Whole-Genome Analysis of Mycobacterium neoaurum DSM 1381 and the Validation of Two Key Enzymes Affecting C22 Steroid Intermediates in Sterol Metabolism.

Mycobacterium neoaurum DSM 1381 originated from Mycobacterium neoaurum ATCC 25790 by mutagenesis screening is a strain of degrading phytosterols and accumulating important C22 steroid intermediates, including 22-hydroxy-23, 24-bisnorchola-4-en-3-one (4-HP) and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (HPD). However, the metabolic mechanism of these C22 products in M. neoaurum DSM 1381 remains unknown. Therefore, the whole-genome sequencing and comparative genomics analysis of M. neoaurum DSM 1381 and its parent strain M. neoaurum ATCC 25790 were performed to figure out the mechanism. As a result, 28 nonsynonymous single nucleotide variants (SNVs), 17 coding region Indels, and eight non-coding region Indels were found between the genomes of the two strains. When the wild-type 3-ketosteroid-9α-hydroxylase subunit A1 (KshA1) and ß-hydroxyacyl-CoA dehydrogenase (Hsd4A) were overexpressed in M. neoaurum DSM 1381, the steroids were transformed into the 4-androstene-3, 17- dione (AD) and 1,4-androstadiene-3,17-dione (ADD) instead of C22 intermediates. This result indicated that 173N of KshA1 and 171K of Hsd4A are indispensable to maintaining their activity, respectively. Amino acid sequence alignment analysis show that both N173D in KshA1 and K171E in Hsd4A are conservative sites. The 3D models of these two enzymes were predicted by SWISS-MODEL and AlphaFold2 to understand the inactivation of the two key enzymes. These results indicate that K171E in Hsd4A may destroy the inaction between the NAD+ with the NH3+ and N173D in KshA1 and may disrupt the binding of the catalytic domain to the substrate. A C22 steroid intermediates-accumulating mechanism in M. neoaurum DSM 1381 is proposed, in which the K171E in Hsd4A leads to the enzyme's inactivation, which intercepts the C19 sub-pathways and accelerates the C22 sub-pathways, and the N173D in KshA1 leads to the enzyme's inactivation, which blocks the degradation of C22 intermediates. In conclusion, this study explained the reasons for the accumulation of C22 intermediates in M. neoaurum DSM 1381 by exploring the inactivation mechanism of the two key enzymes.

Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment.

BACKGROUND: Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. RESULTS: The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a ß-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. CONCLUSION: KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

First case report of prosthetic valve endocarditis caused by Mycobacterium wolinskyi.

To date, only 26 cases of Mycobacterium wolinskyi infections have been reported in humans. We herein report a first case of prosthetic valve endocarditis due to this organism after cardiovascular surgery. An 82-year-old man presented with repeat episodes of syncope and fever after aortic valve replacement, mitral valve replacement, left atrial appendage closure, and pulmonary vein isolation. Blood cultures maintained in aerobic bottles were repeatedly positive after 90-100 hours, and Gallium scan revealed abnormal accumulations in the sternum and left testis. While colonies formed by culturing the fluid of the parasternal area and blood cultures revealed gram-positive rods, we could not analyze the colony using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF). M. wolinskyi was finally identified on 16S rRNA, hsp65, and rpoB gene sequencing. We treated the patient with multiple antimycobacterial drugs, i.e., amikacin, imipenem, and clarithromycin for 6 weeks, which was changed to oral ciprofloxacin and minocycline for 12 months. This case highlights the need to consider rapidly growing mycobacteria, including M. wolinskyi, if chronic fever persists from weeks to months after surgery, the blood culture is positive, and the organism is not identified. In addition, sequencing the 16S rRNA, hsp65, and rpoB genes is essential for diagnosis.

A Dual Role Reductase from Phytosterols Catabolism Enables the Efficient Production of Valuable Steroid Precursors.

4-Androstenedione (4-AD) and progesterone (PG) are two of the most important precursors for synthesis of steroid drugs, however their current manufacturing processes suffer from low efficiency and severe environmental issues. In this study, we decipher a dual-role reductase (mnOpccR) in the phytosterols catabolism, which engages in two different metabolic branches to produce the key intermediate 20-hydroxymethyl pregn-4-ene-3-one (4-HBC) through a 4-e reduction of 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA) and 2-e reduction of 3-oxo-4-pregnene-20-carboxyl aldehyde (3-OPA), respectively. Inactivation or overexpression of mnOpccR in the Mycobacterium neoaurum can achieve exclusive production of either 4-AD or 4-HBC from phytosterols. By utilizing a two-step synthesis, 4-HBC can be efficiently converted into PG in a scalable manner (100 gram scale). This study deciphers a pivotal biosynthetic mechanism of phytosterol catabolism and provides very efficient production routes of 4-AD and PG.

Structure of a robust bacterial protein cage and its application as a versatile biocatalytic platform through enzyme encapsulation.

Using a newly discovered encapsulin from Mycolicibacterium hassiacum, several biocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produce as recombinant protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of 60 protomers (diameter of 23 nm) with narrow pores. By developing an effective coexpression and isolation procedure, the effect of packaging a variety of biocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stability of the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in the encapsulin. Due to the restricted diameters of the encapsulin pores (5-9 Å), the protein cage protects the encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuable tools to tune the properties of biocatalysts such as stability and substrate specificity.

A genuine mycobacterial thermophile: Mycobacterium hassiacum growth, survival and GpgS stability at near-pasteurization temperatures.

Mycobacterium hassiacum is so far the most thermophilic among mycobacteria as it grows optimally at 50 °C and up to 65 °C in a glycerol-based medium, as verified in this study. Since this and other nontuberculous mycobacteria (NTM) thrive in diverse natural and artificial environments, from where they may access and infect humans, we deemed essential to probe M. hassiacum resistance to heat, a strategy routinely used to control microbial growth in water-supply systems, as well as in the food and drink industries. In addition to possibly being a threat in its own right in rare occasions, M. hassiacum is also a good surrogate for studying other NTM species more often associated with opportunistic infection, namely Mycobacterium avium and Mycobacterium abscessus as well as their strictly pathogenic counterparts Mycobacterium tuberculosis and Mycobacterium leprae. In this regard, this thermophilic species is likely to be useful as a source of stable proteins that may provide more detailed structures of potential drug targets. Here, we investigate M. hassiacum growth at near-pasteurization temperatures and at different pHs and also characterize its thermostable glucosyl-3-phosphoglycerate synthase (GpgS), an enzyme considered essential for M. tuberculosis growth and associated with both nitrogen starvation and thermal stress in different NTM species.

Whole-genome and enzymatic analyses of an androstenedione-producing Mycobacterium strain with residual phytosterol-degrading pathways.

Mycobacterium neoaurum strains can transform phytosterols to 4-androstene-3,17-dione (4-AD), a key intermediate for the synthesis of advanced steroidal medicines. In this work, we presented the complete genome sequence of the M. neoaurum strain HGMS2, which transforms ß-sitosterol to 4-AD. Through genome annotation, a phytosterol-degrading pathway in HGMS2 was predicted and further shown to form a 9,10-secosteroid intermediate by five groups of enzymes. These five groups of enzymes included three cholesterol oxidases (ChoM; group 1: ChoM1, ChoM2 and Hsd), two monooxygenases (Mon; group 2: Mon164 and Mon197), a set of enzymes for side-chain degradation (group 3), one 3-ketosteroid-1,2-dehydrogenase (KstD; group 4: KstD211) and three 3-ketosteroid-9a-hydroxylases (Ksh; group 5: KshA226, KshA395 and KshB122). A gene cluster encoding Mon164, KstD211, KshA226, KshB122 and fatty acid ß-oxidoreductases constituted one integrated metabolic pathway, while genes encoding other key enzymes were sporadically distributed. All key enzymes except those from group 3 were prepared as recombinant proteins and their activities were evaluated, and the proteins exhibited distinct activities compared with enzymes identified from other bacterial species. Importantly, we found that the KstD211 and KshA395 enzymes in the HGMS2 strain retained weak activities and caused the occurrence of two major impurities, i.e., 1,4-androstene-3,17-dione (ADD) and 9-hydroxyl-4-androstene-3,17-dione (9OH-AD) during ß-sitosterol fermentation. The concurrence of these two 4-AD analogs not only lowered 4-AD production yield but also hampered 4-AD purification. HGMS2 has the least number of genes encoding KstD and Ksh enzymes compared with current industrial strains. Therefore, HGMS2 could be a potent strain by which the 4-AD production yield could be enhanced by disabling the KstD211 and KshA395 enzymes. Our work also provides new insight into the engineering of the HGMS2 strain to produce ADD and 9OH-AD for industrial application.

Endophthalmitis caused by Mycobacterium houstonense: case report.

BACKGROUND: Mycobacterium houstonense is rapidly growing mycobacteria (RGM) that belongs to M. fortuitum group. So far, there have been few associated reports of human diseases induced by M. houstonense worldwide. CASE PRESENTATION: We present a delayed-onset postoperative endophthalmitis caused by M. houstonense after glaucoma drainage implant (GDI) surgery. The ocular infection lasted for 2 months without appropriate treatment that developed into endophthalmitis and the patient underwent an emergency enucleation. CONCLUSION: Implant erosion and a delay in diagnosis of ocular infection could lead to irreversible damage as observed in our case. Ophthalmologists should be alert for ocular RGM infection, and prompt laboratory diagnosis with initiation of effective multidrug therapy might prevent loss of vision.

Fluorescence-based high-throughput screening system for R-ω-transaminase engineering and its substrate scope extension.

ω-Transaminase (ω-TA) is an attractive alternative to metal catalysts for the stereoselective amination of prochiral ketones. The narrow substrate scope of an R-ω-transaminase from Mycobacterium vanbaalenii (MvTA) limits its application in R-amine synthesis. A fluorescence-based TA activity screening system was developed to extend its substrate scope. The reactions were conducted in microtiter plates (MTPs) and displayed low background interference, high sensitivity (µM magnitude), and a wide dynamic range (ɀ-factor > 0.9). A KnowVolution campaign was performed on this enzyme, and screening ~ 8000 clones with this fluorescence-based screening system resulted in two beneficial substitutions (G68Y and F129A) and three improved variants (M3, M4, and M5). The best variant, MvTA M5 (WT+G68Y+F129A), achieved the highest catalytic efficiency (toward fluorogenic substrate NMA) which was 3.2-fold higher than that of the WT enzyme. MvTA M5 exhibited significantly enhanced activity toward six different prochiral ketones with e.e. > 99% (R). The specific activity of MvTA M5 was more than 100 times higher than that of the WT enzyme toward acetonaphthone (M5: 8.1 U/mg, WT: ~ 0.07 U/mg), and it showed the highest activity on acetonaphthone, p-ethylacetophenone, and phenylacetone.

Analysis of microbial contamination of household water purifiers.

Household water purifiers are increasingly used to treat drinking water at the household level, but their influence on the microbiological safety of drinking water has rarely been assessed. In this study, representative purifiers, based on different filtering processes, were analyzed for their impact on effluent water quality. The results showed that purifiers reduced chemical qualities such as turbidity and free chlorine. However, a high level of bacteria (102-106 CFU/g) was detected at each stage of filtration using a traditional culture-dependent method, whereas quantitative PCR with propidium monoazide (PMA) treatment showed 106-108 copies/L of total viable bacteria in effluent water, indicating elevated microbial contaminants after purifiers. In addition, high-throughput sequencing revealed a diverse microbial community in effluents and membranes. Proteobacteria (22.06-97.42%) was the dominant phylum found in all samples, except for purifier B, in which Melainabacteria was most abundant (65.79%). For waterborne pathogens, Escherichia coli (100-106 copies/g) and Pseudomonas aeruginosa (100-105 copies/g) were frequently detected by qPCR. Sequencing also demonstrated the presence of E. coli (0-6.26%), Mycobacterium mucogenicum (0.01-3.46%), and P. aeruginosa (0-0.16%) in purifiers. These finding suggest that water from commonly used household purifiers still impose microbial risks to human health.

Lacisediminihabitans profunda gen. nov., sp. nov., a member of the family Microbacteriaceae isolated from freshwater sediment.

A novel Gram-stain-positive bacterial strain, CHu50b-6-2T, was isolated from a 67-cm-long sediment core collected from the Daechung Reservoir at a water depth of 17 m, Daejeon, Republic of Korea. The cells of strain CHu50b-6-2T were aerobic non-motile and formed yellow colonies on R2A agar. The phylogenetic analysis based on 16S rRNA gene sequencing indicated that the strain formed a separate lineage within the family Microbacteriaceae, exhibiting 98.0%, 97.7% and 97.6% 16S rRNA gene sequence similarities to Glaciihabitans tibetensis KCTC 29148T, Frigoribacterium faeni KACC 20509T and Lysinibacter cavernae DSM 27960T, respectively. The phylogenetic trees revealed that strain CHu50b-6-2T did not show a clear affiliation to any genus within the family Microbacteriaceae. The chemotaxonomic results showed B1α type peptidoglacan containg 2, 4-diaminobutyric acid (DAB) as the diagnostic diamino acid, MK-10 as the predominant respiratory menaquinone, diphosphatidylglycerol, phosphatidylglycerol, and an unidentified glycolipid as the major polar lipids, anteiso-C15:0, iso-C16:0, and anteiso-C17:0 as the major fatty acids, and a DNA G + C content of 67.3 mol%. The combined genotypic and phenotypic data showed that strain CHu50b-6-2T could be distinguished from all genera within the family Microbacteriaceae and represents a novel genus, Lacisediminihabitans gen. nov., with the name Lacisediminihabitans profunda sp. nov., in the family Microbacteriaceae. The type strain is CHu50b-6-2T (= KCTC 49081T = JCM 32673T).

Pulmonary Infection Associated with Mycobacterium canariasense in Suspected Tuberculosis Patient, Iran.

Mycobacterium canariasense had only been isolated in humans from blood and contaminated catheters. We report a case of pulmonary disease associated with M. canariasense infection that was identified by multilocus sequence analysis; the illness was initially ascribed to M. tuberculosis. M. canariasense should be considered a cause of respiratory infection.

Crystal structure and biochemical characterization of O-acetylhomoserine acetyltransferase from Mycobacterium smegmatis ATCC 19420.

Mycobacterium smegmatis is a good model for studying the physiology and pathogenesis of Mycobacterium tuberculosis due to its genetic similarity. As methionine biosynthesis exists only in microorganisms, the enzymes involved in methionine biosynthesis can be a potential target for novel antibiotics. Homoserine O-acetyltransferase from M. smegmatis (MsHAT) catalyzes the transfer of acetyl-group from acetyl-CoA to homoserine. To investigate the molecular mechanism of MsHAT, we determined its crystal structure in apo-form and in complex with either CoA or homoserine and revealed the substrate binding mode of MsHAT. A structural comparison of MsHAT with other HATs suggests that the conformation of the α5 to α6 region might influence the shape of the dimer. In addition, the active site entrance shows an open or closed conformation and might determine the substrate binding affinity of HATs.

Characterization and engineering control of the effects of reactive oxygen species on the conversion of sterols to steroid synthons in Mycobacterium neoaurum.

The conversion of sterols to steroid synthons by engineered mycobacteria comprises one of the basic ways for the production of steroid medications in the pharmaceutical industry. Here, we revealed that high amounts of reactive oxygen species (ROS) generate during the conversion process of sterols, which impairs the cell viability of mycobacterial cells and thus hinders the conversion of sterols to steroid synthons. Accordingly, the endogenous antioxidants for detoxifying ROS in mycobacteria, ROS scavenging enzymes and low molecular weight thiols, were examined. The results revealed that three antioxidants, catalase (CAT), mycothiol (MSH), and ergothioneine (EGT), demonstrated efficacy toward neutralizing the excessive ROS produced during sterol metabolism. CAT overexpression or MSH or EGT augmentation enhanced the conversion of phytosterols to 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) by 18.9%, 23.8%, and 32.1%, respectively, and also enhanced the cell viability, indicating the benefits of these antioxidants in reducing ROS-induced stress. Further combinatorial augmentation of CAT, MSH, and EGT demonstrated enhanced effects toward intracellular ROS scavenging, resulting in 54.2% greater cell viability and 47.5% enhancement in 4-HBC production. These findings indicated that the excessive ROS induces cell stress, in turn limiting the conversion of sterols, whereas neutralization of the excessive ROS by combined control of CAT, MSH, and EGT serves as an effective strategy to boost the conversion productivity of sterols to steroid synthons.

ß-Aminopeptidases: Insight into Enzymes without a Known Natural Substrate.

ß-Aminopeptidases have the unique capability to hydrolyze N-terminal ß-amino acids, with varied preferences for the nature of ß-amino acid side chains. This unique capability makes them useful as biocatalysts for synthesis of ß-peptides and to kinetically resolve ß-peptides and amides for the production of enantiopure ß-amino acids. To date, six ß-aminopeptidases have been discovered and functionally characterized, five from Gram-negative bacteria and one from a fungus, Aspergillus Here we report on the purification and characterization of an additional four ß-aminopeptidases, one from a Gram-positive bacterium, Mycolicibacterium smegmatis (BapAMs), one from a yeast, Yarrowia lipolytica (BapAYlip), and two from Gram-negative bacteria isolated from activated sludge identified as Burkholderia spp. (BapABcA5 and BapABcC1). The genes encoding ß-aminopeptidases were cloned, expressed in Escherichia coli, and purified. The ß-aminopeptidases were produced as inactive preproteins that underwent self-cleavage to form active enzymes comprised of two different subunits. The subunits, designated α and ß, appeared to be tightly associated, as the active enzyme was recovered after immobilized-metal affinity chromatography (IMAC) purification, even though only the α-subunit was 6-histidine tagged. The enzymes were shown to hydrolyze chromogenic substrates with the N-terminal l-configurations ß-homo-Gly (ßhGly) and ß3-homo-Leu (ß3hLeu) with high activities. These enzymes displayed higher activity with H-ßhGly-p-nitroanilide (H-ßhGly-pNA) than previously characterized enzymes from other microorganisms. These data indicate that the new ß-aminopeptidases are fully functional, adding to the toolbox of enzymes that could be used to produce ß-peptides. Overexpression studies in Pseudomonas aeruginosa also showed that the ß-aminopeptidases may play a role in some cellular functions.IMPORTANCE ß-Aminopeptidases are unique enzymes found in a diverse range of microorganisms that can utilize synthetic ß-peptides as a sole carbon source. Six ß-aminopeptidases have been previously characterized with preferences for different ß-amino acid substrates and have demonstrated the capability to catalyze not only the degradation of synthetic ß-peptides but also the synthesis of short ß-peptides. Identification of other ß-aminopeptidases adds to this toolbox of enzymes with differing ß-amino acid substrate preferences and kinetics. These enzymes have the potential to be utilized in the sustainable manufacture of ß-amino acid derivatives and ß-peptides for use in biomedical and biomaterial applications. This is important, because ß-amino acids and ß-peptides confer increased proteolytic resistance to bioactive compounds and form novel structures as well as structures similar to α-peptides. The discovery of new enzymes will also provide insight into the biological importance of these enzymes in nature.

A genomic analysis of Mycobacterium immunogenum strain CD11_6 and its potential role in the activation of T cells against Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis (Mtb) is an etiological agent of tuberculosis (TB). Tuberculosis is a mounting problem worldwide. The only available vaccine BCG protects the childhood but not adulthood form of TB. Therefore, efforts are made continuously to improve the efficacy of BCG by supplementing it with other therapies. Consequently, we explored the possibility of employing Mycobacterium immunogenum (Mi) to improve BCG potential to protect against Mtb. RESULTS: We report here the genome mining, comparative genomics, immunological and protection studies employing strain CD11_6 of Mi. Mycobacterium immunogenum was isolated from duodenal mucosa of a celiac disease patient. The strain was whole genome sequenced and annotated for identification of virulent genes and other traits that may make it suitable as a potential vaccine candidate. Virulence profile of Mi was mapped and compared with two other reference genomes i.e. virulent Mtb strain H37Rv and vaccine strain Mycobacterium bovis (Mb) AFF2122/97. This comparative analysis revealed that Mi is less virulent, as compared to Mb and Mtb, and contains comparable number of genes encoding for the antigenic proteins that predict it as a probable vaccine candidate. Interestingly, the animals vaccinated with Mi showed significant augmentation in the generation of memory T cells and reduction in the Mtb burden. CONCLUSION: The study signifies that Mi has a potential to protect against Mtb and therefore can be a future vaccine candidate against TB.

Intracellular Environment Improvement of Mycobacterium neoaurum for Enhancing Androst-1,4-Diene-3,17-Dione Production by Manipulating NADH and Reactive Oxygen Species Levels.

As one of the most significant steroid hormone precursors, androst-1,4-diene-3,17-dione (ADD) could be used to synthesize many valuable hormone drugs. The microbial transformation of sterols to ADD has received extensive attention in recent years. In a previous study, Mycobacterium neoaurum JC-12 was isolated and converted sterols to the major product, ADD. In this work, we enhanced ADD yield by improving the cell intracellular environment. First, we introduced a nicotinamide adenine dinucleotide (NADH) oxidase from Bacillus subtilis to balance the intracellular NAD+ availability in order to strengthen the ADD yield. Then, the catalase gene from M. neoaurum was also over-expressed to simultaneously scavenge the generated H2O2 and eliminate its toxic effects on cell growth and sterol transformation. Finally, using a 5 L fermentor, the recombinant strain JC-12yodC-katA produced 9.66 g/L ADD, which increased by 80% when compared with the parent strain. This work shows a promising way to increase the sterol transformation efficiency by regulating the intracellular environment.

Hoyosella rhizosphaerae sp. nov., a halotolerant actinobacterium isolated from rhizosphere soil of Suaeda salsa, and emended description of the genus Hoyosella.

A halotolerant actinobacterium, designated J12GA03T, was isolated from a rhizosphere soil sample of Suaeda salsa collected from a dried saline lake in Hebei Province, China. Cells were Gram-staining-positive, non-motile and non-spore-forming cocci. Strain J12GA03T grew optimally at 28‒37 °C, 0‒3 % NaCl (w/v) and pH 6.5‒7.5. It contained meso-diaminopimelic acid as the diagnostic diamino acid and arabinose, galactose and ribose as the diagnostic whole-cell sugars. MK-8 and MK-7 were detected as predominant menaquinones. Major fatty acids were C17 : 1ω8c, C16 : 0 and C17 : 0. Polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol, phosphoglycolipids, glycolipids, unidentified phospholipids and additional lipids. The muramyl residue was acetyl. Mycolic acids (34-38 carbon atoms) were present. The G+C content of the genomic DNA was 55.8 mol%. It shared the highest 16S rRNA gene sequence similarities with Amycolicicoccus subflavus DQS3-9A1T (98.18 %) and Hoyosella altamirensis OFN S31T (97.75 %). Phylogenetic trees showed that strain J12GA03T firmly formed a distinct monophyletic branch in the clade with A.subflavus DQS3-9A1T and H.altamirensis DSM 45258T. The levels of DNA-DNA relatedness with A.subflavus DSM 45089T and H.altamirensis DSM 45258T were 39.7±3.9 % and 35.7±3.0 %, respectively. Combining the evidence from the polyphasic taxonomic study, strain J12GA03T represents a novel species of the genus Hoyosella, for which the name Hoyosella rhizosphaerae sp. nov. is proposed. The type strain is J12GA03T (=DSM 101985T=CGMCC 1.15478T).

Exploring the cellulolytic activity of environmental mycobacteria.

Although studies on non-tuberculous mycobacteria have increased in recent years because they cause a considerable proportion of infections, their cellulolytic system is still poorly studied. This study presents a characterization of the cellulolytic activities of environmental mycobacterial isolates derived from soil and water samples from the central region of Argentina, aimed to evaluate the conservation of the mechanism for the degradation of cellulose in this group of bacteria. The molecular and genomic identification revealed identity with Mycolicibacterium septicum. The endoglucanase and total cellulase activities were assessed both qualitatively and quantitatively and the optimal enzymatic conditions were characterized. A specific protein of around 56 kDa with cellulolytic activity was detected in a zymogram. Protein sequences possibly arising from a cellulase were identified by mass spectrometry-based shotgun proteomics. Results showed that M. septicum encodes for cellulose- and hemicellulose-related degrading enzymes, including at least an active ß-1,4 endoglucanase enzyme that could be useful to improve its survival in the environment. Given the important health issues related to mycobacteria, the results of the present study may contribute to the knowledge of their cellulolytic system, which could be important for their ability to survive in many different types of environments.