Biochemical and molecular characterization of a restriction endonuclease Tvu2HI from Thermoactinomyces vulgaris 2H and study of its R-M system.

A bacterial strain 2H isolated from soil and identified as Thermoactinomyces vulgaris produce a potent Type II restriction endonuclease activity that has been extracted by a PEG/dextran aqueous two-phase system. Optimal temperature for the restriction endonuclease activity was 55-65°C. Specific DNA cleavage was obtained at pH range 7-10 and 10-20mM MgCl2. Restriction cleavage analysis followed by sequencing confirms GG^CC as the recognition sequence. This enzyme, named Tvu2HI, is a thermostable isoschizomer of the mesophilic prototype restriction endonuclease HaeIII. Sequencing of the complete Thermoactinomyces vulgaris 2H genome revealed the presence of two adjacent ORFs coding for the restriction endonuclease Tvu2HI and the corresponding methyltransferase; an ORF coding for a putative Vsr nicking enzyme was found close to those coding for the Tvu2HI restriction-modification system. Phylogenetic analysis based on sequence alignment suggests a common origin of Tvu2HI R-M system with HaeIII-like R-M systems. This is the first investigation dealing with a Type II restriction endonuclease identified in a natural isolate of the genus Thermoactinomyces.

Antagonistic Properties of Some Halophilic Thermoactinomycetes Isolated from Superficial Sediment of a Solar Saltern and Production of Cyclic Antimicrobial Peptides by the Novel Isolate Paludifilum halophilum.

This study has focused on the isolation of twenty-three halophilic actinomycetes from two ponds of different salinity and the evaluation of their ability to exert an antimicrobial activity against both their competitors and several other pathogens. From the 23 isolates, 18 strains showed antagonistic activity, while 19 showed activities against one or more of the seven pathogen strains tested. Six strains exhibited consistent antibacterial activity against Gram-negative and Gram-positive pathogens characterized at the physiological and molecular levels. These strains shared only 94-95% 16S rRNA sequence identity with the closely related species of the Thermoactinomycetaceae family. Among them, the potent strain SMBg3 was further characterized and assigned to a new genus in the family for which the name Paludifilum halophilum (DSM 102817T) is proposed. Sequential extraction of the antimicrobial compounds with ethyl acetate revealed that the crude extract from SMBg3 strain had inhibitory effect on the growth of the plant pathogen Agrobacterium tumefaciens and the human pathogens Staphylococcus aureus, Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa. Based on the HRESI-MS spectral data, the cyclic lipopeptide Gramicidin S and four cyclic dipeptides (CDPs) named cyclo(L-4-OH-Pro-L-Leu), cyclo(L-Tyr-L-Pro), cyclo(L-Phe-L-Pro), and cyclo(L-Leu-L-Pro) were detected in the fermentation broth of Paludifilum halophilum. To our knowledge, this is the first report on the isolation of these compounds from members of the Thermoactinomycetaceae family.

Kroppenstedtia pulmonis sp. nov. and Kroppenstedtia sanguinis sp. nov., isolated from human patients.

Three human clinical strains (W9323(T), X0209(T) and X0394) isolated from a lung biopsy, blood and cerebral spinal fluid, respectively, were characterised using a polyphasic taxonomic approach. Comparative analysis of the 16S rRNA gene sequences showed the three strains belong to two novel branches within the genus Kroppenstedtia: 16S rRNA gene sequence analysis of W9323(T) showed close sequence similarity to Kroppenstedtia eburnea JFMB-ATE(T) (95.3 %), Kroppenstedtia guangzhouensis GD02(T) (94.7 %) and strain X0209(T) (94.6 %); sequence analysis of strain X0209(T) showed close sequence similarity to K. eburnea JFMB-ATE(T) (96.4 %) and K. guangzhouensis GD02(T) (96.0 %). Strains X0209(T) and X0394 were 99.9 % similar to each other by 16S rRNA gene sequence analysis. The DNA-DNA relatedness was 94.6 %, confirming that X0209(T) and X0394 belong to the same species. Chemotaxonomic data for strains W9323(T) and X0209(T) were consistent with those described for the members of the genus Kroppenstedtia: the peptidoglycan was found to contain LL-diaminopimelic acid; the major cellular fatty acids were identified as iso-C15 and anteiso-C15; and the major menaquinone was identified as MK-7. Differences in endospore morphology, carbon source utilisation profiles, and cell wall sugar patterns of strains W9323(T) and X0209(T), supported by phylogenetic analysis, enabled us to conclude that the strains each represent a new species within the genus Kroppenstedtia, for which the names Kroppenstedtia pulmonis sp. nov. (type strain W9323(T) = DSM 45752(T) = CCUG 68107(T)) and Kroppenstedtia sanguinis sp. nov. (type strain X0209(T) = DSM 45749(T) = CCUG 38657(T)) are proposed.

Thermoactinomyces khenchelensis sp. nov., a filamentous bacterium isolated from soil sediment of a terrestrial hot spring.

A novel thermophilic filamentous bacterium, designated strain T36(T), was isolated from soil sediment sample from a hot spring source collected in Khenchela province, Algeria. Strain T36(T) was identified as a member of the genus Thermoactinomyces by a polyphasic approach. Strain T36(T) was observed to form white aerial mycelium and non-coloured to pale yellow substrate mycelium, both producing endospores, sessile or borne by short sporophores. The optimum growth temperature and pH were found to be 37-55 °C and 7.0-9.0, respectively and the optimum NaCl concentration for growth was found to be 0-7 % (w/v). The diagnostic diamino acid in the cell wall peptidoglycan was identified as meso-diaminopimelic acid. The predominant menaquinone of strain T36(T) was identified as MK-7 (H0). The major fatty acids were found to be iso-C15:0 and iso-C17:0. The phospholipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphoglycolipid. The chemotaxonomic properties of strain T36(T) are consistent with those shared by members of the genus Thermoactinomyces. 16S rRNA gene sequence analysis indicated that the sequence similarities between strain T36(T) and Thermoactinomyces species with validly published names were less than 98 %. Based on the combined genotypic and phenotypic evidence, it is proposed that strain T36(T) should be classified as representative of a novel species, for which the name Thermoactinomyces khenchelensis sp. nov. is proposed. The type strain is T36(T) (=DSM 45951(T) = CECT 8579(T)).

A 68-Year-Old Musician With Cough, Wheezing, and a Lung Mass.

A 68-year-old man was referred to the pulmonary clinic for evaluation of cough and a 5-cm right upper lobe mass. He was in his usual state of health until 1 year prior when he developed intermittent cough, wheezing, and sinus congestion. He denied any sputum production or hemoptysis. He also denied any fevers, chills, or weight loss. He had received various treatments within the prior 6 months, including short courses of oral prednisone, levofloxacin, and bronchodilators, without any relief of his symptoms.

Thermoactinomyces guangxiensis sp. nov., a thermophilic actinomycete isolated from mushroom compost.

A novel thermophilic actinomycete, designated strain CD-1(T), was isolated from mushroom compost in Nanning, Guangxi province, China. The strain grew at 37-55 °C (optimum 45-50 °C), pH 6.0-11.0 (optimum pH 7.0-9.0) and with 0-2.0% NaCl (optimum 0-1.0%), formed well-developed white aerial mycelium and pale-yellow vegetative mycelium, and single endospores (0.8-1.0 µm diameter) were borne on long sporophores (2-3 µm length). The endospores were spherical-polyhedron in shape with smooth surface. Based on its phenotypic and phylogenetic characteristics, strain CD-1(T) is affiliated to the genus Thermoactinomyces. It contained meso-diaminopimelic acid as the diagnostic diamino acid; the whole-cell sugars were ribose and glucose. Major fatty acids were iso-C15 :  0, C16 : 0, anteiso-C15  : 0 and iso-C17  : 0. MK-7 was the predominant menaquinone. The polar phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylethanolamine containing hydroxylated fatty acids, ninhydrin-positive glycophospholipid, an unknown phospholipid and glycolipids. The G+C content of the genomic DNA was 48.8%. 16S rRNA gene sequence analysis showed that the organism was closely related to Lihuaxuella thermophila YIM 77831(T) (95.69% sequence similarity), Thermoactinomyces daqus H-18(T) (95.49%), Laceyella putida KCTC 3666(T) (95.05%), Thermoactinomyces vulgaris KCTC 9076(T) (95.01%) and Thermoactinomyces intermedius JCM 3312(T) (94.55%). Levels of DNA-DNA relatedness between strain CD-1T and Lihuaxuella thermophila JCM 18059(T), Thermoactinomyces daqus DSM 45914(T), Laceyella putida JCM 8091(T), Thermoactinomyces vulgaris JCM 3162(T) and Thermoactinomyces intermedius JCM 3312(T) were low (22.8, 33.3, 24.7, 29.4 and 30.0%, respectively). A battery of phenotypic, genotypic and DNA-DNA relatedness data indicated that strain CD-1T represented a novel species of the genus Thermoactinomyces, for which the name Thermoactinomyces guangxiensis sp. nov. is proposed. The type strain is CD-1(T) ( = ATCC BAA-2630(T) = CGMCC 4.7156(T)).

Degradation of intact chicken feathers by Thermoactinomyces sp. CDF and characterization of its keratinolytic protease.

Thermoactinomyces is known for its resistance to extreme environmental conditions and its ability to digest a wide range of hard-to-degrade compounds. Here, Thermoactinomyces sp. strain CDF isolated from soil was found to completely degrade intact chicken feathers at 55 °C, with the resulting degradation products sufficient to support growth as the primary source of both carbon and nitrogen. Although feathers were not essential for the expression of keratinase, the use of this substrate led to a further 50-300 % increase in enzyme production level under different nutrition conditions, with extracellular keratinolytic activity reaching its highest level (∼400 U/mL) during the late-log phase. Full degradation of feathers required the presence of living cells, which are thought to supply reducing agents necessary for the cleavage of keratin disulfide bonds. Direct contact between the hyphae and substrate may enhance the reducing power and protease concentrations present in the local microenvironment, thereby facilitating keratin degradation. The gene encoding the major keratinolytic protease (protease C2) of strain CDF was cloned, revealing an amino acid sequence identical to that of subtilisin-like E79 protease from Thermoactinomyces sp. E79, albeit with significant differences in the upstream flanking region. Exogenous expression of protease C2 in Escherichia coli resulted in the production of inclusion bodies with proteolytic activity, which could be solubilized to an alkaline solution to produce mature protease C2. Purified protease C2 was able to efficiently hydrolyze α- and ß-keratins at 60-80 °C and pH 11.0, representing a promising candidate for enzymatic processing of hard-to-degrade proteins such as keratinous wastes.

Prevalence of thermoduric bacteria and spores on 10 Midwest dairy farms.

Thermoduric bacteria (TDB), including sporeformers and their spores, can be present in milk and dairy products even after pasteurization. They have the potential to adversely affect the quality and shelf life of products. The objectives of this study were to identify the origin and common species of heat-resistant bacteria occurring during summer and winter on Midwest dairy farms. Bulk tank milk samples were taken from 10 dairy farms located along the South Dakota section of Interstate 29, with herd sizes ranging from 650 to 3,500 lactating dairy cows. Milk samples were profiled for the prevalence of TDB and spore counts (SC). Corn silage samples and swabs of the milking clusters were also taken at the dairies to further profile the potential sources of TDB and SC. The samples were taken 3 times during 2 seasons [winter (January-March) and summer (June-August)] to track seasonal changes in the farm bacterial flora. During winter, the average TDB counts in bulk tank milk were 2.61 log compared with 2.76 log TDB counts in the summer. The SC was 1.08 log in the winter, which was half the 2.06 log SC present in the summer season. Corn silage sampled in winter contained a 7.57 log TDB count compared with an increased 10.77 log TDB count during summer sampling. Concentrations of SC in corn silage reached an average of 6.3 log in winter compared with 11.81 log for summer. The seasonal effect was evident with an increase in summer counts across the board for TDB and SC, both in the feed and bulk tank milk samples. Bacillus licheniformis was the predominant species identified in 62.4% of winter (85 total) and 49.4% of summer (83 total) samples. Bacillus subtilis made up 9.4% of the remaining winter isolates, followed by Bacillus sonorensis at 8.2%. Conversely, B. sonorensis made up 12% of the summer isolates followed by Bacillus pumilus at 10.8%. Bacillus licheniformis is a ubiquitous microbe and was isolated from both TDB and sporeformer categories in all 3 sample types. There were larger increases in SC than TDB, indicating that summer temperatures and conditions may favor proliferation of sporeforming bacteria over that of TDB. In conclusion, samples from bulk tank milk, milking cluster swabs, and corn silage samples at each of the 10 sites indicated that B. licheniformis was the major contaminant species, regardless of season. In this experiment, corn silage was the major environmental source of both TDB and SC with higher concentrations in summer when compared with winter.

Detection of thermoactinomyces species in selected agricultural substrates from Queensland.

Selected overheated substrates commercially available for public use in sub-tropical Queensland, Australia were screened for the presence of Thermoactinomyces species using an air sampler. All substrates with the exception of tea tree mulch were found to contain Thermoactinomyces species. Subsequent 16S rDNA oligonucleotide sequencing of the selected eight isolates indicated that some of these species were closely related to previously reported allergenic Thermoactinomyces vulgaris and Laceyella sacchari. In view of this, the isolates were tested to determine their adhesion ability and cytotoxicity to human lung cells (calu-3 cells). The results indicated that all eight isolates were highly adherent and showed cytotoxicity to this cell line. These findings might indicate that the presence of such species in overheated agricultural materials may constitute a public health risk if storage and handling conditions are not optimal and do not meet criteria defined for sub-tropical climates.

Thermoactinomyces daqus sp. nov., a thermophilic bacterium isolated from high-temperature Daqu.

Daqu is a fermentation starter used in the production of Chinese liquors. A thermophilic bacterium, designated strain H-18(T), was isolated from a high-temperature Daqu sample collected from the manufacturing process of a sesame-flavoured liquor in Shandong province, China. It was investigated in a taxonomic study using a polyphasic approach. Strain H-18(T) formed white aerial mycelium and greyish-yellow substrate mycelium, bearing single endospores on aerial and substrate hyphae or on unbranched short sporophores. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The major fatty acids were iso-C15 : 0 and iso-C17 : 0. The predominant menaquinone was MK-7. These chemotaxonomic properties are similar to those of members of the genus Thermoactinomyces. The G+C content of the genomic DNA was 49.1 mol%. 16S rRNA gene sequence comparisons indicated that strain H-18(T) was most closely related to Thermoactinomyces vulgaris KCTC 9076(T) (96.42 % similarity), Thermoactinomyces intermedius KCTC 9646(T) (96.06 %), Laceyella putida KCTC 3666(T) (96.32 %) and Laceyella sacchari KCTC 9790(T) (95.55 %). Strain H-18(T) showed low DNA-DNA relatedness (40.8, 33.4, 20.0 and 14.4 %) with the above strains. Based on morphological and chemotaxonomic characteristics, DNA-DNA hybridization data and physiological properties, strain H-18(T) represents a novel species of the genus Thermoactinomyces, for which the name Thermoactinomyces daqus sp. nov. is proposed. The type strain is H-18(T) ( = DSM 45914(T) = CICC 10681(T)).

Purification and characterization of a thermolysin like protease from Thermoactinomyces thalpophilus MCMB-380.

The extracellular thermolysin like protease (TLP) was purified and characterized from Thermoactinomyces thalpophilus MCMB-380 (Genbank Accession No. EF397000). The enzyme was purified to homogeneity by successive ultra filtration steps using 50 kDa and 10 kDa membrane filters followed by anion exchange chromatography. The molecular mass and isoelectric point of the enzyme were found to be 34.4 kDa and 9.5, respectively. The proteolytic activity was inhibited by EDTA and the enzyme required Ca2+ to show the full activity as well as thermostability. The T50 of the enzyme at 80 °C was 1 h and the activation energy was estimated to be 11.02 Kcal / mol. Atomic absorption spectrophotometric analysis revealed the presence of Zn2+ ion in the protein core indicating that it is a metalloprotease. This protease has commercial potential in catalyzing the condensation reaction of two amino acids for production of the dipeptide aspartame, an artificial sweetener. The one hour time-frame is significantly faster than that of the enzyme thermolysin from Bacillus thermoproteolyticus. Moreover the TLP was stable at 80°C for one hour which makes it industrially robust. The Zn2+ ion in the T. thalpophilus protease appears to be necessary for maintaining the active conformation of the enzyme molecule.

Development and evaluation of a method for the quantification of airborne Thermoactinomyces vulgaris by real-time PCR.

Actinomycetes are ubiquitous and some can be potentially pathogenic for humans when present in the air of some working areas. It's notably the case for Thermoactinomyces vulgaris in composting facilities where aerial concentrations can reach high values of more than 10(7) CFU·m(-3). Workers exposure to these inhalable bioaerosols can be the source of various diseases. The literature reveals a lack of knowledge about risk assessment: there is neither dose-effects relationship for most agents, or threshold limit value. The objectives of this study were to develop and standardize a method to quantify workers exposure to bioaerosols. We have developed and evaluated a method to quantify airborne T. vulgaris based on DNA extraction of aerial microbial communities and qPCR. Four DNA extraction protocols were compared, and primers and a hydrolysis probe were designed for specific amplification of the target species (gyrB gene). This method was compared to traditional methods based on viable or cultivable counting by epifluorescence microscopy or plating on selective media. The method was applied on environmental bioaerosols sampled under real exposure conditions in composting plants. We demonstrate that the method to quantify T. vulgaris in bioaerosols is specific, sensitive and repeatable. We demonstrate the occurrence and quantified T. vulgaris in the atmosphere of composting facilities with concentrations ranging from 3×10(2) to 3×10(6)×m(-3).