The genome of Mycobacterium tuberculosis harbors four copies of a cluster of genes termed mce operons. Despite extensive research that has demonstrated the importance of these operons on infection outcome, their physiological function remains obscure. Expanding databases of complete microbial genome sequences facilitate a comparative genomic approach that can provide valuable insight into the role of uncharacterized proteins.

In the beginning of the twenty-first century, humanity faces great challenges regarding diseases and health-related quality of life. A drastic rise in bacterial antibiotic resistance, in the number of cancer patients, in the obesity epidemics and in chronic diseases due to life expectation extension are some of these challenges. The discovery of novel therapeutics is fundamental and it may come from underexplored environments, like marine habitats, and microbial origin. Actinobacteria are well-known as treasure chests for the discovery of novel natural compounds. In this study, eighteen Actinomycetales isolated from marine sponges of three Erylus genera collected in Portuguese waters were tested for bioactivities with the main goal of isolating and characterizing the responsible bioactive metabolites. The screening comprehended antimicrobial, anti-fungal, anti-parasitic, anti-cancer and anti-obesity properties. Fermentations of the selected strains were prepared using ten different culturing media. Several bioactivities against the fungus Aspergillus fumigatus, the bacteria Staphylococcus aureus methicillin-resistant (MRSA) and the human liver cancer cell line HepG2 were obtained in small volume cultures. Screening in higher volumes showed consistent anti-fungal activity by strain Dermacoccus sp. #91-17 and Micrococcus luteus Berg02-26. Gordonia sp. Berg02-22.2 showed anti-parasitic (Trypanosoma cruzi) and anti-cancer activity against several cell lines (melanoma A2058, liver HepG2, colon HT29, breast MCF7 and pancreatic MiaPaca). For the anti-obesity assay, Microbacterium foliorum #91-29 and #91-40 induced lipid reduction on the larvae of zebrafish (Danio rerio). Dereplication of the extracts from several bacteria showed the existence of a variety of secondary metabolites, with some undiscovered molecules. This work showed that Actinomycetales are indeed good candidates for drug discovery.

Phosphopantetheinyl transferases (PPTases) are a superfamily of essential enzymes required for the synthetic processes of many compounds including fatty acid, polyketide, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways via the transfer of a phosphopantetheinyl moiety to a serine residue in the conserved motif of carrier proteins. Since many Actinomycetales microorganisms produce a number of polyketide and nonribosomal peptide metabolites, the distribution of PPTase genes was investigated in these microorganisms. PPTases were found in bacterial protein databases using a hidden Markov model search with the PF01648 (4'-phosphopantetheinyl transferase superfamily) model. Actinomycetales microorganisms harbor several genes encoding AcpS-type and Sfp-type PPTases in individual genomes, many of which were associated with the biosynthetic gene cluster for polyketide or nonribosomal peptide metabolites. The properties of these PPTases were evaluated in the heterologous expression system using the biosynthetic gene clusters and genes encoding PPTases found in the present study. Sfp-type PPTases were classified into two subgroups, and although the substrate specificities of the enzymes in one subgroup were wide, the catalytic activities of enzymes in the other subgroup were low. SAV_1784 of Streptomyces avermitilis possessed the most characteristic broad-range activity against several type I polyketide synthases and nonribosomal peptide synthetases.

Bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we found that C. glutamicum cells are at least diploid under all of the conditions tested and that these organisms have overlapping C periods during replication, with both origins initiating replication simultaneously. On the basis of experimental data, we propose growth rate-dependent cell cycle models for C. glutamicumIMPORTANCE Bacterial cell cycles are known for few model organisms and can vary significantly between species. Here, we studied the cell cycle of Corynebacterium glutamicum, an emerging cell biological model organism for mycolic acid-containing bacteria, including mycobacteria. Our data suggest that C. glutamicum carries two pole-attached chromosomes that replicate with overlapping C periods, thus initiating a new round of DNA replication before the previous one is terminated. The newly replicated origins segregate to midcell positions, where cell division occurs between the two new origins. Even after long starvation or under extremely slow-growth conditions, C. glutamicum cells are at least diploid, likely as an adaptation to environmental stress that may cause DNA damage. The cell cycle of C. glutamicum combines features of slow-growing organisms, such as polar origin localization, and fast-growing organisms, such as overlapping C periods.

Actinomycetes are a heterogeneous group of gram positive filamentous bacteria that have been found to produce a wide range of valuable bioactive secondary metabolites, particularly antibiotics. Moreover, actinomycetes isolated from unexplored environments show an unprecedented potential to generate novel active compounds. Hence, in order to search for novel antibiotics, we isolated and characterized actinomycetes strains from plant samples collected from a mangrove in Macau. Within the class of actinobacteria, fourteen actinomycetes isolates have been isolated and identified belonging to the genus of Streptomyces, Micromonospora, Mycobacterium, Brevibacterium, Curtobacterium and Kineococcus based on their 16S rRNA sequences. Further whole genome sequencing analysis of one of the isolated Streptomyces sp., which presented 99.13% sequence similarity with Streptomyces parvulus strain 2297, showed that it consisted of 118 scaffolds, 8,348,559 base pairs and had a 72.28% G + C content. In addition, genome-mining revealed that the isolated Streptomyces sp. contains 109 gene clusters responsible for the biosynthesis of known and/or novel secondary metabolites, including different types of terpene, T1pks, T2pks, T3pks, Nrps, indole, siderophore, bacteriocin, thiopeptide, phosphonate, lanthipeptide, ectoine, butyrolactone, T3pks-Nrps, and T1pks-Nrps. Meanwhile, the small molecules present in ethyl acetate extract of the fermentation broth of this strain were analyzed by LC-MS. Predicted secondary metabolites of melanin and desferrioxamine B were identified and both of them were firstly found to be produced by the Streptomyces parvulus strain. Our study highlights that combining genome mining is an efficient method to detect potentially promising natural products from mangrove-derived actinomycetes.

Purpose: Type 1 diabetes mellitus (T1DM) has dramatically increased in recent years, especially in young people, and limits the life quality of the patients involved. Thus, many researchers are performing extensive studies to find alternative treatments for DM. Methods: Here, we evaluated the improvement effects of the heat-killed Actinomycetales species, including Gordonia bronchialis, and Tsukamurella inchonensis in streptozotocin (STZ)- diabetic rats by biochemical, immunological, and histopathological examinations. Results: The present findings exhibited a dramatic and progressive alteration in the serum levels of interleukin-6 (IL-6), IL-10 and tumor necrosis factor-α (TNF-α) in the diabetic group, which were related to the blood glucose and insulin levels, oxidative stress defense (evaluated by TAC and MDA activities), and the pancreas biochemical indicators (such as amylase and lipase). More importantly, the present results were consistent with the histopathological findings, which included cellular degeneration, vascular congestion, hemorrhage, focal necrosis associated with mononuclear cell infiltration. Interestingly, all of the diabetic changes in the blood serum and tissues improved remarkably in the treated groups by Actinomycetales species. Conclusion: Surprisingly, most of the current diabetic complications effectively attenuated after oral administration of both Actinomycetales species, particularly with a high dose of T. inchonensis. Thus, it is concluded that the heat-killed Actinomycetales species can prevent and improve the progression of T1DM and its various complications profoundly.

Recent metagenomic studies have revealed that microbial diversity in the atmosphere rivals that of surface environments. This indicates that the atmosphere may be worth bioprospecting in for novel microorganisms, especially those selected for by harsh atmospheric conditions. This is interesting in light of the antibiotic resistance crisis and renewed interests in bioprospecting for members of the Actinomycetales, which harbor novel secondary metabolite-producing pathways and produce spores that make them well suited for atmospheric travel. The latter leads to the hypothesis that the atmosphere may be a promising environment in which to search for novel Actinomycetales. Although ubiquitous in soils, where bioprospecting efforts for Actinomycetales have been and are largely still focused, we present novel data indicating that culturable members of this taxonomic order are 3-5.6 times more abundant in air samples collected at 1.5, 4.5, 7.5, and 18 m above the ground, than in the underlying soil. These results support the hypothesis that mining the vast and readily accessible lower atmosphere for novel Actinomycetales in the search for undescribed secondary metabolites could prove fruitful.

Anti-virulence agents are non-bacteriostatic and non-bactericidal emerging therapeutic options which hamper the production of virulence factors in pathogenic flora. In Staphylococcus aureus and Enterococcus faecalis, regulation of virulence genes' expression occurs through the cyclic peptide-mediated accessory gene regulator (agr) and its ortholog fsr quorum sensing systems, respectively. In the present study, we screened a set of 54 actinomycetales secondary metabolites as novel anti-virulence compounds targeting quorum sensing system of the Gram-positive bacteria. The results indicated that four compounds, Phenalinolactones A-D, BU-4664LMe, 4,5-dehydrogeldamycin, and Questinomycin A, potentially inhibit the agr quorum sensing system and hemolytic activity of S. aureus. On the other hand, Decatromicin A and B, Okilactomycin, Rishirilide A, Abyssomicin I, and Rebeccamycin selectively blocked the fsr quorum sensing system and the gelatinase production in E. faecalis at sub-lethal concentrations. Interestingly, Synerazol uniquely showed the capability to inhibit both fsr and agr quorum sensing systems. Further, in silico molecular docking studies were performed which provided closer insights into the mode of action of these compounds and proposed that the inhibitory activity of these compounds could be attributed to their potential ability to bind to the ATP-active site of S. aureus AgrA. Taken together, our study highlights the potential of actinomycetales secondary metabolites with diverse structures as anti-virulence quorum sensing inhibitors.

Kutznerides 2 and 8 of the cyclic hexadepsipeptide family of antifungal natural products from the soil actinomycete Kutzneria sp. 744 contain two sets of chlorinated residues, a 6,7-dichlorohexahydropyrroloindole moiety derived from dichlorotryptophan and a 5-chloropiperazate moiety, as well as a methylcyclopropylglycine residue that may arise from isoleucine via a cryptic chlorination pathway. Previous studies identified KtzD, KtzQ, and KtzR as three halogenases in the kutzneride pathway but left no candidate for installing the C5 chlorine on piperazate. On the basis of analysis of the complete genome sequence of Kutzneria, we now identify a fourth halogenase in the pathway whose gene is separated from the defined kutzneride cluster by 12 open reading frames. KthP (kutzneride halogenase for piperazate) is a mononuclear nonheme iron halogenase that acts on the piperazyl ring tethered by a thioester linkage to the holo forms of thiolation domains. MS analysis of the protein-bound product confirmed chlorination of the piperazate framework from the (3S)- but not the (3R)-piperazyl-S-pantetheinyl thiolation proteins. After thioesterase-mediated release, nuclear magnetic resonance was used to assign the free imino acid as (3S,5S)-5-chloropiperazate, distinct from the 3S,5R stereoisomer reported in the mature kutznerides. These results demonstrate that a fourth halogenase, KthP, is active in the kutzneride biosynthetic pathway and suggest further processing of the (3S,5S)-5-chloropiperazate during subsequent incorporation into the kutzneride depsipeptide frameworks.

Lung transplant recipients have an increased risk for actinomycetales infection secondary to immunosuppressive regimen.

The complete genome sequences of two highly arsenite-resistant Actinomycetales isolates are presented. Both genomes are G+C rich and consist of a single chromosome containing homologs of known arsenite resistance genes.

The human gastrointestinal (GI) tract microbiota is characterised by an abundance of uncultured bacteria most often assigned in phyla Firmicutes and Bacteroidetes. Diversity of this microbiota, even though approached with culture independent techniques in several studies, still requires more elucidation. The main purpose of this work was to study whether the genomic percent guanine and cytosine (%G+C) -based profiling and fractioning prior to 16S rRNA gene sequence analysis reveal higher microbiota diversity, especially with high G+C bacteria suggested to be underrepresented in previous studies.

Cellulosimicrobium species, formely known as Oerskovia species, are gram-positive bacilli belonging to the order Actinomycetales. They rarely cause human infections. The genus comprises two pathogenic species in humans: C. cellulans and C. funkei. Based on a case report, we provide a review of the literature of infections caused by Cellulosimicrobium/Oerskovia, in order to improve our knowledge of this unusual infection.

The pSETT4 vector integrates into the Actinoplanes sp. SE50/110 chromosome via the bacteriophage φC31 integrase and allows cloning of a gene of interest by Golden Gate assembly (BsaI). T4 terminators surround the expression cassette to isolate the transcriptional unit and to prevent antisense transcription. The system can be used in other Actinomycetales by exchanging the promoter.

Actinomyces and Mycobacterium avium-intracellulare are facultative intracellular organisms, members of the bacterial order actinomycetales. Although Actinomyces can behave as copathogen when anatomic barriers are compromised, its coinfection with Mycobacterium avium-intracellulare has not previously been reported. We present the first reported case of palatal actinomycosis co-infection with disseminated MAC, in an HIV-infected subject with Kaposi sarcoma and diabetes. We discuss the pathogenesis of the complex condition of this subject.

Streptomyces spp. are aerobic, Gram-positive bacteria of the order Actinomycetales, known for their ability to produce antimicrobial molecules such as streptomycin. Pneumonia due to Streptomyces is considered to be rare and limited to immunocompromised patients. Streptomyces spp. are only rarely associated with invasive systemic infections. To our knowledge, we report the first documented case of community-acquired Streptomyces atratus bacteremic pneumonia in an immunocompetent patient.

Functional characterization of genes in important pathogenic bacteria such as Mycobacterium tuberculosis is imperative. Rv2135c, which was originally annotated as conserved hypothetical, has been found to be associated with membrane protein fractions of H37Rv strain. The gene appears to contain histidine phosphatase motif common to both cofactor-dependent phosphoglycerate mutases and acid phosphatases in the histidine phosphatase superfamily. The functions of many of the members of this superfamily are annotated based only on similarity to known proteins using automatic annotation systems, which can be erroneous. In addition, the motif at the N-terminal of Rv2135c is 'RHA' unlike 'RHG' found in most members of histidine phosphatase superfamily. These necessitate the need for its experimental characterization. The crystal structure of Rv0489, another member of the histidine phosphatase superfamily in M. tuberculosis, has been previously reported. However, its biochemical characteristics remain unknown. In this study, Rv2135c and Rv0489 from M. tuberculosis were cloned and expressed in Escherichia coli with 6 histidine residues tagged at the C terminal.

Endolithic microbial communities survive nutrient and energy deficient conditions while contributing to the weathering of their mineral substrate. This study examined the mineral composition and microbial communities of fully serpentinized weathered rock from 0.1 to 6.5 m depth at a site within the Khalilovsky massif, Orenburg Region, Southern Ural Mountains, Russia. The mineral composition includes a major content of serpentinite family (mostly consisting of lizardite and chrysotile), magnesium hydrocarbonates (hydromagnesite with lesser amounts of hydrotalcite and pyroaurite) concentrated in the upper layers, and clay minerals. We found that the deep-seated weathered serpentinites are chrysotile-type minerals, while the middle and surface serpentinites mostly consist of lizardite and chrysotile types. Microbial community analysis, based on 16S rRNA gene sequencing, showed a similar diversity of phyla throughout the depth profile. The dominant bacterial phyla were the Actinobacteria (of which unclassified genera in the orders Acidimicrobiales and Actinomycetales were most numerous), Chloroflexi (dominated by an uncultured P2-11E order) and the Proteobacteria (predominantly class Betaproteobacteria). Densities of several groups of bacteria were negatively correlated with depth. Occurrence of the orders Actinomycetales, Gaiellales, Solirubrobacterales, Rhizobiales and Burkholderiales were positively correlated with depth. Our findings show that endolithic microbial communities of the Khalilovsky massif have similar diversity to those of serpentine soils and rocks, but are substantially different from those of the aqueous environments of actively serpentinizing systems.

Strain Marseille-P3248т is a new species from the order Actinomycetales that was isolated from the urine sample of a girl aged 20 months with rotavirus gastroenteritis. It is a facultative anaerobic Gram-positive rod-shaped bacterium. Strain Marseille-P3248т exhibits 94.73% sequence similarity with Arcanobacterium pluranimalium strain M430/94/2, a phylogenetically related species with standing in nomenclature. Its genome size is 1 667 964 bp with 49.1% G + C content. Strain Marseille-P3248т (= CSURP3248) is the type strain of the new species Arcanobacterium urinimassiliense sp. nov.

Corynebacterium glutamicum is a high-GC Gram-positive soil bacterium of great biotechnological importance for the production of amino acids. To facilitate the rational design of sulphur amino acid-producing strains, the pathway for assimilatory sulphate reduction providing the necessary reduced sulfur moieties has to be known. Although this pathway has been well studied in Gram-negative bacteria like Escherichia coli and low-GC Gram-positives like Bacillus subtilis, little is known for the Actinomycetales and other high-GC Gram-positive bacteria.