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1.
Artigo em Inglês | MEDLINE | ID: mdl-33608292

RESUMO

Bacterial proline-alanine-alanine-arginine (PAAR) proteins are located at the top of the type VI secretion system (T6SS) nanomachine and carry and deliver effectors into neighboring cells. Many PAAR proteins are fused with a variable C-terminal extended domain (CTD). Here, we report that two paar-ctd genes (MXAN_RS08765 and MXAN_RS36995) located in two homologous operons are involved in different ecological functions of Myxococcus xanthus MXAN_RS08765 inhibited the growth of plant pathogenic fungi, while MXAN_RS36995 was associated with the colony-merger incompatibility of M. xanthus cells. These two PAAR-CTD proteins were both toxic to Escherichia coli cells, while MXAN_RS08765, rather than MXAN_RS36995, was also toxic to Saccharomyces cerevisiae cells. Their downstream adjacent genes, i.e., MXAN_RS08760 and MXAN_RS24590, protected against the toxicities. The MXAN_RS36995 protein was demonstrated to have nuclease activity, and the activity was inhibited by the presence of MXAN_RS24590. Our results highlight that the PAAR proteins diversify the CTDs to play divergent roles in M. xanthus IMPORTANCE The type VI secretion system (T6SS) is a bacterial cell contact-dependent weapon capable of delivering protein effectors into neighboring cells. The PAAR protein is located at the top of the nanomachine and carries an effector for delivery. Many PAAR proteins are extended with a diverse C-terminal sequence with an unknown structure and function. Here, we report two paar-ctd genes located in two homologous operons involved in different ecological functions of Myxococcus xanthus: one has antifungal activity, and the other is associated with the self-identification phenotype. The PAAR-CTD proteins and the proteins encoded by their downstream genes form two toxin-immunity protein pairs. We demonstrated that the C-terminal diversification of the PAAR-CTD proteins enriches the ecological functions of bacterial cells.

2.
Comput Struct Biotechnol J ; 18: 3723-3733, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33304467

RESUMO

Toxic effectors secreted by the type VI secretion system (T6SS) facilitate interbacterial warfare, as well as pathogenesis toward humans, animals and plants. However, systematically predicting T6SS effectors remains challenging due to their sequence and functional diversity. In this study, we systematically identified putative T6SS toxic effectors in prokaryotic genomes on the basis of the observation that genes encoding adaptor proteins and genes encoding cognate effector proteins are generally adjacent in the genome. Adaptor proteins are mediators that help to load their cognate effectors onto the T6SS spike complex. The contextual genes of the known adaptor proteins (DUF1795, DUF2169 or DUF4123) all exhibited a high proportion of encoding T6SS spike complex protein (VgrG or PAAR) and effector proteins. On the basis of the genomic context, we found that PRK06147 might be a novel adaptor protein. These four adaptors are widely distributed among the bacterial genomes. From neighbors of 5297 adaptor genes, we identified 1356 putative effector genes from 92 different families, and two-thirds were currently annotated as hypothetical proteins or as having unknown functions. Our results indicate that each class of adaptors can be used as an effective marker to identify T6SS toxic effectors, moreover, this approach can promote the discovery of new effectors.

3.
Artigo em Inglês | MEDLINE | ID: mdl-33118924

RESUMO

A Gram-stain-negative, strictly aerobic, non-motile, orange-coloured bacterium, designated YR1-1T, was isolated from a soil sample collected from the Yellow River Delta wetlands (PR China). Growth was observed at a salinity of 1.0-15.0 % NaCl, 4-45 °C and pH 6.0-9.0. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that YR1-1T represented a member of the genus Psychroflexus, with the highest sequence similarity to Psychroflexus sediminis YIM-C238T (97.9 %), followed by Psychroflexus aestuariivivens (97.1 %) and Psychroflexus torquis (96.4 %). The average nucleotide identity and digital DNA-DNA hybridization values between YR1-1T and other closely related type strains of species of the genus Psychroflexus were 68.7-86.3% and 17.8-30.9 %. The genome of the strain was 2 899 374 bp in length with 39.8 % DNA G+C content. The predominant fatty acids (>10 %) were iso-C15 : 0 and anteiso-C15 : 0. The major respiratory quinone was menaquinone-6 (MK-6) and the major polar lipids were phosphatidylethanolamine, phospholipid, diphosphatidylglycerol, two unidentified aminolipids and four unidentified lipids. The combined genotypic and phenotypic data indicate that YR1-1T represents a novel species within the genus Psychroflexus, for which the name Psychroflexus aurantiacus sp. nov., is proposed. The type strain is YR1-1T (=KCTC 72794T=CGMCC 1.17458T).

4.
Nat Prod Rep ; 2020 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-32895676

RESUMO

Covering: up to 2020As a main bioactive component of the Chinese, Indian, and American Podophyllum species, the herbal medicine, podophyllotoxin (PTOX) exhibits broad spectrum pharmacological activity, such as superior antitumor activity and against multiple viruses. PTOX derivatives (PTOXs) could arrest the cell cycle, block the transitorily generated DNA/RNA breaks, and blunt the growth-stimulation by targeting topoisomerase II, tubulin, or insulin-like growth factor 1 receptor. Since 1983, etoposide (VP-16) is being used in frontline cancer therapy against various cancer types, such as small cell lung cancer and testicular cancer. Surprisingly, VP-16 (ClinicalTrials NTC04356690) was also redeveloped to treat the cytokine storm in coronavirus disease 2019 (COVID-19) in phase II in April 2020. The treatment aims at dampening the cytokine storm and is based on etoposide in the case of central nervous system. However, the initial version of PTOX was far from perfect. Almost all podophyllotoxin derivatives, including the FDA-approved drugs VP-16 and teniposide, were seriously limited in clinical therapy due to systemic toxicity, drug resistance, and low bioavailability. To meet this challenge, scientists have devoted continuous efforts to discover new candidate drugs and have developed drug strategies. This review focuses on the current clinical treatment of PTOXs and the prospective analysis for improving druggability in the rational design of new generation PTOX-derived drugs.

5.
Appl Microbiol Biotechnol ; 104(21): 9125-9134, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32940736

RESUMO

The macrolactone rapamycin (RAP) presents a broad range of bioactivities, but its clinical applications are compromised due to the poor water solubility and low bioavailability, which could probably be overcome by glycosylation. In this study, we tested a set of promiscuous glycosyltransferases (GTs) to modify rapamycin with four different sugar donors. BsGT-1 displayed the best glycosylation activity with a preference for UDP-glucose, and the glycosylation happened at C-28 or C-40 of rapamycin, producing rapamycin-40-O-ß-D-glucoside (RG1), and two new compounds rapamycin-28-O-ß-D-glucoside (RG2) and rapamycin-28,40-O-ß-D-diglucoside (RG3). The glycosylation remarkably improved water solubility and almost completely abolished cytotoxicity but simultaneously attenuated the antifungal, antitumor, and immunosuppression bioactivities of rapamycin. We found the glycosylation at C-40 had less effect on the bioactivities than that at C-28. The molecular docking analysis revealed that the glycosylation, especially the glycosylation at C-28, weakened the hydrophobic and hydrogen bonding contacts between the rapamycin glucosides and the binding proteins: the FK506-binding protein (FKBP12) and the FKBP12-rapamycin binding (FRB) domain. This study highlights a succinct approach to expand the chemical diversity of the therapeutically important molecule rapamycin by using promiscuous glycosyltransferases. Moreover, the fact that glycosyl moieties at different positions of rapamycin affect bioactivity to different extents inspires further glycosylation engineering to improve properties of rapamycin. KEY POINTS: • Rapamycin was glycosylated efficiently by some promiscuous GTs. • Glycosylation improved water solubility, attenuated cytotoxicity, and bioactivities. • Glycosylation affected the interactions between ligand and binding proteins.

6.
Int J Syst Evol Microbiol ; 70(10): 5373-5381, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32886596

RESUMO

A Gram-stain-negative, strictly aerobic, non-motile, rod-shaped bacterium, designated CWB-1T, was isolated from a haloalkaline lake sediment sample collected from the bottom of Chaiwopu Lake, Urumchi, Xinjiang Province, PR China. Strain CWB-1T grew at 4-40 °C (optimum, 30-35 °C), pH 6.5-9.0 (optimum, pH 6.5-7.0) and with 0.5-5.5 % (w/v) NaCl (optimum, 2.5-3.0 %). Phylogenetic analyses based on the 16S rRNA gene sequence and the whole genome sequence both revealed that strain CWB-1T belonged to the family Flavobacteriaceae. The strain had the highest similarity of the 16S rRNA gene sequence to Psychroserpens jangbogonensis PAMC 27130T (92.8 %). The genome of strain CWB-1T was 3 548 011 bp long with 36.3 % DNA G+C content. The predominant fatty acids (>10 %) in the CWB-1T cells were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 1 (iso-C15 : 1 H/C13 : 0 3-OH). The major respiratory quinone was menaquinone-6 and the major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and two unidentified lipids. Based on the phylogenetic analyses, as well as the phenotypic characteristics, a novel genus and species of the family Flavobacteriaceae, Paucihalobacter ruber gen. nov., sp. nov., is proposed. The type strain is CWB-1T (=KCTC 72450T=CGMCC 1.17149T).


Assuntos
Flavobacteriaceae/classificação , Sedimentos Geológicos/microbiologia , Lagos/microbiologia , Filogenia , Álcalis , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Ácidos Graxos/química , Flavobacteriaceae/isolamento & purificação , Concentração de Íons de Hidrogênio , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/química
7.
Int J Syst Evol Microbiol ; 70(9): 4993-5000, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32776869

RESUMO

Strain SDU3-2T was isolated from a soil sample collected in Shandong Province, PR China. Cells of SDU3-2T were spherical, Gram-stain-positive, aerobic and non-motile. Cellular growth of the strain occurred at 25-45 °C, pH 5.5-8.5 and with 0-1.5 % (w/v) of NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain SDU3-2T was closest to the type strain Deinococcus murrayi ALT-1bT with a similarity of 95.2 %. The draft genome was 3.49 Mbp long with 69.2 mol% G+C content. Strain SDU3-2T exhibited high resistance to gamma radiation (D10 >12 kGy) and UV (D10 >900 J m-2). The strain encoded many genes for resistance to radiation and oxidative stress, which were highly conserved with other Deinococcus species, but possessed interspecific properties. The major fatty acids of SDU3-2T cells were C15 : 1 ω6c, C16 : 1 ω7c/C16 : 1 ω6c, and C17 : 1 ω8c, the major menaquinone was menaquinone-8, and the major polar lipids were an unidentified phosphoglycolipid, four unidentified glycolipids and an unidentified phospholipid. The average nucleotide identity and DNA-DNA hybridization results further indicated that strain SDU3-2T represents a new species in the genus Deinococcus, for which the name Deinococcus terrestris sp. nov. is proposed. The type strain is SDU3-2T (=CGMCC 1.17147T=KCTC 43098T).


Assuntos
Deinococcus/classificação , Filogenia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Deinococcus/isolamento & purificação , Deinococcus/efeitos da radiação , Ácidos Graxos/química , Raios gama , Glicolipídeos/química , Hibridização de Ácido Nucleico , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Raios Ultravioleta , Vitamina K 2/análogos & derivados , Vitamina K 2/química
8.
ACS Synth Biol ; 9(8): 2009-2022, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32603592

RESUMO

Epothilones, as a new class of microtubule-stabilizing anticancer drugs, exhibit strong bioactivity against taxane-resistant cells and show clinical activity for the treatment of advanced breast cancer. Additionally, they also show great potential for a central nervous system injury and Alzheimer's disease. However, due to the long fermentation period of the original producer and challenges of genetic engineering of nonribosomal peptide/polyketide (NRP/PK) megasynthase genes, the application of epothilones is severely limited. Here, we addressed these problems by reassembling a novel 56-kb epothilone biosynthetic gene cluster, optimizing the promoter of each gene based on RNA-seq profiling, and completing precursor synthetic pathways in engineered Schlegella brevitalea. Furthermore, we debottlenecked the cell autolysis by optimizing culture conditions. Finally, the yield of epothilones in shake flasks was improved to 82 mg/L in six-day fermentation. Overall, we not only constructed epothilone overproducers for further drug development but also provided a rational strategy for high-level NRP/PK compound production.

9.
mSystems ; 5(3)2020 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518198

RESUMO

Vegetation represents probably the most crucial step for the ecosystem functions of wetlands, but it is unclear how microbial populations and functions shift along with vegetation. In this study, we found that the richness and diversity of soil bacteria increased with vegetation levels and that the community composition was distinctly shifted from bare to vegetative places. The bare land displayed an extremely high abundance of Cyanobacteria as a monospecies genus, while a Gemmatimonadetes genus was predominant as multiple species in all the vegetative wetlands, suggesting their important ecosystem functions and potential mechanisms. Expression of the genes related to photosynthesis was enriched exclusively in bare land. Genes involved in biological organic carbon metabolism and the cycling of main elements (C, N, S, and P) were highly expressed in vegetative wetlands and were mostly included in the metagenome-assembled genome (MAG) of Gemmatimonadetes Some compounds identified from soil metabolomic results also corresponded to pathways involving these key active genes. Cyanobacteria is thus responsible for the carbon sink in early infertile wetlands, and Gemmatimonadetes plays a crucial role in ecosystem functions in vegetative wetlands. Our results highlight that the soil microbial populations execute ecosystem functions for wetlands and that vegetation is the determinant for the population and functional shifts in the coastal estuarine wetland of the Yellow River Delta.IMPORTANCE Vegetation probably represents the most crucial step for the ecosystem functions of wetlands, but it is unclear how microbial populations and functions shift in pace with the colonization and succession of vegetation. In this study, we found that a Cyanobacteria monospecies genus and a Gemmatimonadetes multispecies genus are fastidiously predominant in the bare and vegetative wetlands of the Yellow River Delta, respectively. Consistently, photosynthesis genes were enriched exclusively in bare land, while genes involved in biological organic carbon metabolism and the cycling of main elements were highly expressed in vegetative wetlands, were mostly included in the MAG of Gemmatimonadetes, and were consistent with soil metabolomic results. Our results provide insight into the adaptive succession of predominant bacterial species and their ecosystem functions in response to the presence of vegetation.

10.
Front Microbiol ; 11: 140, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32117159

RESUMO

Myxococcus xanthus DK1622 has two RecA genes, recA1 (MXAN_1441) and recA2 (MXAN_1388), with unknown functional differentiation. Herein, we showed that both recA genes were induced by ultraviolet (UV) irradiation but that the induction of recA1 was more delayed than that of recA2. Deletion of recA1 did not affect the growth but significantly decreased the UV-radiation survival, homologous recombination (HR) ability, and induction of LexA-dependent SOS genes. In contrast, the deletion of recA2 markedly prolonged the lag phase of bacterial growth and increased the sensitivity to DNA damage caused by hydrogen peroxide but did not change the UV-radiation resistance or SOS gene inducibility. Protein activity analysis demonstrated that RecA1, but not RecA2, catalyzed DNA strand exchange (DSE) and LexA autocleavage in vitro. Transcriptomic analysis indicated that RecA2 has evolved mainly to regulate gene expression for cellular transportation and antioxidation. This is the first report of functional divergence of duplicated bacterial recA genes. The results highlight the evolutionary strategy of M. xanthus cells for DNA HR and genome sophistication.

11.
J Med Chem ; 63(6): 2877-2893, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32084316

RESUMO

As an FDA-approved drug, teniposide, was utilized in cancer treatment but was accompanied by a strong side effect in long-term clinical trials. This work discovered potential candidate drugs with low toxicity by modifying the molecule structure of teniposide through a structure-guided drug design approach. The IC50 value of novel 4,6-O-thenylidene-ß-d-glucopyranoside-(2″-acetamido, 3″-acetyl-di-S-5-fluorobenzothizole/5-fluorobenzoxazole)-4'-demethylepipodophyllotoxin (compounds 15 and 16) was 120.4-125.1 µM, which was significantly improved by around 10 times more than teniposide (11.5-22.3 µM) against healthy human cells (i.e., HL-7702, H8, MRC-5, and HMEC). In vivo studies demonstrated compounds 15 and 16 significantly suppressed the tumor growth in the HepG2 cell xenograft model without exhibiting obvious toxicity (LD50 values of 208.45 and 167.52 mg/kg), which was lower than that of teniposide (LD50 = 46.12 mg/kg). Compounds 15 and 16 caused mild γH2AX phosphorylation for low DNA toxicity and less inhibition of PI3K/Akt. Compounds 15 and 16 might be potential antitumor drugs with low toxicity.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Podofilotoxina/análogos & derivados , Teniposídeo/análogos & derivados , Teniposídeo/farmacologia , Animais , Antineoplásicos/toxicidade , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Células Hep G2 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/química , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/toxicidade , Podofilotoxina/química , Podofilotoxina/farmacologia , Podofilotoxina/toxicidade , Teniposídeo/toxicidade
12.
FEMS Microbiol Ecol ; 96(3)2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31917409

RESUMO

Many endogenous plasmids carry no noticeable benefits for their bacterial hosts, and the persistence of these 'cryptic plasmids' and their functional impacts are mostly unclear. In this study, we investigated these uncertainties using the social bacterium Myxococcus fulvus 124B02 and its endogenous plasmid pMF1. pMF1 possesses diverse genes that originated from myxobacteria, suggesting a longstanding co-existence of the plasmid with various myxobacterial species. The curing of pMF1 from 124B02 had almost no phenotypic effects on the host. Laboratory evolution experiments showed that the 124B02 strain retained pMF1 when subcultured on dead Escherichia coli cells but lost pMF1 when subcultured on living E. coli cells or on casitone medium; these results indicated that the persistence of pMF1 in 124B02 was environment-dependent. Curing pMF1 caused the mutant to lose the ability to predate and develop fruiting bodies more quickly than the pMF1-containing strain after they were subcultured on dead E. coli cells, which indicated that the presence of pMF1 in M. fulvus 124B02 has some long-term effects on its host. The results provide some new insights into the persistence and impacts of cryptic plasmids in their natural bacterial cells.


Assuntos
Myxococcus , Escherichia coli/genética , Myxococcus/genética , Plasmídeos/genética
13.
ACS Synth Biol ; 8(12): 2718-2725, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31774653

RESUMO

The 4-O-ß-d-glucopyranoside of DMEP ((-)-4'-desmethylepipodophyllotoxin) (GDMEP), a natural product from Podophyllum hexandrum, is the direct precursor to the topoisomerase inhibitor etoposide, used in dozens of chemotherapy regimens for various malignancies. The biosynthesis pathway for DMEP has been completed, while the enzyme for biosynthesizing GDMEP is still unclear. Here, we report the enzymatic O-glycosylation of DMEP with 53% conversion by exploring the substrate promiscuity and entrances of glycosyltransferases. Notably, we found 6 essential amino acid residues surrounding the putative substrate entrances exposed to the protein surface in UGT78D2, CsUGT78D2, and CsUGT78D2-like, and these residues may determine substrate specificity and high O-glycosylation activity toward DMEP. Our results provide an effective route for one-step synthesis of GDMEP. Identification of the key residues and entrances of glycosyltransferases will promote precise identification of glycosyltransferase biocatalysts for novel substrates and provide a rational basis for glycosyltransferase engineering.


Assuntos
Etoposídeo/metabolismo , Glicosiltransferases/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Arabidopsis/enzimologia , Biocatálise , Glicosilação , Glicosiltransferases/química , Filogenia , Podofilotoxina/química , Podofilotoxina/metabolismo , Especificidade por Substrato
14.
Cells ; 8(6)2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31163575

RESUMO

Two unrecognizable strains of the same bacterial species form a distinct colony boundary. During growth as colonies, Myxococcus xanthus uses multiple factors to establish cooperation between recognized strains and prevent interactions with unrecognized strains of the same species. Here, ΔMXAN_0049 is a mutant strain deficient in immunity for the paired nuclease gene, MXAN_0050, that has a function in the colony-merger incompatibility of Myxococcus xanthus DK1622. With the aim to investigate the factors involved in boundary formation, a proteome and metabolome study was employed. Visualization of the boundary between DK1622 and ΔMXAN_0049 was done scanning electron microscope (SEM), which displayed the presence of many damaged cells in the boundary. Proteome analysis of the DK1622- boundary disclosed many possible proteins, such as cold shock proteins, cell shape-determining protein MreC, along with a few pathways, such as RNA degradation, phenylalanine, tyrosine and tryptophan biosynthesis, and Type VI secretion system (T6SS), which may play major roles in the boundary formation. Metabolomics studies revealed various secondary metabolites that were significantly produced during boundary formation. Overall, the results concluded that multiple factors participated in the boundary formation in M. xanthus, leading to cellular damage that is helpful in solving the mystery of the boundary formation mechanism.


Assuntos
Metabolômica/métodos , Myxococcus xanthus/crescimento & desenvolvimento , Myxococcus xanthus/metabolismo , Proteômica/métodos , Sistemas de Secreção Bacterianos , Contagem de Colônia Microbiana , Regulação para Baixo , Viabilidade Microbiana , Myxococcus xanthus/ultraestrutura , Mapas de Interação de Proteínas , Proteoma/metabolismo , Metabolismo Secundário , Regulação para Cima
15.
Microb Biotechnol ; 12(4): 763-774, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31069998

RESUMO

Glycosylation of natural products can influence their pharmacological properties, and efficient glycosyltransferases (GTs) are critical for this purpose. The polyketide epothilones are potent anti-tumour compounds, and YjiC is the only reported GT for the glycosylation of epothilone. In this study, we phylogenetically analysed 8261 GTs deposited in CAZy database and revealed that YjiC locates in a subbranch of the Macrolide I group, forming the YjiC-subbranch with 160 GT sequences. We demonstrated that the YjiC-subbranch GTs are normally efficient in epothilone glycosylation, but some showed low glycosylation activities. Sequence alignment of YjiC-subbranch showed that the 66th and 77th amino acid residues, which were close to the catalytic cavity in molecular docking model, were conserved in five high-active GTs (Q66 and P77) but changed in two low-efficient GTs. Site-directed residues swapping at the two positions in the two low-active GTs (BssGT and BamGT) and the high-active GT BsGT-1 demonstrated that the two amino acid residues played an important role in the catalytic efficiency of epothilone glycosylation. This study highlights that the potent GTs for appointed compounds are phylogenetically grouped with conserved residues for the catalytic efficiency.


Assuntos
Epotilonas/metabolismo , Glicosiltransferases/metabolismo , Moduladores de Tubulina/metabolismo , Biotransformação , Domínio Catalítico , Sequência Conservada , Glicosilação , Glicosiltransferases/classificação , Glicosiltransferases/genética , Cinética , Simulação de Acoplamento Molecular , Filogenia , Alinhamento de Sequência
16.
Biochim Biophys Acta Gene Regul Mech ; 1861(10): 928-937, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30496038

RESUMO

Chaperonin groEL genes are duplicated in approximately 20% of bacteria, and the duplicates are differentially transcribed due to their divergent functions. The coordinated regulation of this differential transcription is as yet undetermined. In this study, we reported that the controlling inverted repeat of chaperone expression (CIRCE) element (the HrcA-binding site located upstream of the promoter) evolved for the transcriptional regulation of duplicate groELs. CIRCE composition and locations were found to be phylogenetically conserved in bacterial taxa. Myxococcus xanthus DK1622 has two CIRCE elements (CIRCE1groESL1 and CIRCE2groESL1) in the promoter region of groESL1 and one CIRCE element (CIRCEgroEL2) before groEL2. We also found that negative HrcA and positive ?32 regulators coordinated the transcription of duplicate groELs, and that the double deletion in DK1622 eliminated transcriptional differences and reduced the heat-shock responses of groELs. In vitro binding assays showed that HrcA protein binding was biased towards CIRCE1groESL1, followed by CIRCEgroEL2, but that HrcA proteins failed to bind with CIRCE2groESL1. Mutation experiments revealed that single-nucleotide mutations in the inverted repeat regions changed the HrcA-binding abilities of CIRCEs. We constructed an in vivo transcription-regulation system in Escherichia coli to pair each of the regulators with a groEL promoter. The results indicated that the transcriptional regulation performed by HrcA and ?32 was biased towards the groEL2 and groEL1 promoters, respectively. Based on promoter-sequence characteristics, we proposed a model of the coordinated regulation of the transcription of duplicate groELs in M. xanthus DK1622.


Assuntos
Proteínas de Bactérias/genética , Chaperonina 60/genética , Regulação Bacteriana da Expressão Gênica , Genes Duplicados , Regiões Promotoras Genéticas , Proteínas de Bactérias/biossíntese , Chaperonina 60/biossíntese , Proteínas de Choque Térmico/metabolismo , Myxococcus xanthus/genética , Filogenia , Proteínas Repressoras/metabolismo , Fator sigma/metabolismo , Transcrição Genética
17.
Biomolecules ; 8(4)2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30404219

RESUMO

Myxococcus xanthus DK1622 is a rich source of novel secondary metabolites, and it is often used as an expression host of exogenous biosynthetic gene clusters. However, the frequency of obtaining large genome-deletion variants by using traditional strategies is low, and progenies generated by homologous recombination contain irregular deletions. The present study aims to develop an efficient genome-engineering system for this bacterium based on the Cre/loxP system. We first verified the functionality of the native cre system that was integrated into the chromosome with an inducible promoter PcuoA. Then we assayed the deletion frequency of 8-bp-spacer-sequence mutants in loxP by Cre recombinase which was expressed by suicide vector pBJ113 or self-replicative vector pZJY41. It was found that higher guanine content in a spacer sequence had higher deletion frequency, and the self-replicative vector was more suitable for the Cre/loxP system, probably due to the leaky expression of inducible promoter PcuoA. We also inspected the effects of different antibiotics and the native or synthetic cre gene. Polymerase chain reaction (PCR) and sequencing of new genome joints confirmed that the Cre/loxP system was able to delete a 466 kb fragment in M. xanthus. This Cre/loxP-mediated recombination could serve as an alternative genetic manipulation method.


Assuntos
Edição de Genes , Genoma Bacteriano , Integrases/metabolismo , Myxococcus xanthus/genética , Recombinação Genética/genética , Antibacterianos/farmacologia , Sequência de Bases , Cromossomos Bacterianos/genética , Deleção de Genes , Família Multigênica , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Recombinases/metabolismo , Sideróforos/metabolismo
18.
Int J Syst Evol Microbiol ; 68(11): 3518-3522, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30222097

RESUMO

A pale yellow bacterial strain, designated SDU1-6T, was isolated from a soil sample collected in the Jinan campus of Shandong University, Shandong Province, PR China. The strain was aerobic, motile, Gram-stain-negative and rod-shaped. Cellular growth occurred at 13-37 °C, pH 5.0-10.0 and with 0-0.1 % (w/v) NaCl, and the optimal growth was at 25 °C and pH 7.5. SDU1-6T had the highest 16S rRNA gene similarity of 95.42 % with Chryseolinea serpens DSM 24574T. The genome was 6 542 746 bp in length with 43.5 mol% DNA G+C content. The major fatty acids of SDU1-6T were C16 : 1ω5 and iso-C15 : 0, the major menaquinone was menaquinone-7 (MK-7) and the major polar lipid was phosphatidylethanolamine. On the basis of the polyphasic evidence and the 16S rRNA gene sequence, SDU1-6T represents a novel species of the genus Chryseolinea, for which the name Chryseolineaflava sp. nov. is proposed. The type strain is SDU1-6T (=CGMCC 1.13492T=JCM 32520T).


Assuntos
Bacteroidetes/classificação , Filogenia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Composição de Bases , China , DNA Bacteriano/genética , Ácidos Graxos/química , Fosfatidiletanolaminas/química , Pigmentação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/química
19.
Artigo em Inglês | MEDLINE | ID: mdl-30131946

RESUMO

Although plasmids provide additional functions for cellular adaptation to the environment, they also create a metabolic burden, which causes the host cells to be less competitive with their siblings. Low-copy-number plasmids have thus evolved several mechanisms for their long-term maintenance in host cells. pMF1, discovered in Myxococcus fulvus 124B02, is the only endogenous autonomously replicated plasmid yet found in myxobacteria. Here we report that a post-segregational killing system, encoded by a co-transcriptional gene pair of pMF1.19 and pMF1.20, is involved in maintaining the pMF1 plasmid in its host cells. We demonstrate that the protein encoded by pMF1.20 is a new kind of nuclease, which is able to cleave DNA in vitro. The nuclease activity can be neutralized by the protein encoded by pMF1.19 through protein-protein interaction, suggesting that the protein is an immune protein for nuclease cleavage. We propose that the post-segregational killing mechanism of the nuclease toxin and immune protein pair encoded by pMF1.20 and pMF1.19 is helpful for the stable maintenance of pMF1 in M. fulvus cells.


Assuntos
Transporte Biológico , Divisão Celular , Viabilidade Microbiana , Myxococcus/genética , Myxococcus/fisiologia , Plasmídeos/metabolismo , Sistemas Toxina-Antitoxina , Replicação do DNA , Genes Bacterianos
20.
Front Microbiol ; 9: 1200, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29922269

RESUMO

Due to the high similarity in their requirements for space and food, close bacterial relatives may be each other's strongest competitors. Close bacterial relatives often form visible boundaries to separate their swarming colonies, a phenomenon termed colony-merger incompatibility. While bacterial species are known to have many incompatible strains, it is largely unclear which traits lead to multiple incompatibilities and the interactions between multiple incompatible siblings. To investigate the competitive interactions of closely related incompatible strains, we mutated Myxococcus xanthus DK1622, a predatory bacterium with complex social behavior. From 3392 random transposon mutations, we obtained 11 self-identification (SI) deficient mutants that formed unmerged colony boundaries with the ancestral strain. The mutations were at nine loci with unknown functions and formed nine independent SI mutants. Compared with their ancestral strain, most of the SI mutants showed reduced growth, swarming and development abilities, but some remained unchanged from their monocultures. When pairwise mixed with their ancestral strain for co-cultivation, these mutants exhibited improved, reduced or unchanged competitive abilities compared with the ancestral strain. The sporulation efficiencies were affected by the DK1622 partner, ranging from almost complete inhibition to 360% stimulation. The differences in competitive growth between the SI mutants and DK1622 were highly correlated with the differences in their sporulation efficiencies. However, the competitive efficiencies of the mutants in mixture were inconsistent with their growth or sporulation abilities in monocultures. We propose that the colony-merger incompatibility in M. xanthus is associated with multiple independent genetic loci, and the incompatible strains hold competitive interaction abilities, which probably determine the complex relationships between multiple incompatible M. xanthus strains and their co-existence strategies.

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