RESUMO
SignificanceAlthough most studies of the genetic regulation of genome stability involve an analysis of mutations within the coding sequences of genes required for DNA replication or DNA repair, recent studies in yeast show that reduced levels of wild-type enzymes can also produce a mutator phenotype. By whole-genome sequencing and other methods, we find that reduced levels of the wild-type DNA polymerase ε in yeast greatly increase the rates of mitotic recombination, aneuploidy, and single-base mutations. The observed pattern of genome instability is different from those observed in yeast strains with reduced levels of the other replicative DNA polymerases, Pol α and Pol δ. These observations are relevant to our understanding of cancer and other diseases associated with genetic instability.
Assuntos
DNA Polimerase II , Saccharomyces cerevisiae , DNA Polimerase II/metabolismo , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Mutação , Saccharomyces cerevisiae/metabolismoRESUMO
Genomic alterations including single-base mutations, deletions and duplications, translocations, mitotic recombination events, and chromosome aneuploidy generate genetic diversity. We examined the rates of all of these genetic changes in a diploid strain of Saccharomyces cerevisiae by whole-genome sequencing of many independent isolates (n = 93) subcloned about 100 times in unstressed growth conditions. The most common alterations were point mutations and small (<100 bp) insertion/deletions (n = 1,337) and mitotic recombination events (n = 1,215). The diploid cells of most eukaryotes are heterozygous for many single-nucleotide polymorphisms (SNPs). During mitotic cell divisions, recombination can produce derivatives of these cells that have become homozygous for the polymorphisms, termed loss-of-heterozygosity (LOH) events. LOH events can change the phenotype of the cells and contribute to tumor formation in humans. We observed two types of LOH events: interstitial events (conversions) resulting in a short LOH tract (usually less than 15 kb) and terminal events (mostly cross-overs) in which the LOH tract extends to the end of the chromosome. These two types of LOH events had different distributions, suggesting that they may have initiated by different mechanisms. Based on our results, we present a method of calculating the probability of an LOH event for individual SNPs located throughout the genome. We also identified several hotspots for chromosomal rearrangements (large deletions and duplications). Our results provide insights into the relative importance of different types of genetic alterations produced during vegetative growth.
Assuntos
Cromossomos Fúngicos/genética , Mutação/genética , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , Diploide , Conversão Gênica/genética , Rearranjo Gênico/genética , Perda de Heterozigosidade/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Saccharomyces cerevisiae/citologiaRESUMO
Oxidative stress has been implicated in a variety of human diseases. One plausible mechanism is that reactive active species can induce DNA damages and jeopardize genome integrity. To explore how oxidative stress results in global genomic instability in cells, our current study examined the genomic alterations caused by H2O2 exposure at the whole genome level in yeast. Using SNP microarrays and genome sequencing, we mapped H2O2-induced genomic alterations in the yeast genome ranging from point mutations and mitotic recombination to chromosomal aneuploidy. Our results suggested most H2O2-induced mitotic recombination events were the result of DNA double-stand breaks generated by hydroxyl radicals. Moreover, the mutagenic effect of H2O2 was shown to be largely dependent on DNA polymerase ζ. Lastly, we showed that H2O2 exposure allows rapid phenotypic evolution in yeast strains. Our findings indicate DNA lesions resulting from H2O2 may be general factors that drive genome instability and phenotypic evolution in organisms.
Assuntos
Dano ao DNA/efeitos dos fármacos , Instabilidade Genômica/genética , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/genética , Aneuploidia , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Instabilidade Genômica/efeitos dos fármacos , Humanos , Mitose/efeitos dos fármacos , Mitose/genética , Mutagênese/genética , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genética , Saccharomyces cerevisiae/genética , Sequenciamento Completo do GenomaRESUMO
Furfural is an important renewable precursor for multiple commercial chemicals and fuels; a main inhibitor existing in cellulosic hydrolysate, which is used for bioethanol fermentation; and a potential carcinogen, as well. Using a genetic system in Saccharomyces cerevisiae that allows detection of crossover events, we observed that the frequency of mitotic recombination was elevated by 1.5- to 40-fold when cells were treated with 0.1 g/liter to 20 g/liter furfural. Analysis of the gene conversion tracts associated with crossover events suggested that most furfural-induced recombination resulted from repair of DNA double-strand breaks (DSBs) that occurred in the G1 phase. Furfural was incapable of breaking DNA directly in vitro but could trigger DSBs in vivo related to reactive oxygen species accumulation. By whole-genome single nucleotide polymorphism (SNP) microarray and sequencing, furfural-induced genomic alterations that range from single base substitutions, loss of heterozygosity, and chromosomal rearrangements to aneuploidy were explored. At the whole-genome level, furfural-induced events were evenly distributed across 16 chromosomes but were enriched in high-GC-content regions. Point mutations, particularly the C-to-T/G-to-A transitions, were significantly elevated in furfural-treated cells compared to wild-type cells. This study provided multiple novel insights into the global effects of furfural on genomic stability.IMPORTANCE Whether and how furfural affects genome integrity have not been clarified. Using a Saccharomyces cerevisiae model, we found that furfural exposure leads to in vivo DSBs and elevation in mitotic recombination by orders of magnitude. Gross chromosomal rearrangements and aneuploidy events also occurred at a higher frequency in furfural-treated cells. In a genome-wide analysis, we show that the patterns of mitotic recombination and point mutations differed dramatically in furfural-treated cells and wild-type cells.
Assuntos
Carcinógenos , Divisão Celular/efeitos dos fármacos , Furaldeído/efeitos adversos , Genoma Fúngico/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Cromossomos Fúngicos/efeitos dos fármacos , Cromossomos Fúngicos/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Genoma Fúngico/genética , Saccharomyces cerevisiae/genéticaRESUMO
DNA replication stress (DRS)-induced genomic instability is an important factor driving cancer development. To understand the mechanisms of DRS-associated genomic instability, we measured the rates of genomic alterations throughout the genome in a yeast strain with lowered expression of the replicative DNA polymerase δ. By a genetic test, we showed that most recombinogenic DNA lesions were introduced during S or G2 phase, presumably as a consequence of broken replication forks. We observed a high rate of chromosome loss, likely reflecting a reduced capacity of the low-polymerase strains to repair double-stranded DNA breaks (DSBs). We also observed a high frequency of deletion events within tandemly repeated genes such as the ribosomal RNA genes. By whole-genome sequencing, we found that low levels of DNA polymerase δ elevated mutation rates, both single-base mutations and small insertions/deletions. Finally, we showed that cells with low levels of DNA polymerase δ tended to accumulate small promoter mutations that increased the expression of this polymerase. These deletions conferred a selective growth advantage to cells, demonstrating that DRS can be one factor driving phenotypic evolution.
Assuntos
Replicação do DNA/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Instabilidade Genômica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Aneuploidia , Cromossomos Fúngicos/genética , Variações do Número de Cópias de DNA , DNA Polimerase III/metabolismo , Humanos , Mutação INDEL , Perda de Heterozigosidade , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA , Estresse Fisiológico , Sequências de Repetição em TandemRESUMO
Yeast Saccharomyces cerevisiae strains isolated from different sources generally show extensive genetic and phenotypic diversity. Understanding how genomic variations influence phenotypes is important for developing strategies with improved economic traits. The diploid S. cerevisiae strain NY1308 is used for cellulosic bioethanol production. Whole genome sequencing identified an extensive amount of single nucleotide variations and small insertions/deletions in the genome of NY1308 compared with the S288c genome. Gene annotation of the assembled NY1308 genome showed that 43 unique genes are absent in the S288c genome. Phylogenetic analysis suggested most of the unique genes were obtained through horizontal gene transfer from other species. RNA-Seq revealed that some unique genes were not functional in NY1308 due to unidentified intron sequences. During bioethanol fermentation, NY1308 tends to flocculate when certain inhibitors (derived from the pretreatment of cellulosic feedstock) are present in the fermentation medium. qRT-PCR and genetic manipulation confirmed that the novel gene, NYn43, contributed to the flocculation ability of NY1308. Deletion of NYn43 resulted in a faster fermentation rate for NY1308. This work disclosed the genetic characterization of a bioethanol-producing S. cerevisiae strain and provided a useful paradigm showing how the genetic diversity of the yeast population would facilitate the personalized development of desirable traits.
Assuntos
Etanol/metabolismo , Saccharomyces cerevisiae/genética , Diploide , Fermentação , Genoma Fúngico , Anotação de Sequência Molecular , Fenótipo , Filogenia , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Polyploidy is common in Saccharomyces cerevisiae strains, but the physiological and phenotypic effects of ploidy changes have not been fully clarified. Here, isogenic diploid, triploid, and tetraploid S. cerevisiae strains were constructed from a haploid strain, CEN.PK2-1C. Stress tolerance and ethanol fermentation performance of the four euploid strains were compared. Each euploid strain had strengths and weaknesses in tolerance to certain stressors, and no single strain was tolerant of all stressors. The diploid had higher ethanol production than the other strains in normal fermentation medium, while the triploid strain showed the fastest fermentation rate in the presence of inhibitors found in lignocellulosic hydrolysate. Physiological determination revealed diverse physiological attributes, such as trehalose, ergosterol, glutathione, and anti-oxidative enzymes among the strains. Our analyses suggest that both ploidy parity and number of chromosome sets contribute to changes in physiological status. Using qRT-PCR, different expression patterns of genes involved in the regulation of cell morphology and the biosynthesis of key physiological attributes among strains were determined. Our data provide novel insights into the multiple effects of genome duplication on yeast cells and are a useful reference for breeding excellent strains used in specific industrial applications.
Assuntos
Duplicação Gênica , Genoma Fúngico , Microbiologia Industrial , Saccharomyces cerevisiae/genética , Ergosterol/metabolismo , Etanol/metabolismo , Fermentação , Expressão Gênica , Fenótipo , Poliploidia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico/genética , Trealose/metabolismoRESUMO
Genomic structural variation (GSV) is a ubiquitous phenomenon observed in the genomes of Saccharomyces cerevisiae strains with different genetic backgrounds; however, the physiological and phenotypic effects of GSV are not well understood. Here, we first revealed the genetic characteristics of a widely used industrial S. cerevisiae strain, ZTW1, by whole genome sequencing. ZTW1 was identified as an aneuploidy strain and a large-scale GSV was observed in the ZTW1 genome compared with the genome of a diploid strain YJS329. These GSV events led to copy number variations (CNVs) in many chromosomal segments as well as one whole chromosome in the ZTW1 genome. Changes in the DNA dosage of certain functional genes directly affected their expression levels and the resultant ZTW1 phenotypes. Moreover, CNVs of large chromosomal regions triggered an aneuploidy stress in ZTW1. This stress decreased the proliferation ability and tolerance of ZTW1 to various stresses, while aneuploidy response stress may also provide some benefits to the fermentation performance of the yeast, including increased fermentation rates and decreased byproduct generation. This work reveals genomic characters of the bioethanol S. cerevisiae strain ZTW1 and suggests that GSV is an important kind of mutation that changes the traits of industrial S. cerevisiae strains.
Assuntos
Etanol/metabolismo , Variação Estrutural do Genoma , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Dosagem de Genes , Fenótipo , Ploidias , Análise de Sequência de DNARESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent. The aim of this study is to produce large quantities of highly pure and bioactive recombinant human TRAIL. Here, TRAIL was expressed in soluble form by pH-stat fed-batch cultivation and purified using a rapid and simple two-step chromatographic procedure. To improve the soluble yield, expression of TRAIL in Escherichia coli was induced with low IPTG concentration (0.1 mM) at low temperature (28 °C) supplemented with ZnSO4 (0.5 mM), using glycerol as carbon source. Under the optimized conditions, 4.14 ± 0.19 g/L of TRAIL in soluble form was achieved at 19 h without pure oxygen. To purify the recombinant TRAIL, we developed an efficient two-step chromatographic procedure including affinity chromatography and cation-exchange chromatography, especially improved the cation-exchange chromatography using a combination of pH and NaCl gradients strategy. Consequently, 4313.5 mg of target protein with high purity (98.1%) was obtained from 2.3 L of cell broth. Our results also showed that the purified TRAIL was with ordered secondary and tertiary structures, in homogeneous form and with strong cytotoxicity.
Assuntos
Ligante Indutor de Apoptose Relacionado a TNF , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificaçãoRESUMO
OBJECTIVES: To improve tolerance to acetic acid that is present in lignocellulosic hydrolysates and affects bioethanol production by Saccharomyces cerevisiae. RESULTS: Saccharomyces cerevisiae strains with improved tolerance to acetic acid were obtained through deletion of the JJJ1 gene. The lag phase of the JJJ1 deletion mutant BYΔJJJ1 was ~16 h shorter than that of the parent strain, BY4741, when the fermentation medium contained 4.5 g acetic acid/l. Additionally, the specific ethanol production rate of BYΔJJJ1 was increased (0.057 g/g h) compared to that of the parent strain (0.051 g/g h). Comparative transcription and physiological analyses revealed higher long chain fatty acid, trehalose, and catalase contents might be critical factors responsible for the acetic acid resistance of JJJ1 knockout strains. CONCLUSIONS: JJJ1 deletion improves acetic acid tolerance and ethanol fermentation performance of S. cerevisiae.
Assuntos
Ácido Acético/farmacologia , Etanol/metabolismo , Saccharomyces cerevisiae/metabolismo , Fermentação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Xylose is described as a component of bacterial exopolysaccharides in only a limited number of bacterial strains. A bacterial strain, Paenibacillus elgii, B69 was shown to be efficient in producing a xylose-containing exopolysaccharide. Sequence analysis was performed to identify the genes encoding the uridine diphosphate (UDP)-glucuronic acid decarboxylase required for the synthesis of UDP-xylose, the precursor of the exopolysaccharide. Two sequences, designated as Peuxs1 and Peuxs2, were found as the candidate genes for such enzymes. The activities of the UDP-glucuronic acid decarboxylases were proven by heterologous expression and real-time nuclear magnetic resonance analysis. The intracellular activity and effect of these genes on the synthesis of exopolysaccharide were further investigated by developing a thymidylate synthase based knockout system. This system was used to substitute the conventional antibiotic resistance gene system in P. elgii, a natural multi-antibiotic resistant strain. Results of intracellular nucleotide sugar analysis showed that the intracellular UDP-xylose and UDP-glucuronic acid levels were affected in Peuxs1 or Peuxs2 knockout strains. The knockout of either Peuxs1 or Peuxs2 reduced the polysaccharide production and changed the monosaccharide ratio. No polysaccharide was found in the Peuxs1/Peuxs2 double knockout strain. Our results show that P. elgii can be efficient in forming UDP-xylose, which is then used for the synthesis of xylose-containing exopolysaccharide.
Assuntos
Carboxiliases/metabolismo , Paenibacillus/metabolismo , Polissacarídeos/biossíntese , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Farmacorresistência Bacteriana/genética , Técnicas de Inativação de Genes , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutação , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Uridina Difosfato Xilose/metabolismo , Xilose/química , Xilose/metabolismoRESUMO
Baker's yeast (Saccharomyces cerevisiae) is the common yeast used in the fields of bread making, brewing, and bioethanol production. Growth rate, stress tolerance, ethanol titer, and byproducts yields are some of the most important agronomic traits of S. cerevisiae for industrial applications. Here, we developed a novel method of constructing S. cerevisiae strains for co-producing bioethanol and ergosterol. The genome of an industrial S. cerevisiae strain, ZTW1, was first reconstructed through treatment with an antimitotic drug followed by sporulation and hybridization. A total of 140 mutants were selected for ethanol fermentation testing, and a significant positive correlation between ergosterol content and ethanol production was observed. The highest performing mutant, ZG27, produced 7.9 % more ethanol and 43.2 % more ergosterol than ZTW1 at the end of fermentation. Chromosomal karyotyping and proteome analysis of ZG27 and ZTW1 suggested that this breeding strategy caused large-scale genome structural variations and global gene expression diversities in the mutants. Genetic manipulation further demonstrated that the altered expression activity of some genes (such as ERG1, ERG9, and ERG11) involved in ergosterol synthesis partly explained the trait improvement in ZG27.
Assuntos
Ergosterol/biossíntese , Etanol/metabolismo , Engenharia Genética , Microbiologia Industrial , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fermentação , Variação Genética , Fenótipo , Proteômica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Whole-genome shuffling (WGS) is a powerful technology of improving the complex traits of many microorganisms. However, the molecular mechanisms underlying the altered phenotypes in isolates were less clarified. Isolates with significantly enhanced stress tolerance and ethanol titer under very-high-gravity conditions were obtained after WGS of the bioethanol Saccharomyces cerevisiae strain ZTW1. Karyotype analysis and RT-qPCR showed that chromosomal rearrangement occurred frequently in genome shuffling. Thus, the phenotypic effects of genomic structural variations were determined in this study. RNA-Seq and physiological analyses revealed the diverse transcription pattern and physiological status of the isolate S3-110 and ZTW1. Our observations suggest that the improved stress tolerance of S3-110 can be largely attributed to the copy number variations in large DNA regions, which would adjust the ploidy of yeast cells and expression levels of certain genes involved in stress response. Overall, this work not only constructed shuffled S. cerevisiae strains that have potential industrial applications but also provided novel insights into the molecular mechanisms of WGS and enhanced our knowledge on this useful breeding strategy.
Assuntos
Embaralhamento de DNA , Variação Estrutural do Genoma , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cariotipagem , Reação em Cadeia da Polimerase em Tempo RealRESUMO
An understanding of the genetic basis underlying the phenotypic variations of yeast strains would guide the breeding of this useful microorganism. Here, comparative functional genomics (CFG) of two bioethanol Saccharomyces cerevisiae strains (YJS329 and ZK2) with different stress tolerances and ethanol fermentation performances were performed. Our analysis indicated that different patterns of gene expression in the central carbon metabolism, antioxidative factors, and membrane compositions of these two strains are the main contributors to their various traits. Some of the differently expressed genes were directly caused by the genomic structural variations between YJS329 and ZK2. Moreover, CFG of these two strains also led to novel insights into the mechanism of stress tolerance in yeast. For example, it was found that more oleic acid in the plasma membrane contributes to the acetic acid tolerance of yeast. Based on the genetic information particular to each strain, strategies to improve their adaptability and ethanol fermentation performances were designed and confirmed. Thus, CFG could not only help reveal basis of phenotypic diversities but also guide the genetic breeding of industrial microorganisms.
Assuntos
Etanol/metabolismo , Genômica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Embaralhamento de DNA/métodos , Perfilação da Expressão Gênica , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genéticaRESUMO
BACKGROUND: Environmental stresses and inhibitors encountered by Saccharomyces cerevisiae strains are the main limiting factors in bioethanol fermentation. Strains with different genetic backgrounds usually show diverse stress tolerance responses. An understanding of the mechanisms underlying these phenotypic diversities within S. cerevisiae populations could guide the construction of strains with desired traits. RESULTS: We explored the genetic characteristics of the bioethanol S. cerevisiae strain YJS329 and elucidated how genetic variations in its genome were correlated with specified traits compared to similar traits in the S288c-derived strain, BYZ1. Karyotypic electrophoresis combined with array-comparative genomic hybridization indicated that YJS329 was a diploid strain with a relatively constant genome as a result of the fewer Ty elements and lack of structural polymorphisms between homologous chromosomes that it contained. By comparing the sequence with the S288c genome, a total of 64,998 SNPs, 7,093 indels and 11 unique genes were identified in the genome of YJS329-derived haploid strain YJSH1 through whole-genome sequencing. Transcription comparison using RNA-Seq identified which of the differentially expressed genes were the main contributors to the phenotypic differences between YJS329 and BYZ1. By combining the results obtained from the genome sequences and the transcriptions, we predicted how the SNPs, indels and chromosomal copy number variations may affect the mRNA expression profiles and phenotypes of the yeast strains. Furthermore, some genetic breeding strategies to improve the adaptabilities of YJS329 were designed and experimentally verified. CONCLUSIONS: Through comparative functional genomic analysis, we have provided some insights into the mechanisms underlying the specific traits of the bioenthanol strain YJS329. The work reported here has not only enriched the available genetic resources of yeast but has also indicated how functional genomic studies can be used to improve genetic breeding in yeast.
Assuntos
Variação Genética , Genoma Fúngico , Microbiologia Industrial , Saccharomyces cerevisiae/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Etanol/metabolismo , Fermentação , Genômica/métodos , Mutação INDEL , Fases de Leitura Aberta , Fenótipo , Polimorfismo de Nucleotídeo Único , Saccharomyces cerevisiae/fisiologia , Análise de Sequência de DNA , Estresse FisiológicoRESUMO
Hospital-acquired infections caused by drug-resistant bacteria are a significant challenge to patient safety. Numerous clinical isolates resistant to almost all commercially available antibiotics have emerged. Thus, novel antimicrobial agents, specifically those for multidrug-resistant Gram-negative bacteria, are urgently needed. In the current study, we report the isolation, structure elucidation, and preliminary biological characterization of a new cationic lipopeptide antibiotic, battacin or octapeptin B5, produced from a Paenibacillus tianmuensis soil isolate. Battacin kills bacteria in vitro and has potent activity against Gram-negative bacteria, including multidrug-resistant and extremely drug-resistant clinical isolates. Hospital strains of Escherichia coli and Pseudomonas aeruginosa are the pathogens most sensitive to battacin, with MICs of 2 to 4 µg/ml. The ability of battacin to disrupt the outer membrane of Gram-negative bacteria is comparable to that of polymyxin B, the last-line therapy for infections caused by antibiotic-resistant Gram-negative bacteria. However, the capacity of battacin to permeate bacterial plasma membranes is less extensive than that of polymyxin B. The bactericidal kinetics of battacin correlate with the depolarization of the cell membrane, suggesting that battacin kills bacteria by disrupting the cytoplasmic membrane. Other studies indicate that battacin is less acutely toxic than polymyxin B and has potent in vivo biological activity against E. coli. Based on the findings of the current study, battacin may be considered a potential therapeutic agent for the treatment of infections caused by antibiotic-resistant Gram-negative bacteria.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/efeitos dos fármacos , Lipopeptídeos/farmacologia , Paenibacillus/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Transporte Biológico/efeitos dos fármacos , Permeabilidade da Membrana Celular , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla , Escherichia coli/crescimento & desenvolvimento , Fermentação , Células HEK293 , Hemólise , Humanos , Cinética , Dose Letal Mediana , Lipopeptídeos/biossíntese , Lipopeptídeos/isolamento & purificação , Camundongos , Testes de Sensibilidade Microbiana , Polimixina B/farmacologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Espectrometria de Massas em TandemRESUMO
BACKGROUND: The recent increase in bacterial resistance to antibiotics has promoted the exploration of novel antibacterial materials. As a result, many researchers are undertaking work to identify new lantibiotics because of their potent antimicrobial activities. The objective of this study was to provide details of a lantibiotic-like gene cluster in Paenibacillus elgii B69 and to produce the antibacterial substances coded by this gene cluster based on culture screening. RESULTS: Analysis of the P. elgii B69 genome sequence revealed the presence of a lantibiotic-like gene cluster composed of five open reading frames (elgT1, elgC, elgT2, elgB, and elgA). Screening of culture extracts for active substances possessing the predicted properties of the encoded product led to the isolation of four novel peptides (elgicins AI, AII, B, and C) with a broad inhibitory spectrum. The molecular weights of these peptides were 4536, 4593, 4706, and 4820 Da, respectively. The N-terminal sequence of elgicin B was Leu-Gly-Asn-Tyr, [corrected] which corresponded to the partial sequence of the peptide ElgA encoded by elgA. Edman degradation suggested that the product elgicin B is derived from ElgA. By correlating the results of electrospray ionization-mass spectrometry analyses of elgicins AI, AII, and C, these peptides are deduced to have originated from the same precursor, ElgA. CONCLUSIONS: A novel lantibiotic-like gene cluster was shown to be present in P. elgii B69. Four new lantibiotics with a broad inhibitory spectrum were isolated, and these appear to be promising antibacterial agents.
Assuntos
Antibacterianos/biossíntese , Bacteriocinas/biossíntese , Bacteriocinas/genética , Genes Bacterianos/genética , Paenibacillus/genética , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bacteriocinas/química , Bacteriocinas/farmacologia , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Família Multigênica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
BACKGROUND: Pelgipeptin, a potent antibacterial and antifungal agent, is a non-ribosomally synthesised lipopeptide antibiotic. This compound consists of a ß-hydroxy fatty acid and nine amino acids. To date, there is no information about its biosynthetic pathway. RESULTS: A potential pelgipeptin synthetase gene cluster (plp) was identified from Paenibacillus elgii B69 through genome analysis. The gene cluster spans 40.8 kb with eight open reading frames. Among the genes in this cluster, three large genes, plpD, plpE, and plpF, were shown to encode non-ribosomal peptide synthetases (NRPSs), with one, seven, and one module(s), respectively. Bioinformatic analysis of the substrate specificity of all nine adenylation domains indicated that the sequence of the NRPS modules is well collinear with the order of amino acids in pelgipeptin. Additional biochemical analysis of four recombinant adenylation domains (PlpD A1, PlpE A1, PlpE A3, and PlpF A1) provided further evidence that the plp gene cluster involved in pelgipeptin biosynthesis. CONCLUSIONS: In this study, a gene cluster (plp) responsible for the biosynthesis of pelgipeptin was identified from the genome sequence of Paenibacillus elgii B69. The identification of the plp gene cluster provides an opportunity to develop novel lipopeptide antibiotics by genetic engineering.
Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Lipopeptídeos/biossíntese , Família Multigênica , Paenibacillus/genética , Paenibacillus/metabolismo , Biologia Computacional , DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Dados de Sequência Molecular , Fases de Leitura Aberta , Peptídeo Sintases/genética , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
Here, we report the draft genome sequence of Paenibacillus elgii B69, which was isolated from soil and has broad-spectrum antimicrobial activity. As far as we know, the P. elgii genome is the largest of the Paenibacillus genus for which genome sequences are available. Multiple sets of genes related to antibiotic biosynthetic pathways have been found in the genome.
Assuntos
Anti-Infecciosos/metabolismo , Genoma Bacteriano , Paenibacillus/genética , Sequência de Bases , Dados de Sequência Molecular , Família Multigênica , Paenibacillus/metabolismo , Microbiologia do SoloRESUMO
Bacteria belonging to the genus Paenibacillus are recognized as rich sources of bioactive natural products. To date, there are few characterized siderophores from this genus. Here, through genome analysis, we identified a non-ribosomal peptide biosynthetic gene cluster (pae) responsible for siderophore assembly in Paenibacillus elgii B69. The 12.8 kb gene cluster comprises six open reading frames encoding proteins similar to the components of the bacillibactin biosynthetic machinery and bacillibactin esterase. To examine the product of the pae gene cluster, we cultured P. elgii B69 in iron-deficient medium for siderophore expression. A novel siderophore structurally similar to bacillibactin, designated paenibactin, was purified and characterized. Its structure was determined as a cyclic trimeric lactone of 2,3-dihydroxybenzoyl-alanine-threonine. The involvement of the pae gene cluster in paenibactin biosynthesis was confirmed by the biochemical assay of adenylation domain specificity. Furthermore, we demonstrated that the pae gene cluster evolves from an ancestral bacillibactin biosynthetic gene cluster via sequence and phylogenetic analyses. The structural difference between paenibactin and bacillibactin may stem from a mutation-induced change in the adenylation domain specificity. Based on these findings and published models for bacillibactin, we proposed models for paenibactin biosynthesis, ferric-paenibactin uptake and paenibactin-bounded iron release.