province, Thailand, clearly demonstrated that this strain belongs to the genus Micromonospora. 16S rDNA sequence analysis for the strain supported the assignment of the strain to the genus Micromonospora and the similarity value of sequences between this strain and the closely related species, Micromonospora mirobrigensis was 99.1%, and M. carbonacea and M. matsumotoense were 98.8%. The DNA-DNA hybridization result and some physiological and biochemical properties indicated that strain TT2-4T was distinguished from the phylogenetically closest relatives. Based on these genotypic and phenotypic data, strain TT2-4T merits a new species in the genus Micromonospora and the name Micromonospora siamensis sp. nov. is proposed for the strain. The type strain is strain TT2-4T (=JCM 12769T =PCU 266T =TISTR 1554T).

Eight new species of the genus Micromonospora, Micromonospora citrea sp. nov., Micromonospora echinaurantiaca sp. nov., Micromonospora echinofusca sp. nov. Micromonospora fulviviridis sp. nov., Micromonospora inyonensis sp. nov., Micromonospora peucetia sp. nov., Micromonospora sagamiensis sp. nov., and Micromonospora viridifaciens sp. nov.

A previous phylogenetic study on type strains of the genus Micromonospora and Micromonospora species bearing non-validly published names has pointed towards the species status of several of latter strains. Subsequent studies on morphological, cultural, chemotaxonomic, metabolic, and genomic properties, and on whole cell mass spectrometric analyses by matrix adsorbed laser desorption/ionization time-of-flight (MALDI-TOF) confirmed the species status, leading to the proposal of eight new Micromonospora species: Micromonospora citrea sp. nov., type strain DSM 43903T, Micromonospora echinaurantiaca sp. nov., type strain DSM 43904T, Micromonospora echinofusca sp. nov., type strain DSM 43913T, Micromonospora fulviviridis sp. nov., type strain DSM 43906T, Micromonospora inyonensis sp. nov., type strain DSM 46123T, Micromonospora peucetia sp. nov., type strain DSM 43363T, Micromonospora sagamiensis sp. nov., type strain DSM 43912T and Micromonospora viridifaciens sp. nov., type strain DSM 43909T.

Antibiotic production, accumulation of intracellular carbon reserves, and sporulation in Micromonospora echinospora (ATCC 15837).

The physiology of the actinomycete Micromonospora echinospora was examined during growth. Biphasic accumulation of glycogen occurred, initially during the early exponential growth phase, and again following the onset of sporulation at 120 h. Lipid levels increased during growth eventually representing 25% of the cell mass. A significant proportion of the lipid was found to be in the form of triacylglycerols, which were found to accumulate markedly during the sporulation phase. The disaccharide trehalose was also found to accumulate during growth with levels rising to 5% of the dry weight during the mycelial production phase, then remaining constant during sporulation. Antibiotic was produced transiently by the cultures over the period preceding sporulation.

Response of Micromonospora echinospora (NCIMB 12744) spores to heat treatment with evidence of a heat activation phenomenon.

The effects of heat treatment on spores of the actinomycete Micromonospora echinospora were investigated. The percentage of culturable spores in untreated spore stocks was found to be approximately 20%. A 60 degrees C treatment of spores in phosphate buffer for 10 min led to an approximately five-fold increase in the number of culturable units. This indicated that a large proportion of the spores were constitutively dormant. Within 10 min and in the absence of an external energy-yielding substrate, the heat treatment was found to stimulate spore respiration suggesting that endogenous storage compounds were being utilized. Heating spores at 70 degrees C shortened the time period required for activation; holding times greater than 10 min, however, resulted in a reduction of culturable cells. Classic thermal death characteristics were seen at temperatures of 80 degrees C and above with D-values of 21.43, 2.67, 0.45 and 0.09 min being recorded at 70, 80, 90 and 100 degrees C, respectively. Spores of this organism, while being weakly heat resistant in comparison with bacterial endospores, are significantly more resistant than vegetative cells.

Fermentation studies of rustmicin production by a Micromonospora sp.

The antifungal antibiotic rustmicin was detected in the fermentation broth of the actinomycete MA 7094 as a specific inhibitor of sphingolipid biosynthesis in Candida albicans and as a potent fungicidal agent against Cryptococcus neoformans. Taxonomic characterization by both classical means and PCR fingerprinting supported the assignment of the producing culture to the genus Micromonospora. Fermentation medium optimization studies showed that the concentration of tomato paste in the medium was critical to increased production of rustmicin by MA 7094. The stimulatory effect of tomato paste in the medium on rustmicin production appeared to be related to the maintenance of pH at or below a value of 6.0. Addition of the antifoam agent P-2000 to the fermentation was found to dramatically reduce the rustmicin titer, while substitution of another antifoam agent, UCON-LB625, resulted in a 100% increase in the amount of rustmicin detected. After fermentation optimization studies and the generation of a non-sporulating mutant of MA 7094, the rustmicin titer was increased from an initial titer of 10mg/liter to 145 mg/liter.

Characteristics of airborne actinomycete spores.

Airborne actinomycete spores, important contaminants in occupational and residential environments, were studied with respect to their (i) release into the air, (ii) aerodynamic and physical size while airborne, and (iii) survival after collection onto agar with an impactor. Three actinomycete species were selected for the tests to exemplify the three main spore types: Streptomyces albus for arthrospores, Micromonospora halophytica for aleuriospores, and Thermoactinomyces vulgaris for endospores. The results show that the incubation conditions (temperature, time, and nutrients) needed for the development of spores for their release into air are different from the conditions that are needed for colony growth only. Additional drying of M. halophytica and T. vulgaris cultures was needed before spores could be released from the culture. The aerodynamic sizes of the spores, measured with an aerodynamic particle sizer, ranged from 0.57 (T. vulgaris) to 1.28 micron (M. halophytica). The physical sizes of the spores, when measured with a microscope and an image analysis system, were found to be smaller than previously reported in the literature. The relative recovery of the spores on agar media ranged from 0.5 (T. vulgaris) to 35% (S. albus). The results indicate that the culturability of the collected airborne actinomycete spores varies widely and is affected by several variables, such as the species and the sampling flow rate. Therefore, alternatives to commonly used cultivation methods need to be developed for the enumeration of actinomycete spores.