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1.
Microb Cell Fact ; 18(1): 162, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581942

RESUMO

BACKGROUND: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. RESULTS: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. CONCLUSIONS: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.


Assuntos
Edição de Genes/métodos , Genoma Bacteriano , Zymomonas/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Francisella/enzimologia , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Zymomonas/metabolismo
2.
Microb Cell Fact ; 18(1): 163, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581944

RESUMO

BACKGROUND: Sustainable production of microbial fatty acids derivatives has the potential to replace petroleum based equivalents in the chemical, cosmetic and pharmaceutical industry. Most fatty acid sources for production oleochemicals are currently plant derived. However, utilization of these crops are associated with land use change and food competition. Microbial oils could be an alternative source of fatty acids, which circumvents the issue with agricultural competition. RESULTS: In this study, we generated a chimeric microbial production system that features aspects of both prokaryotic and eukaryotic fatty acid biosynthetic pathways targeted towards the generation of long chain fatty acids. We redirected the type-II fatty acid biosynthetic pathway of Escherichia coli BL21 (DE3) strain by incorporating two homologues of the beta-ketoacyl-[acyl carrier protein] synthase I and II from the chloroplastic fatty acid biosynthetic pathway of Arabidopsis thaliana. The microbial clones harboring the heterologous pathway yielded 292 mg/g and 220 mg/g DCW for KAS I and KAS II harboring plasmids respectively. Surprisingly, beta-ketoacyl synthases KASI/II isolated from A. thaliana showed compatibility with the FAB pathway in E. coli. CONCLUSION: The efficiency of the heterologous plant enzymes supersedes the overexpression of the native enzyme in the E. coli production system, which leads to cell death in fabF overexpression and fabB deletion mutants. The utilization of our plasmid based system would allow generation of plant like fatty acids in E. coli and their subsequent chemical or enzymatic conversion to high end oleochemical products.


Assuntos
Arabidopsis/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Engenharia Metabólica , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/síntese química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/síntese química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Ácido Graxo Sintases/genética , Ácidos Graxos/química , Isoenzimas/síntese química , Isoenzimas/genética , Isoenzimas/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo
3.
Rev Soc Bras Med Trop ; 52: e20190237, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31508785

RESUMO

INTRODUCTION: The increased use of colistin against infections caused by Acinetobacter baumannii and Pseudomonas aeruginosa has resulted in colistin resistance. The purpose of this study was to detect plasmid-mediated mcr-1 gene in colistin-resistant A. baumannii and P. aeruginosa isolates. METHODS: A total of 146 clinical isolates of A. baumannii (n = 62) and P. aeruginosa (n = 84) were collected from the four largest tertiary care hospitals in Peshawar, Pakistan. All bacterial isolates were phenotypically screened for multidrug resistance using the Kirby-Baur disc diffusion method. The minimum inhibitory concentration (MIC) of colistin in all isolates was phenotypically performed using dilution methods. mcr-1 gene was detected through polymerase chain reaction and the nucleotide sequence of amplicon was determined using Sanger sequencing. RESULTS: Approximately 96.7% A. baumannii and 83.3% P. aeruginosa isolates were resistant to multiple antibiotics. Colistin resistance was found in 9.6% (6/62) of A. baumannii and 11.9% (10/84) of P. aeruginosa isolates. Among 16 colistin resistant isolates, the mcr-1 gene was detected in one A. baumannii (1.61% of total isolates; 16.6% of colistin resistant isolates) and one P. aeruginosa strain (1.19% of total isolates; 10% of colistin resistant isolates). Nucleotide BLAST showed 98-99% sequence similarity to sequences of the mcr-1 gene in GenBank. CONCLUSIONS: Our study reports, for the first time, the emergence of plasmid-mediated mcr-1-encoded colistin resistance in multidrug resistant strains of A. baumannii and P. aeruginosa. Further large scales studies are recommended to investigate the prevalence of this mode of resistance in these highly pathogenic bacteria.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Acinetobacter baumannii/efeitos dos fármacos , Farmacorresistência Bacteriana , Humanos , Testes de Sensibilidade Microbiana , Paquistão , Plasmídeos/genética , Pseudomonas aeruginosa/efeitos dos fármacos
4.
Acta Virol ; 63(3): 301-308, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507196

RESUMO

Transmissible gastroenteritis virus (TGEV) causes great economic loss to swine industry worldwide. Vaccination is an important method to control the TGEV infection. In this study, a TGEV oral vaccine was generated by transferring a eukaryotic expression recombinant plasmid carrying the SAD (A and D antigenic sites of the S protein) epitope of TGEV into a swine-origin Lactobacillus acidophilus (L. acidophilus). In orally immunized BALB/c mice, the TGEV L. acidophilus oral vaccine induced significantly higher level of SIgA antibodies specific to TGEV compared with the mice immunized with a commercial inactivated TGEV vaccine and similar levels of IgG specific to TGEV as the inactivated vaccine. Furthermore, the TGEV L. acidophilus oral vaccine induced higher levels of IFN-γ, which suggested that the vaccine was able to induce immune response. In brief, this novel TGEV L. acidophilus oral vaccine could induce high levels of both mucosal and humoral immune responses, which has a potential to be used in the pig industries in the future. Keywords: transmissible gastroenteritis virus (TGEV); live L. acidophilus oral vaccine; SIgA antibody; IgG antibody; IFN-γ; IL-4.


Assuntos
Anticorpos Antivirais , Epitopos , Gastroenterite Suína Transmissível , Lactobacillus acidophilus , Vírus da Gastroenterite Transmissível , Vacinas Virais , Administração Oral , Animais , Anticorpos Antivirais/sangue , Epitopos/genética , Epitopos/imunologia , Gastroenterite Suína Transmissível/imunologia , Gastroenterite Suína Transmissível/patologia , Imunogenicidade da Vacina/imunologia , Lactobacillus acidophilus/genética , Lactobacillus acidophilus/virologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Suínos , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
5.
Epidemiol Mikrobiol Imunol ; 68(2): 99-102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31398983

RESUMO

The increasing incidence of multiresistant bacterial strains is currently a serious health concern. These pathogens are often the cause of nosocomial infections with limited treatment options and high fatality rates. A case report is presented of an uncommon detection of four different species (Citrobacter freundii, Klebsiella pneumoniae, Escherichia coli, and Morganella morganii) producing the same type of carbapenemase, KPC-2, in a female patient during her complicated long-term hospital stay. Resistance was probably spread to other species by horizontal transmission of plasmids carrying the blaKPC-2 genes. The implementation of strict anti-epidemic measures prevented further spread of these carbapenem-resistant bacteria.


Assuntos
Antibacterianos , Bactérias , Infecções Bacterianas , Infecção Hospitalar , beta-Lactamases , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/genética , Coinfecção/microbiologia , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana/genética , Feminino , Transferência Genética Horizontal , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/genética , beta-Lactamases/genética
6.
Mol Biol (Mosk) ; 53(4): 600-612, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397434

RESUMO

A new plasmid, pSM22, was isolated from Serratia marcescens and sequenced. Its 43 190-bp sequence with an average GC-content of 58% contains 31 open reading frames (ORFs) which form replication, conjugation, stability, and adaptive modules. The replication module includes a site of initiation of leading-strand synthesis in plasmid replication, a replication termination site (terC), the rep A (=repA1) and repA4 genes, and the copA sequence, which codes for an antisense RNA (asRNA). These structures are functionally integrated in an FII replicon (incompatibility group IncFII). Based on the significant differences between the FII replicon and the canonical sequences of the R plasmids R1 and NR1 (=R100=R222), pSM22 was assigned to a new subtype. The conjugation module includes 13 genes with a high identity to the genes responsible for conjugation of the F plasmid. A comparative genomic analysis showed that the conjugation modules of pSM22 and F are structurally similar. By the conjugation system and the presence of three conserved motifs in relaxase (TraI), pSM22 belongs to the F12 clade of the MOBF type. The stability module includes the resD and parA genes, which are responsible for the resolution of multimeric plasmid forms and their subsequent segregation between daughter cells. The adaptive module contains the microcin H47 (MccH47) secretion/processing and UV resistance genes. The mosaic structure of pSM22 and reductive evolution of its modules suggest high genomic plasticity for the genus Serratia. An analysis of the architecture of the pSM22 modules clarifies the evolutionary relationships among IncF/MOBF12 group plasmids in bacteria of the family Enterobacteriaceae and opens a novel avenue for further comparative genomic studies of Serratia plasmids.


Assuntos
Evolução Molecular , Genômica , Plasmídeos/classificação , Plasmídeos/genética , Replicação do DNA , Genes Bacterianos , Genoma Bacteriano/genética , Replicon/genética , Serratia marcescens/genética
7.
World J Microbiol Biotechnol ; 35(8): 119, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332541

RESUMO

The microalgal genus of Nannochloropsis is considered one of the most promising organisms for the production of biofuels due to their high lipid content. Transformation systems for marine Nannochloropsis species have been established in the recent decade, however, genetic manipulation of Nannochloropsis limnetica, the only known freshwater species in this genus, is not yet available. Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. For the selection of transformants in N. limnetica on plates, 0.07 µg mL-1 of zeocin or 5 µg mL-1 of hygromycin B was proved sufficient, and the transformation efficiency was < 2 × 10-8 with a single pulse ranging from 2200 to 2600 V using 2-mm electroporation cuvettes. Pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times, and the highest transformation efficiency of 10-11 × 10-6 was obtained with an electric field strength of 12,000 V/cm. Our results help to expand the biotechnological applications of this freshwater species and provide means for successful electrotransformation of other microalgae as well. High-efficiency transformation of freshwater Nannochloropsis pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times.


Assuntos
Eletroporação , Água Doce/microbiologia , Microalgas/metabolismo , Estramenópilas/metabolismo , Acetatos , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microalgas/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Estramenópilas/genética , Transformação Genética
8.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(6): 557-562, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31292061

RESUMO

Objective To construct a random mutagenesis library of 3E1D7, a chimerical antibody against c-mesenchymal epithelial transition factor (c-Met), using mammalian cell surface display. Methods Antibody genes with randomly mutated complementarity-determining region 3 (CDR3) part were inserted into the mammalian expression plasmid pSZI-CD to construct the random mutagenesis library using double enzyme digestion. Reconstructed plasmids were then cloned into CHO cells by transfection. The expression level of antibodies on the surface of CHO cells was checked by C6 PLUS flow cytometry. Results 3E1D7 random mutagenesis library was successfully constructed with a volume of 5.52×106 in diversity on gene level. Sequence analysis showed that all 20 clones randomly picked from the library coded for 20 different mutated amino acid sequences in open reading frames. After transfection, the expression of full-length antibodies on CHO cell surfaces could be detected by flow cytometry. Conclusion A random mutagenesis library of a certain anti-c-Met antibody has been successfully constructed with an exhibitable diversity of 5.52×106, which would be a useful platform for further screening of therapeutic antibodies.


Assuntos
Biblioteca Gênica , Vetores Genéticos , Mutagênese , Sequência de Aminoácidos , Animais , Anticorpos/química , Cricetinae , Cricetulus , Fases de Leitura Aberta , Plasmídeos/genética , Transfecção
9.
Vet Microbiol ; 235: 143-150, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282372

RESUMO

Porcine parvovirus (PPV) is one of the major pathogens that bring about reproductive failure of pregnant sows. However, the study of the pathogenesis mechanism is circumscribed due to the lack of efficient genetic manipulation method. Infectious clone is a powerful tool for further studying the genetic mechanisms of PPV. In the present study, the gene fragment (157-4812) of PPV was amplified by PPV China isolate strain as a template, and PPV DNA fragments (1-182) forming Y-structure within in 5' end and (4788-5074) forming U-structure in 3' end were synthesized. And then, the above three fragments were inserted into plasmid pKQLL to congregate a PPV full-length recombinant plasmid by means of In-Fusion cloning technology. After the successful sequencing identification of the recombinant plasmid, the EcoR I restriction site was brought out as a genetic marker by nonsense mutation (A3058 T) to produce plasmid Y-PPV, which was transfected into PK-15 cells for rescue of virus. The rescued viral particles were observed under transmission electron microscopy, and the sequencing analysis showed that Y-PPV could stably carry the genetic marker. It could be seen that Y-PPV has similar replicate capability and pathogenicity as the wild-type parental PPV strain by cellular and animal experiments. These results confirmed that Y-PPV maintain similar biological characteristics with wild-type parental PPV strain. Infectious clone could be a valuable tool for studying the individual genes of PPV and applications in gene deletion or live vector vaccines.


Assuntos
Genoma Viral , Parvovirus Suíno/genética , Parvovirus Suíno/patogenicidade , Animais , Linhagem Celular , China , Clonagem Molecular , Marcadores Genéticos , Vetores Genéticos , Rim/citologia , Rim/virologia , Plasmídeos/genética , Suínos
10.
Nat Commun ; 10(1): 2763, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235751

RESUMO

Multidrug resistant (MDR) Acinetobacter baumannii poses a growing threat to global health. Research on Acinetobacter pathogenesis has primarily focused on pneumonia and bloodstream infections, even though one in five A. baumannii strains are isolated from urinary sites. In this study, we highlight the role of A. baumannii as a uropathogen. We develop the first A. baumannii catheter-associated urinary tract infection (CAUTI) murine model using UPAB1, a recent MDR urinary isolate. UPAB1 carries the plasmid pAB5, a member of the family of large conjugative plasmids that represses the type VI secretion system (T6SS) in multiple Acinetobacter strains. pAB5 confers niche specificity, as its carriage improves UPAB1 survival in a CAUTI model and decreases virulence in a pneumonia model. Comparative proteomic and transcriptomic analyses show that pAB5 regulates the expression of multiple chromosomally-encoded virulence factors besides T6SS. Our results demonstrate that plasmids can impact bacterial infections by controlling the expression of chromosomal genes.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/patogenicidade , Infecções Relacionadas a Cateter/microbiologia , Plasmídeos/genética , Pneumonia Bacteriana/microbiologia , Infecções Urinárias/microbiologia , Infecções por Acinetobacter/epidemiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções Relacionadas a Cateter/epidemiologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Humanos , Camundongos , Pneumonia Bacteriana/epidemiologia , Proteômica , Estudos Retrospectivos , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Cateteres Urinários/efeitos adversos , Cateteres Urinários/microbiologia , Sistema Urinário/microbiologia , Infecções Urinárias/epidemiologia , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
11.
Sci Total Environ ; 685: 419-427, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31176227

RESUMO

Peracetic acid (PAA) is an emerging disinfectant with a low disinfection by-product formation potential, but how PAA destroys gene function after killing bacteria remains to be studied. Bacterial plasmid DNA is a mobile genetic element that often harbors undesirable genes encoding antibiotic resistance and virulence factors. Even though PAA efficiently kills bacteria, bacterial plasmids and other mobile genetic elements might still be intact and functional after PAA disinfection, posing potential public health and environmental risks. This study evaluated the impact of PAA disinfection on the functionality of plasmid DNA in vivo and compared the results with those from chlorination. We delivered a plasmid DNA harboring two antibiotic resistance genes to Escherichia coli TOP10 to form an antibiotic-resistant bacterium (ARB). The planktonic ARB was treated with PAA and chlorine to find the minimum doses inhibiting the regrowth of the strain. PAA and chlorine stopped the regrowth at 8 ±â€¯1 mg PAA·L-1 and 20 ±â€¯9 mg Cl2·L-1, respectively. The functionality of the plasmid DNA after PAA and chlorine disinfection was then determined at higher doses in vivo. Neither PAA nor chlorine completely destroyed the plasmid DNA. However, chlorine was more efficient than PAA in eliminating the plasmid DNA. PAA at 25 mg PAA·L-1 reduced the transforming activity of the plasmid DNA by less than 0.3 log10 units, whereas chlorine at 25 mg Cl2·L-1 reduced the transforming activity by approximately 1.7 log10 units. Chlorine had a more pronounced impact on the functionality of the plasmid DNA because it oxidizes or destroys bacterial components including plasmid DNA faster than PAA. In addition, environmental scanning electron microscopy shows that chlorination desiccated the cells resulting in the flat cellular structure and possibly more complete loss of plasmid DNA, whereas PAA disinfection had a less impact on cell structure and morphology. This study demonstrates that more plasmid DNA remains functional in water after PAA disinfection than after chlorination. These functional genetic elements could be acquired by other microorganisms via horizontal gene transfer to pose potential public health and environmental risks.


Assuntos
Desinfetantes/análise , Desinfecção/métodos , Ácido Peracético/análise , Purificação da Água/métodos , DNA Bacteriano , Halogenação , Plasmídeos/genética
12.
J Microbiol Biotechnol ; 29(6): 962-972, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31216839

RESUMO

Non-typhoidal Salmonella (NTS) is one of the most frequent causes of bacterial foodborne illnesses. Considering that the main reservoir of NTS is the intestinal tract of livestock, foods of animal origin are regarded as the main vehicles of Salmonella infection. In particular, poultry colonized with Salmonella Typhimurium (S. Typhimurium), a dominant serotype responsible for human infections, do not exhibit overt signs and symptoms, thereby posing a potential health risk to humans. In this study, comparative genomics approaches were applied to two S. Typhimurium strains, ST1539 and ST1120, isolated from a duck slaughterhouse and a pig farm, respectively, to characterize their virulence and antimicrobial resistance-associated genomic determinants. ST1539 containing a chromosome (4,905,039 bp; 4,403 CDSs) and a plasmid (93,876 bp; 96 CDSs) was phylogenetically distinct from other S. Typhimurium strains such as ST1120 and LT2. Compared to the ST1120 genome (previously deposited in GenBank; CP021909.1 and CP021910.1), ST1539 possesses more virulence determinants, including ST64B prophage, plasmid spv operon encoding virulence factors, genes encoding SseJ effector, Rck invasin, and biofilm-forming factors (bcf operon and pefAB). In accordance with the in silico prediction, ST1539 exhibited higher cytotoxicity against epithelial cells, better survival inside macrophage cells, and faster mice-killing activity than ST1120. However, ST1539 showed less resistance against antibiotics than ST1120, which may be attributed to the multiple resistanceassociated genes in the ST1120 chromosome. The accumulation of comparative genomics data on S. Typhimurium isolates from livestock would enrich our understanding of strategies Salmonella employs to adapt to diverse host animals.


Assuntos
Farmacorresistência Bacteriana , Genoma Bacteriano/genética , Aves Domésticas , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Matadouros , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Sobrevivência Celular , Farmacorresistência Bacteriana/genética , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Plasmídeos/genética , Prófagos/genética , República da Coreia , Salmonella typhimurium/classificação , Salmonella typhimurium/isolamento & purificação , Sorogrupo , Suínos , Virulência/genética
13.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 847-856, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223003

RESUMO

Pectobacterium carotovorum subsp. carotovorum is one of the world's top ten plant pathogens, mainly infecting cruciferous economic crops and ornamental flowers. In this study, an antibacterial gene cpxP (Gene ID: 29704421) was cloned from the genome of Pectobacterium carotovorum subsp. carotovorum, and constructed on the prokaryotic expression plasmid pET-15b, and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3), then stability and bacteriostatic experiments of the purified CpxP protein were performed. The final concentration of IPTG was 1 mmol/L, obtaining high-efficiency exogenous expression of the CpxP protein. There was no other protein after purification, and the destined protein exhibited good thermal stability and pH stability. The antibacterial test results showed that the inhibition rate of the CpxP protein on carrot slice was 44.89% while the inhibition rate on potato slice was 59.41%. To further explain its antibacterial mechanism, studying the spatial structure of this protein can provide new ideas for the control of soft rot and new protein pesticide targets.


Assuntos
Bactérias , Proteínas de Bactérias , Proteínas de Membrana , Pectobacterium carotovorum , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Escherichia coli/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/farmacologia , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/metabolismo , Plasmídeos/genética
14.
Nat Commun ; 10(1): 2595, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197163

RESUMO

Plasmid acquisition is an important mechanism of rapid adaptation and niche expansion in prokaryotes. Positive selection for plasmid-coded functions is a major driver of plasmid evolution, while plasmids that do not confer a selective advantage are considered costly and expected to go extinct. Yet, plasmids are ubiquitous in nature, and their persistence remains an evolutionary paradox. Here, we demonstrate that non-mobile plasmids persist over evolutionary timescales without selection for the plasmid function. Evolving a minimal plasmid encoding for antibiotics resistance in Escherichia coli, we discover that plasmid stability emerges in the absence of antibiotics and that plasmid loss is determined by transcription-replication conflicts. We further find that environmental conditions modulate these conflicts and plasmid persistence. Silencing the transcription of the resistance gene results in stable plasmids that become fixed in the population. Evolution of plasmid stability under non-selective conditions provides an evolutionary explanation for the ubiquity of plasmids in nature.


Assuntos
Adaptação Biológica/genética , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Plasmídeos/genética , Antibacterianos/farmacologia , Variações do Número de Cópias de DNA/genética , Evolução Molecular Direcionada/métodos , Genoma Bacteriano/genética , Plasmídeos/efeitos dos fármacos , Temperatura Ambiente
15.
Prep Biochem Biotechnol ; 49(8): 800-806, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156029

RESUMO

In this study, azurin, a bacteriocin with anticancer property, was produced by food-grade Lactococcus lactis using the Nisin Controlled Gene Expression (NICE) System. In addition, the antibacterial and cytotoxic properties of recombinant azurin in the culture supernatant were also investigated. Azurin gene from Pseudomonas aeruginosa was cloned into the pNZ8149 vector and the resulting recombinant DNA was transformed into food grade L. lactis NZ3900. The expression of azurin protein was induced by the optimum concentration of nisin for 3 h. Inhibition zones for Escherichia coli and Bacillus cereus were observed at 5.0 and 10 mg/mL concentrations of lyophilized supernatants containing azurin, but no inhibition zone at azurin-free lyophilized supernatants. When HUVEC, HT29, HCT116, and MCF7 cell lines were treated with lyophilized culture supernatants with azurin or without azurin, cell viability decreased with increasing concentrations of the supernatant. Furthermore, the supernatants containing azurin showed more anti-proliferative effect than the azurin-free supernatants. This work provides a practicable method to produce recombinant azurin in the food grade L. lactis strain. As a result, the recombinant L. lactis strain, producing azurin, can be used in the investigation of food biopreservatives and in the development of a therapeutic probiotic.


Assuntos
Azurina/genética , Clonagem Molecular/métodos , Lactococcus lactis/genética , Pseudomonas aeruginosa/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Azurina/farmacologia , Bacillus cereus/efeitos dos fármacos , Linhagem Celular , Escherichia coli/efeitos dos fármacos , Amplificação de Genes , Humanos , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Transformação Genética
16.
BMC Evol Biol ; 19(1): 124, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31215393

RESUMO

BACKGROUND: Mycobacteria occupy various ecological niches and can be isolated from soil, tap water and ground water. Several cause diseases in humans and animals. To get deeper insight into our understanding of mycobacterial evolution focusing on tRNA and non-coding (nc)RNA, we conducted a comparative genome analysis of Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clade members. RESULTS: Genome sizes for Mmuc- and Mneo-clade members vary between 5.4 and 6.5 Mbps with the complete MmucT (type strain) genome encompassing 6.1 Mbp. The number of tRNA genes range between 46 and 79 (including one pseudo tRNA gene) with 39 tRNA genes common among the members of these clades, while additional tRNA genes were probably acquired through horizontal gene transfer. Selected tRNAs and ncRNAs (RNase P RNA, tmRNA, 4.5S RNA, Ms1 RNA and 6C RNA) are expressed, and the levels for several of these are higher in stationary phase compared to exponentially growing cells. The rare tRNAIleTAT isoacceptor and two for mycobacteria novel ncRNAs: the Lactobacillales-derived GOLLD RNA and a homolog to the antisense Salmonella typhimurium phage Sar RNA, were shown to be present and expressed in certain Mmuc-clade members. CONCLUSIONS: Phages, IS elements, horizontally transferred tRNA gene clusters, and phage-derived ncRNAs appears to have influenced the evolution of the Mmuc- and Mneo-clades. While the number of predicted coding sequences correlates with genome size, the number of tRNA coding genes does not. The majority of the tRNA genes in mycobacteria are transcribed mainly from single genes and the levels of certain ncRNAs, including RNase P RNA (essential for the processing of tRNAs), are higher at stationary phase compared to exponentially growing cells. We provide supporting evidence that Ms1 RNA represents a mycobacterial 6S RNA variant. The evolutionary routes for the ncRNAs RNase P RNA, tmRNA and Ms1 RNA are different from that of the core genes.


Assuntos
Genoma Bacteriano , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/genética , RNA Bacteriano/genética , RNA de Transferência/genética , RNA não Traduzido/genética , Aminoacil-tRNA Sintetases/genética , Bacteriófagos/genética , Tamanho do Genoma , Genômica , Anotação de Sequência Molecular , Mycobacterium/classificação , Filogenia , Plasmídeos/genética , RNA não Traduzido/química , Ribonuclease P/genética , Inversão de Sequência
17.
Emerg Microbes Infect ; 8(1): 857-865, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169081

RESUMO

Ciprofloxacin resistance in Salmonella has been increasingly reported due to the emergence and dissemination of multiple Plasmid-Mediated Quinolone Resistance (PMQR) determinants, which are mainly located in non-conjugative plasmids or chromosome. In this study, we aimed to depict the molecular mechanisms underlying the rare phenomenon of horizontal transfer of ciprofloxacin resistance phenotype in Salmonella by conjugation experiments, S1-PFGE and complete plasmid sequencing. Two types of non-conjugative plasmids, namely an IncX1 type carrying a qnrS1 gene, and an IncH1 plasmid carrying the oqxAB-qnrS gene, both ciprofloxacin resistance determinants in Salmonella, were recovered from two Salmonella strains. Importantly, these non-conjugative plasmids could be fused with a novel Incl1 type conjugative helper plasmid, which could target insertion sequence (IS) elements located in the non-conjugative, ciprofloxacin-resistance-encoding plasmid through replicative transcription, eventually forming a hybrid conjugative plasmid transmissible among members of Enterobacteriaceae. Since our data showed that such conjugative helper plasmids are commonly detectable among clinical Salmonella strains, particularly S. Typhimurium, fusion events leading to generation and enhanced dissemination of conjugative ciprofloxacin resistance-encoding plasmids in Salmonella are expected to result in a sharp increase in the incidence of resistance to fluoroquinolone, the key choice for treating life-threatening Salmonella infections, thereby posing a serious public health threat.


Assuntos
Antibacterianos/farmacologia , Ciprofloxacino/farmacologia , Conjugação Genética , Farmacorresistência Bacteriana , Plasmídeos/genética , Salmonella/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/metabolismo , Salmonella/genética , Salmonella/metabolismo , Infecções por Salmonella/microbiologia
18.
Life Sci ; 231: 116531, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31175856

RESUMO

BACKGROUND: The Proteus is one of the most common human and animal pathogens. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR/Cas) are inheritable genetic elements found in a variety of archaea and bacteria in the evolution, providing immune function against foreign invasion. OBJECTIVES: To analyze the characteristics and functions of the CRISPR/Cas system in Proteus genomes, as well as the internal and external factors affecting the system. METHODS: CRISPR loci were identified and divided into groups based on the repeat sequence in 96 Proteus strains by identification. Compared the RNA secondary structure and minimum free energy of CRISPR loci through bioinformatics, the evolution of cas genes, and the effects of related elements were also discussed. RESULTS: 85 CRISPR loci were identified and divided into six groups based on the sequence of repeats, and the more stable the secondary structure of RNA, the smaller the minimum free energy, the fewer base mutations in the repeat, the more stable the CRISPR and the more complete the evolution of the system. In addition, Cas1 gene can be a symbol to distinguish species to some extent. Of all the influencing factors, CRISPR/Cas had the greatest impact on plasmids. CONCLUSIONS: This study examined the diversity of CRISPR/Cas system in Proteus and found statistically significant positive/negative correlations between variety factors (the RNA stability, free energy, etc.) and the CRISPR locus, which played a vital role in regulating the CRISPR/Cas system.


Assuntos
Proteus/genética , Proteus/metabolismo , Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Biologia Computacional/métodos , Bases de Dados Genéticas , Plasmídeos/genética , RNA/genética
19.
J Med Microbiol ; 68(7): 986-990, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31162025

RESUMO

A colistin-resistant Salmonella enterica 4, [5],12:i:- sequence type (ST) 34 harbouring mcr-3.1 was recovered from a patient who travelled to China 2 weeks prior to diarrhoea onset. Genomic analysis revealed the presence of the mcr-3.1 gene located in the globally disseminated IncHI2 plasmid, highlighting the intercontinental dissemination of the colistin-resistant S. enterica 4, [5],12:i:- ST34 pandemic clone.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Plasmídeos/genética , Salmonella enterica/efeitos dos fármacos , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Proteínas de Bactérias/genética , China , Humanos , Salmonella enterica/genética , Salmonella enterica/isolamento & purificação , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Viagem
20.
World J Microbiol Biotechnol ; 35(6): 79, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134410

RESUMO

The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Pichia/genética , Plasmídeos/genética , Transformação Genética , Replicação do DNA , Escherichia coli/genética , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Vetores Genéticos , Instabilidade Genômica , Microbiologia Industrial , Kluyveromyces/genética , Regiões Promotoras Genéticas , RNA Guia , Biologia Sintética
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