RESUMO
In many gram-positive Actinobacteria, including Actinomyces oris and Corynebacterium matruchotii, the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in Corynebacterium diphtheriae, the deletion of mdbA blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontaneous thermotolerant revertants of the mdbA mutant at 37 °C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of tsdA, causing its elevated expression. Strikingly, increased expression of tsdA-via suppressor mutations or a constitutive promoter-rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of mdbA. Structural, genetic, and biochemical analyses demonstrated TsdA is a membrane-tethered thiol-disulfide oxidoreductase with a conserved CxxC motif that can substitute for MdbA in mediating oxidative folding of pilin and toxin substrates. Together with our observation that tsdA expression is upregulated at nonpermissive temperature (40 °C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in C. diphtheriae against thermal stress.
Assuntos
Proteínas de Bactérias , Corynebacterium diphtheriae , Proteína Dissulfeto Redutase (Glutationa) , Dobramento de Proteína , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corynebacterium diphtheriae/enzimologia , Corynebacterium diphtheriae/genética , Estresse Oxidativo , Proteína Dissulfeto Redutase (Glutationa)/genética , Proteína Dissulfeto Redutase (Glutationa)/metabolismoRESUMO
DNA replication fidelity in Streptomyces bacteria, prolific producers of many medically important secondary metabolites, is understudied, while in Escherichia coli it is controlled by DnaQ, the ϵ subunit of DNA polymerase III (DNA PolIII). Manipulation of dnaQ paralogues in Streptomyces lividans TK24, did not lead to increased spontaneous mutagenesis in this bacterium suggesting that S. lividans DNA PolIII uses an alternative exonuclease activity for proofreading. In Mycobacterium tuberculosis, such activity is attributed to the DnaE protein representing α subunit of DNA PolIII. Eight DnaE mutants designed based on the literature data were overexpressed in S. lividans, and recombinant strains overexpressing two of these mutants displayed markedly increased frequency of spontaneous mutagenesis (up to 1000-fold higher compared to the control). One of these 'mutators' was combined in S. lividans with a biosensor specific for antibiotic coelimycin, which biosynthetic gene cluster is present but not expressed in this strain. Colonies giving a positive biosensor signal appeared at a frequency of ca 10-5, and all of them were found to produce coelimycin congeners. This result confirmed that our approach can be applied for chemical- and radiation-free mutagenesis in Streptomyces leading to activation of orphan biosynthetic gene clusters and discovery of novel bioactive secondary metabolites.
Assuntos
Técnicas Biossensoriais , DNA Polimerase III/genética , Replicação do DNA/genética , Proteínas de Escherichia coli/genética , Antibacterianos/química , Antibacterianos/isolamento & purificação , DNA/química , DNA Polimerase III/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Regulação Enzimológica da Expressão Gênica/genética , Inativação Gênica , Mycobacterium tuberculosis , Streptomyces/enzimologiaRESUMO
Bacterial specialized metabolites are increasingly recognized as important factors in animal-microbiome interactions: for example, by providing the host with chemical defenses. Even in chemically rich animals, such compounds have been found to originate from individual members of more diverse microbiomes. Here, we identified a remarkable case of a moderately complex microbiome in the sponge host Mycale hentscheli in which multiple symbionts jointly generate chemical diversity. In addition to bacterial pathways for three distinct polyketide families comprising microtubule-inhibiting peloruside drug candidates, mycalamide-type contact poisons, and the eukaryotic translation-inhibiting pateamines, we identified extensive biosynthetic potential distributed among a broad phylogenetic range of bacteria. Biochemical data on one of the orphan pathways suggest a previously unknown member of the rare polytheonamide-type cytotoxin family as its product. Other than supporting a scenario of cooperative symbiosis based on bacterial metabolites, the data provide a rationale for the chemical variability of M. hentscheli and could pave the way toward biotechnological peloruside production. Most bacterial lineages in the compositionally unusual sponge microbiome were not known to synthesize bioactive metabolites, supporting the concept that microbial dark matter harbors diverse producer taxa with as yet unrecognized drug discovery potential.
Assuntos
Bactérias/metabolismo , Microbiota/fisiologia , Poríferos/microbiologia , Animais , Citotoxinas/metabolismo , Genoma Bacteriano , SimbioseRESUMO
Endophytic fungi have been recognized as prolific producers of chemically diverse secondary metabolites. In this work, we describe a new representative of the order Helotiales isolated from the medicinal plant Bergenia pacumbis. Several bioactive secondary metabolites were produced by this Helotiales sp. BL 73 isolate grown on rice medium, including cochlioquinones and isofusidienols. Sequencing and analysis of the approximately 59-Mb genome revealed at least 77 secondary metabolite biosynthesis gene clusters, of which several could be associated with detected compounds or linked to previously reported molecules. Four terpene synthase genes identified in the BL73 genome were codon optimized and expressed, together with farnesyl-, geranyl-, and geranylgeranyl-pyrophosphate synthases, in Streptomyces spp. An analysis of recombinant strains revealed the production of linalool and its oxidized form, terpenoids typically associated with plants, as well as a yet unidentified terpenoid. This study demonstrates the importance of a complex approach to the investigation of the biosynthetic potential of endophytic fungi using both conventional methods and genome mining. IMPORTANCE Endophytic fungi represent an as yet underexplored source of secondary metabolites, of which some may have industrial and medical applications. We isolated a slow-growing fungus belonging to the order Helotiales from the traditional medicinal plant Bergenia pacumbis and characterized its potential to biosynthesize secondary metabolites. We used cultivation of the isolate with a subsequent analysis of compounds produced, bioinformatics-based mining of the genome, and heterologous expression of several terpene synthase genes. Our study revealed that this Helotiales isolate has enormous potential to produce structurally diverse natural products, including polyketides, nonribosomally synthesized peptides, terpenoids, and ribosomally synthesized and posttranslationally modified peptides (RiPPs). Identification of meroterpenoids and xanthones, along with establishing a link between these molecules and their putative biosynthetic genes, sets the stage for investigation of the respective biosynthetic pathways. The heterologous production of terpenoids suggests that this approach can be used for the discovery of new compounds belonging to this chemical class using Streptomyces bacteria as hosts.
Assuntos
Ascomicetos , Streptomyces , Ascomicetos/genética , Vias Biossintéticas/genética , Família Multigênica , Metabolismo Secundário , Streptomyces/genéticaRESUMO
Bacterial symbionts of marine invertebrates are rich sources of novel, pharmaceutically relevant natural products that could become leads in combatting multidrug-resistant pathogens and treating disease. In this study, the bioactive potential of the marine invertebrate symbiont Thalassomonas actiniarum was investigated. Bioactivity screening of the strain revealed Gram-positive specific antibacterial activity as well as cytotoxic activity against a human melanoma cell line (A2058). The dereplication of the active fraction using HPLC-MS led to the isolation and structural elucidation of cholic acid and 3-oxo cholic acid. T. actiniarum is one of three type species belonging to the genus Thalassomonas. The ability to generate cholic acid was assessed for all three species using thin-layer chromatography and was confirmed by LC-MS. The re-sequencing of all three Thalassomonas type species using long-read Oxford Nanopore Technology (ONT) and Illumina data produced complete genomes, enabling the bioinformatic assessment of the ability of the strains to produce cholic acid. Although a complete biosynthetic pathway for cholic acid synthesis in this genus could not be determined based on sequence-based homology searches, the identification of putative penicillin or homoserine lactone acylases in all three species suggests a mechanism for the hydrolysis of conjugated bile acids present in the growth medium, resulting in the generation of cholic acid and 3-oxo cholic acid. With little known currently about the bioactivities of this genus, this study serves as the foundation for future investigations into their bioactive potential as well as the potential ecological role of bile acid transformation, sterol modification and quorum quenching by Thalassomonas sp. in the marine environment.
Assuntos
Antibacterianos , Humanos , Ácido Cólico , Filogenia , DNA Bacteriano , Antibacterianos/farmacologia , Análise de Sequência de DNARESUMO
Benzoxazolinate is a rare bis-heterocyclic moiety that interacts with proteins and DNA and confers extraordinary bioactivities on natural products, such as C-1027. However, the biosynthetic gene responsible for the key cyclization step of benzoxazolinate remains unclear. Herein, we show a putative acyl AMP-ligase responsible for the last cyclization step. We used the enzyme as a probe for genome mining and discovered that the orphan benzobactin gene cluster in entomopathogenic bacteria prevails across Proteobacteria and Firmicutes. It turns out that Pseudomonas chlororaphis produces various benzobactins, whose biosynthesis is highlighted by a synergistic effect of two unclustered genes encoding enzymes on boosting benzobactin production; the formation of non-proteinogenic 2-hydroxymethylserine by a serine hydroxymethyltransferase; and the types I and II NRPS architecture for structural diversity. Our findings reveal the biosynthetic potential of a widespread benzobactin gene cluster.
Assuntos
Produtos Biológicos , Produtos Biológicos/metabolismo , Bactérias/metabolismo , Família Multigênica , Peptídeo Sintases/metabolismoRESUMO
BACKGROUND: Magnetosome formation in the alphaproteobacterium Magnetospirillum gryphiswaldense is controlled by more than 30 known mam and mms genes clustered within a large genomic region, the 'magnetosome island' (MAI), which also harbors numerous mobile genetic elements, repeats, and genetic junk. Because of the inherent genetic instability of the MAI caused by neighboring gene content, the elimination of these regions and their substitution by a compact, minimal magnetosome expression cassette would be important for future analysis and engineering. In addition, the role of the MAI boundaries and adjacent regions are still unclear, and recent studies indicated that further auxiliary determinants for magnetosome biosynthesis are encoded outside the MAI. However, techniques for large-scale genome editing of magnetic bacteria are still limited, and the full complement of genes controlling magnetosome formation has remained uncertain. RESULTS: Here we demonstrate that an allelic replacement method based on homologous recombination can be applied for large-scale genome editing in M. gryphiswaldense. By analysis of 24 deletion mutants covering about 167 kb of non-redundant genome content, we identified genes and regions inside and outside the MAI irrelevant for magnetosome biosynthesis. A contiguous stretch of ~ 100 kb, including the scattered mam and mms6 operons, could be functionally substituted by a compact and contiguous ~ 38 kb cassette comprising all essential biosynthetic gene clusters, but devoid of interspersing irrelevant or problematic gene content. CONCLUSIONS: Our results further delineate the genetic complement for magnetosome biosynthesis and will be useful for future large-scale genome editing and genetic engineering of magnetosome biosynthesis.
Assuntos
Genoma Bacteriano , Magnetossomos/metabolismo , Magnetospirillum/genética , Magnetospirillum/metabolismo , Família Multigênica , Genes Bacterianos , Genômica , Mutação , ÓperonRESUMO
Owing to the increasing demand for amino acids and valuable commodities that can be produced by Corynebacterium glutamicum, there is a pressing need for new rapid genome engineering tools that improve the speed and efficiency of genomic insertions, deletions, and mutations. Recombineering using the λ Red system in Escherichia coli has proven very successful at genetically modifying this organism in a quick and efficient manner, suggesting that optimizing a recombineering system for C. glutamicum will also improve the speed for genomic modifications. Here, we maximized the recombineering efficiency in C. glutamicum by testing the efficacy of seven different recombinase/exonuclease pairs for integrating single-stranded DNA and double-stranded DNA (dsDNA) into the genome. By optimizing the homologous arm length and the amount of dsDNA transformed, as well as eliminating codon bias, a dsDNA recombineering efficiency of 13,250 transformed colonies/109 viable cells was achieved, the highest efficiency currently reported in the literature. Using this optimized system, over 40,000 bp could be deleted in one transformation step. This recombineering strategy will greatly improve the speed of genetic modifications in C. glutamicum and assist other systems, such as clustered regularly interspaced short palindromic repeats and multiplexed automated genome engineering, in improving targeted genome editing.
Assuntos
Corynebacterium glutamicum/genética , Engenharia Genética , DNA de Cadeia Simples/genética , Exonucleases/genética , Edição de Genes , Engenharia Genética/métodos , Microrganismos Geneticamente Modificados , Recombinases/genéticaRESUMO
Actinobacteria provide a rich spectrum of bioactive natural products and therefore display an invaluable source towards commercially valuable pharmaceuticals and agrochemicals. Here, we studied the use of inorganic talc microparticles (hydrous magnesium silicate, 3MgO·4SiO2 ·H2 O, 10 µm) as a general supplement to enhance natural product formation in this important class of bacteria. Added to cultures of recombinant Streptomyces lividans, talc enhanced production of the macrocyclic peptide antibiotic bottromycin A2 and its methylated derivative Met-bottromycin A2 up to 109 mg L-1 , the highest titer reported so far. Hereby, the microparticles fundamentally affected metabolism. With 10 g L-1 talc, S. lividans grew to 40% smaller pellets and, using RNA sequencing, revealed accelerated morphogenesis and aging, indicated by early upregulation of developmental regulator genes such as ssgA, ssgB, wblA, sigN, and bldN. Furthermore, the microparticles re-balanced the expression of individual bottromycin cluster genes, resulting in a higher macrocyclization efficiency at the level of BotAH and correspondingly lower levels of non-cyclized shunt by-products, driving the production of mature bottromycin. Testing a variety of Streptomyces species, talc addition resulted in up to 13-fold higher titers for the RiPPs bottromycin and cinnamycin, the alkaloid undecylprodigiosin, the polyketide pamamycin, the tetracycline-type oxytetracycline, and the anthramycin-analogs usabamycins. Moreover, talc addition boosted production in other actinobacteria, outside of the genus of Streptomyces: vancomycin (Amycolatopsis japonicum DSM 44213), teicoplanin (Actinoplanes teichomyceticus ATCC 31121), and the angucyclinone-type antibiotic simocyclinone (Kitasatospora sp.). For teicoplanin, the microparticles were even crucial to activate production. Taken together, the use of talc was beneficial in 75% of all tested cases and optimized natural and heterologous hosts forming the substance of interest with clusters under native and synthetic control. Given its simplicity and broad benefits, microparticle-supplementation appears as an enabling technology in natural product research of these most important microbes.
Assuntos
Antibacterianos/biossíntese , Engenharia Metabólica , Microrganismos Geneticamente Modificados , Peptídeos Cíclicos , Streptomyces lividans , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/genética , Streptomyces lividans/genética , Streptomyces lividans/metabolismoRESUMO
A bacterial strain designated 26BT, which had been isolated from the cloaca of a toad-headed turtle, was subjected to a comprehensive taxonomic study. Comparison of 16S rRNA gene sequences demonstrated that strain 26BT is a member of the family Neisseriaceae. Based on highest similarity values, Neisseria animaloris DSM 21642T (95.15â%), Alysiella filiformis ATCC 15532T (95.06â%), Uruburuella testudinis 07_OD624T (94.71â%), Uruburuella suis CCUG 47806T (94.66â%) and Alysiella crassa DSM 2578T (94.64â%) were identified as the closest relatives. Average nucleotide identity values based on the blast algorithm (ANIb) indicated that U. suis (76.10/76.17â%), Neisseria shayeganii 871T (74.34/74.51â%), Stenoxybacter acetivorans (73.30/73.41â%), N. animaloris (72.98/72.80)â%, A. filiformis (71.14/71.21â%) and A. crassa (70.53/71.15â%) are the next closest relatives. Like ANIb, genome-based phylogeny did not suggest the affiliation of strain 26BT with any established genus. The polyamine pattern consisted of the major compounds putrescine, 1,3-diaminopropane and spermidine and the major quinone was ubiquinone Q-8. In the polar lipid profile, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an ornithine lipid were predominant. The fatty acid profile contained predominantly C16â:â1 ω7c, C12â:â0, C14â:â0, C16â:â0 and C12â:â0 3OH. The size of the genome was 2.91 Mbp and the genomic G+C content was 54.0 mol%. Since these data do not demonstrate an unambiguous association with any established genus, we here propose the novel genus Paralysiella with the type species Paralysiella testudinis gen. nov., sp. nov. The type strain is 26BT (=CCM 9137T=LMG 32212T).
Assuntos
Neisseriaceae/classificação , Filogenia , Tartarugas , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Cloaca/microbiologia , DNA Bacteriano/genética , Ácidos Graxos/química , Neisseriaceae/isolamento & purificação , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Tartarugas/microbiologiaRESUMO
Three novel corynebacterial species were isolated from soil sampled at a paddock in Vilsendorf, North Rhine-Westphalia, Germany. The strains were coccoid or irregular rod-shaped, catalase-positive and pale white to yellow-orange in colour. By whole genome sequencing and comparison of the 16S rRNA genes as well as the whole genome structure, it was shown that all three strains represent novel species of the family Corynebacteriaceae, order Corynebacteriales, class Actinobacteria. This project describes the isolation, identification, sequencing, and phenotypic characterization of the three novel Corynebacterium species. We propose the names Corynebacterium kalinowskii sp. nov. (DSM 110639T=LMG 31801T), Corynebacterium comes sp. nov. (DSM 110640T=LMG 31802T), and Corynebacterium occultum sp. nov. (DSM 110642T=LMG 31803T).
Assuntos
Corynebacterium , Filogenia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , Corynebacterium/classificação , Corynebacterium/isolamento & purificação , DNA Bacteriano/genética , Fazendas , Ácidos Graxos/química , Alemanha , Pigmentação , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
BACKGROUND: The demand for biobased polymers is increasing steadily worldwide. Microbial hosts for production of their monomeric precursors such as glutarate are developed. To meet the market demand, production hosts have to be improved constantly with respect to product titers and yields, but also shortening bioprocess duration is important. RESULTS: In this study, adaptive laboratory evolution was used to improve a C. glutamicum strain engineered for production of the C5-dicarboxylic acid glutarate by flux enforcement. Deletion of the L-glutamic acid dehydrogenase gene gdh coupled growth to glutarate production since two transaminases in the glutarate pathway are crucial for nitrogen assimilation. The hypothesis that strains selected for faster glutarate-coupled growth by adaptive laboratory evolution show improved glutarate production was tested. A serial dilution growth experiment allowed isolating faster growing mutants with growth rates increasing from 0.10 h-1 by the parental strain to 0.17 h-1 by the fastest mutant. Indeed, the fastest growing mutant produced glutarate with a twofold higher volumetric productivity of 0.18 g L-1 h-1 than the parental strain. Genome sequencing of the evolved strain revealed candidate mutations for improved production. Reverse genetic engineering revealed that an amino acid exchange in the large subunit of L-glutamic acid-2-oxoglutarate aminotransferase was causal for accelerated glutarate production and its beneficial effect was dependent on flux enforcement due to deletion of gdh. Performance of the evolved mutant was stable at the 2 L bioreactor-scale operated in batch and fed-batch mode in a mineral salts medium and reached a titer of 22.7 g L-1, a yield of 0.23 g g-1 and a volumetric productivity of 0.35 g L-1 h-1. Reactive extraction of glutarate directly from the fermentation broth was optimized leading to yields of 58% and 99% in the reactive extraction and reactive re-extraction step, respectively. The fermentation medium was adapted according to the downstream processing results. CONCLUSION: Flux enforcement to couple growth to operation of a product biosynthesis pathway provides a basis to select strains growing and producing faster by adaptive laboratory evolution. After identifying candidate mutations by genome sequencing causal mutations can be identified by reverse genetics. As exemplified here for glutarate production by C. glutamicum, this approach allowed deducing rational metabolic engineering strategies.
Assuntos
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Evolução Molecular Direcionada , Glutaratos/análise , Glutaratos/metabolismo , Engenharia Metabólica/métodos , Reatores Biológicos , Corynebacterium glutamicum/crescimento & desenvolvimento , Meios de Cultura , Fermentação , Análise do Fluxo Metabólico , Mutação , Genética ReversaRESUMO
BACKGROUND: Pamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis, to enhance pamamycin production in the heterologous host S. albus J1074/R2. RESULTS: Supplemented with L-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 3.5 mg L-1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA thioesters, the building blocks for the polyketide, resulting from L-valine catabolism. Unfavorably, L-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post L-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with L-valine, the novel strain enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663: almost 20-fold more than the wild type. CONCLUSIONS: Our findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.
Assuntos
Macrolídeos/metabolismo , Policetídeos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Fatores de Transcrição/genética , Valina/metabolismo , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , Proteínas de Bactérias/genética , Vias Biossintéticas , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Microbiologia Industrial , Metaboloma , Família Multigênica , MutaçãoRESUMO
BACKGROUND: Because of its tractability and straightforward cultivation, the magnetic bacterium Magnetospirillum gryphiswaldense has emerged as a model for the analysis of magnetosome biosynthesis and bioproduction. However, its future use as platform for synthetic biology and biotechnology will require methods for large-scale genome editing and streamlining. RESULTS: We established an approach for combinatory genome reduction and generated a library of strains in which up to 16 regions including large gene clusters, mobile genetic elements and phage-related genes were sequentially removed, equivalent to ~ 227.6 kb and nearly 5.5% of the genome. Finally, the fragmented genomic magnetosome island was replaced by a compact cassette comprising all key magnetosome biosynthetic gene clusters. The prospective 'chassis' revealed wild type-like cell growth and magnetosome biosynthesis under optimal conditions, as well as slightly improved resilience and increased genetic stability. CONCLUSION: We provide first proof-of-principle for the feasibility of multiple genome reduction and large-scale engineering of magnetotactic bacteria. The library of deletions will be valuable for turning M. gryphiswaldense into a microbial cell factory for synthetic biology and production of magnetic nanoparticles.
Assuntos
Deleção de Genes , Genoma Bacteriano , Magnetossomos , Magnetospirillum , Magnetossomos/genética , Magnetossomos/metabolismo , Magnetospirillum/genética , Magnetospirillum/metabolismoRESUMO
Strain M2T was isolated from the beach of Cuxhaven, Wadden Sea, Germany, in course of a program to attain new producers of bioactive natural products. Strain M2T produces litoralimycin and sulfomycin-type thiopeptides. Bioinformatic analysis revealed a potential biosynthetic gene cluster encoding for the M2T thiopeptides. The strain is Gram-stain-positive, rod shaped, non-motile, spore forming, showing a yellow colony color and forms extensively branched substrate mycelium and aerial hyphae. Inferred from the 16S rRNA gene phylogeny strain M2T affiliates with the genus Streptomonospora. It shows 96.6% 16S rRNA gene sequence similarity to the type species Streptomonospora salina DSM 44593 T and forms a distinct branch with Streptomonospora sediminis DSM 45723 T with 97.0% 16S rRNA gene sequence similarity. Genome-based phylogenetic analysis revealed that M2T is closely related to Streptomonospora alba YIM 90003 T with a digital DNA-DNA hybridisation (dDDH) value of 26.6%. The predominant menaquinones of M2T are MK-10(H6), MK-10(H8), and MK-11(H6) (> 10%). Major cellular fatty acids are iso-C16:0, anteiso C17:0 and C18:0 10-methyl. The polar lipid profile consisted of diphosphatidylglycerol phosphatidyl glycerol, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, three glycolipids, two unknown phospholipids, and two unknown lipids. The genome size of type strain M2T is 5,878,427 bp with 72.1 mol % G + C content. Based on the results obtained from phylogenetic and chemotaxonomic studies, strain M2T (= DSM 106425 T = NCCB 100650 T) is considered to represent a novel species within the genus Streptomonospora for which the name Streptomonospora litoralis sp. nov. is proposed.
Assuntos
Areia , Actinobacteria , DNA Bacteriano/genética , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
Protein secretion is a central biological process in all organisms. Most studies dissecting bacterial secretion mechanisms have focused on Gram-negative cell envelopes such as that of Escherichia coli However, proteomics analyses in Gram negatives is hampered by their outer membrane. Here we studied protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, in which most of the secretome is released in the growth medium. We monitored changes of the secretome as a function of growth phase and medium. We determined distinct protein classes of "house-keeping" secreted proteins that do not change their appearance or abundance in the various media and growth phases. These comprise mainly enzymes involved in cell wall maintenance and basic transport. In addition, we detected significant abundance and content changes to a sub-set of the proteome, as a function of growth in the different media. These did not depend on the media being minimal or rich. Transcriptional regulation but not changes in export machinery components can explain some of these changes. However, additional downstream mechanisms must be important for selective secretome funneling. These observations lay the foundations of using S. lividans as a model organism to study how metabolism is linked to optimal secretion and help develop rational optimization of heterologous protein production.
Assuntos
Proteínas de Bactérias/metabolismo , Meios de Cultura/análise , Proteômica/métodos , Streptomyces lividans/crescimento & desenvolvimento , Técnicas de Cultura Celular por Lotes , Reatores Biológicos/microbiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Essenciais , Modelos Biológicos , Streptomyces lividans/metabolismoRESUMO
Marine sponges are prolific sources of unique bioactive natural products. The sponge Theonella swinhoei is represented by several distinct variants with largely nonoverlapping chemistry. For the Japanese chemotype Y harboring diverse complex polyketides and peptides, we previously provided genomic and functional evidence that a single symbiont, the filamentous, multicellular organism "Candidatus Entotheonella factor," produces almost all of these compounds. To obtain further insights into the chemistry of "Entotheonella," we investigated another phylotype, "Candidatus Entotheonella serta," present in the T. swinhoei WA sponge chemotype, a source of theonellamide- and misakinolide-type compounds. Unexpectedly, considering the lower chemical diversity, sequencing of individual bacterial filaments revealed an even larger number of biosynthetic gene regions than for Ca E. factor, with virtually no overlap. These included genes for misakinolide and theonellamide biosynthesis, the latter assigned by comparative genomic and metabolic analysis of a T. swinhoei chemotype from Israel, and by biochemical studies. The data suggest that both compound families, which were among the earliest model substances to study bacterial producers in sponges, originate from the same bacterium in T. swinhoei WA. They also add evidence that metabolic richness and variability could be a more general feature of Entotheonella symbionts.
Assuntos
Fenômenos Fisiológicos Bacterianos , Simbiose , Theonella/microbiologia , Animais , Bactérias/química , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Genômica , Policetídeos/metabolismo , Theonella/química , Theonella/fisiologiaRESUMO
Artificial laboratory evolution was used to produce mutant strains of Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) able to survive on antimicrobial metallic copper surfaces. These mutants were 12- and 60-fold less susceptible to the copper-mediated contact killing process than their respective parent strains. Growth levels of the mutant and its parent in complex growth medium were similar. Tolerance to copper ions of the mutants was unchanged. The mutant phenotype remained stable over about 250 generations under nonstress conditions. The mutants and their respective parental strains accumulated copper released from the metallic surfaces to similar extents. Nevertheless, only the parental strains succumbed to copper stress when challenged on metallic copper surfaces, suffering complete destruction of the cell structure. Whole-genome sequencing and global transcriptome analysis were used to decipher the genetic alterations in the mutant strains; however, these results did not explain the copper-tolerance phenotypes on the systemic level. Instead, the mutants shared features with those of stressed bacterial subpopulations entering the early or "shallow" persister state. In contrast to the canonical persister state, however, the ability to survive on solid copper surfaces was adopted by the majority of the mutant strain population. This indicated that application of solid copper surfaces in hospitals and elsewhere has to be accompanied by strict cleaning regimens to keep the copper surfaces active and prevent evolution of tolerant mutant strains.IMPORTANCE Microbes are rapidly killed on solid copper surfaces by contact killing. Copper surfaces thus have an important role to play in preventing the spread of nosocomial infections. Bacteria adapt to challenging natural and clinical environments through evolutionary processes, for instance, by acquisition of beneficial spontaneous mutations. We wish to address the question of whether mutants can be selected that have evolved to survive contact killing on solid copper surfaces. We isolated such mutants from Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) by artificial laboratory evolution. The ability to survive on solid copper surfaces was a stable phenotype of the mutant population and not restricted to a small subpopulation. As a consequence, standard operation procedures with strict hygienic measures are extremely important to prevent the emergence and spread of copper-surface-tolerant persister-like bacterial strains if copper surfaces are to be sustainably used to limit the spread of pathogenic bacteria, e.g., to curb nosocomial infections.
Assuntos
Evolução Biológica , Cobre/farmacologia , Escherichia coli/genética , Staphylococcus aureus Resistente à Meticilina/genética , Seleção Genética , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacosRESUMO
Streptomyces spp. are a rich source for natural products with recognized industrial value, explaining the high interest to improve and streamline the performance of in these microbes. Here, we studied the production of pamamycins, macrodiolide homologs with a high activity against multiresistant pathogenic microbes, using recombinant Streptomyces albus J1074/R2. Talc particles (hydrous magnesium silicate, 3MgO·4SiO2 ·H2 O) of micrometer size, added to submerged cultures of the recombinant strain, tripled pamamycin production up to 50 mg/L. Furthermore, they strongly affected morphology, reduced the size of cell pellets formed by the filamentous microbe during the process up to sixfold, and shifted the pamamycin spectrum to larger derivatives. Integrated analysis of transcriptome and precursor (CoA thioester) supply of particle-enhanced and control cultures provided detailed insights into the underlying molecular changes. The microparticles affected the expression of 3,341 genes (56% of all genes), revealing a global and fundamental impact on metabolism. Morphology-associated genes, encoding major regulators such as SsgA, RelA, EshA, Factor C, as well as chaplins and rodlins, were found massively upregulated, indicating that the particles caused a substantially accelerated morphogenesis. In line, the pamamycin cluster was strongly upregulated (up to 1,024-fold). Furthermore, the microparticles perturbed genes encoding for CoA-ester metabolism, which were mainly activated. The altered expression resulted in changes in the availability of intracellular CoA-esters, the building blocks of pamamycin. Notably, the ratio between methylmalonyl CoA and malonyl-CoA was increased fourfold. Both metabolites compete for incorporation into pamamycin so that the altered availability explained the pronounced preference for larger derivatives in the microparticle-enhanced process. The novel insights into the behavior of S. albus in response to talc appears of general relevance to further explore and upgrade the concept of microparticle enhanced cultivation, widely used for filamentous microbes.
Assuntos
Macrolídeos/metabolismo , Engenharia Metabólica , Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Talco/química , Talco/farmacologiaRESUMO
A Gram-stain-positive bacterial isolate, designated LMM-1652T, was isolated from an intrauterine cytobrush sample originating from a postpartum Holstein Friesian dairy cow. The strain had a rod to coccoid-shape, was catalase-positive and oxidase-negative. 16S rRNA gene sequence similarity analyses revealed that its closest relatives were Corynebacterium falsenii (97.05â% similarity), Corynebacterium jeikeium (96.83â%) and Corynebacterium urealyticum (96.82â%). Subsequent whole genome analysis showed that the genome-to-genome distance of strain LMM-1652T to its closest relatives was in the range of 23.2-24.8 %, while the average nucleotide identity values ranged from 73.7 to 74.3%, thus confirming that this isolate represents a novel species. Strain LMM-1652T was characterized by a quinone system mainly consisting of MK-9(H2) and MK-10(H2). The polar lipids profile of the strain consisted mainly of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol-mannoside, as well as one unidentified lipid lacking any functional group. Smaller amounts of four unidentified phospholipids, four unidentified glycolipids, ß-gentiobiosyl diacylglycerol and four unidentified lipids lacking a functional group were also found. The cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan. The fatty acid profile was mainly composed of C18â:â1 ω9c, C18â:â0 and C16â:â0. We propose a novel species of the genus Corynebacterium with the name Corynebacterium urogenitale LMM-1652T (=LMG 31163T=DSM 108747T).