RESUMEN
Microbes hold the key to life. They hold the secrets to our past (as the descendants of the earliest forms of life) and the prospects for our future (as we mine their genes for solutions to some of the planet's most pressing problems, from global warming to antibiotic resistance). However, the piecemeal approach that has defined efforts to study microbial genetic diversity for over 20 years and in over 30,000 genome projects risks squandering that promise. These efforts have covered less than 20% of the diversity of the cultured archaeal and bacterial species, which represent just 15% of the overall known prokaryotic diversity. Here we call for the funding of a systematic effort to produce a comprehensive genomic catalog of all cultured Bacteria and Archaea by sequencing, where available, the type strain of each species with a validly published name (currentlyâ¼11,000). This effort will provide an unprecedented level of coverage of our planet's genetic diversity, allow for the large-scale discovery of novel genes and functions, and lead to an improved understanding of microbial evolution and function in the environment.
Asunto(s)
Genoma Arqueal/genética , Genoma Bacteriano/genética , Genómica , Análisis de Secuencia de ADN , Archaea/clasificación , Archaea/genética , Bacterias/clasificación , Bacterias/genética , Bases de Datos Genéticas , FilogeniaRESUMEN
Three strains of lactic acid bacteria (LAB) were isolated from the faeces of apparently healthy wild Canada geese (Branta canadensis) in 2010 by cultivating faecal LAB on Rogosa SL agar under aerobic conditions. These three isolates were found to share 99.9 % gene sequence similarity of their 16S rRNA, their 16S-23S intergenic transcribed spacer region (ITS), partial 23S rRNA, rpoB, rpoC, rpoA and pheS gene sequences. However, the three strains exhibited lower levels of sequence similarity of these genetic targets to all known LAB, and the phylogenetically closest species to the geese strains were Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus saniviri. In comparison to L. casei ATCC 393(T), L. paracasei ATCC 25302(T), L. rhamnosus ATCC 7469(T) and L. saniviri DSM 24301(T), the novel isolates reacted uniquely in tests for cellobiose, galactose, mannitol, citric acid, aesculin and dextrin, and gave negative results in tests for l-proline arylamidase and l-pyrrolydonyl-arylamidase, and in the Voges-Proskauer test. Biochemical tests for cellobiose, aesculin, galactose, gentiobiose, mannitol, melezitose, ribose, salicin, sucrose, trehalose, raffinose, turanose, amygdalin and arbutin could be used for differentiation between L. saniviri and the novel strains. On the basis of phenotypic and genotypic characteristics, and phylogenetic data, the three isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus brantae sp. nov. is proposed. The type strain is SL1108(T) (= ATCC BAA-2142(T) = LMG 26001(T) = DSM 23927(T)) and two additional strains are SL1170 and SL60106.
Asunto(s)
Gansos/microbiología , Lactobacillus/clasificación , Filogenia , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , Carbohidratos/análisis , ADN Bacteriano/genética , ADN Espaciador Ribosómico/genética , Ácidos Grasos/análisis , Heces/microbiología , Genes Bacterianos , Genotipo , Lactobacillus/genética , Lactobacillus/aislamiento & purificación , Datos de Secuencia Molecular , Fenotipo , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
ATCC 49444, originally designated as Staphylococcus aureus, has been cited as a component strain in the CAMP test for identification of Listeria monocytogenes. A polyphasic study, in which molecular data were combined with cytochemical properties and physiological characteristics, identified this isolate as Staphylococcus pseudintermedius. The nucleotide sequences of the 16S rRNA and sodA genes from ATCC 49444 were determined and found to be identical to those of other S. pseudintermedius strains. Ribotyping analysis of ATCC 49444 indicated a low (0.2) similarity index with S. aureus and Staphylococcus intermedius strains but a similarity index of more than 0.66 with S. pseudintermedius isolates. A functional comparison of ATCC 49444 with S. aureus ATCC 25923 utilizing the CAMP test demonstrated equivalent hemolytic enhancement with L. monocytogenes isolates. While this study demonstrates that ATCC 49444 is a valid component strain for the CAMP test, it is proposed that ATCC 49444 be reclassified as S. pseudintermedius and that users worldwide be alerted to this reclassification and name change.
Asunto(s)
ADN Bacteriano/genética , Filogenia , Staphylococcus/clasificación , Staphylococcus/genética , Técnicas de Tipificación Bacteriana , Secuencia de Bases , ADN Ribosómico/genética , Datos de Secuencia Molecular , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Ribotipificación , Infecciones Estafilocócicas/microbiologíaRESUMEN
The design and synthesis of a fluorescent probe Fl-Sal-AMS 6 based on the tight-binding inhibitor 5'- O-[ N-(salicyl)sulfamoyl]adenosine (Sal-AMS) is described for the aryl acid adenylating enzymes (AAAEs) known as MbtA, YbtE, EntE, VibE, DhbE, and BasE involved in siderophore biosynthesis from Mycobacterium tuberculosis, Yersinia pestis, Escherichia coli, Vibrio cholerae, Bacillus subtilis, and Acinetobacter baumannii, respectively. The probe was successfully used to develop a fluorescence polarization assay for these six AAAEs, and equilibrium dissociation constants were determined in direct binding experiments. Fl-Sal-AMS was effective for AAAEs that utilize salicylic acid or 2,3-dihydroxybenzoic acid as native substrates, with dissociation constants ranging from 9-369 nM, but was ineffective for AsbC, the AAAE from Bacillus anthracis, which activates 3,4-dihydroxybenzoic acid. Competitive binding experiments using a series of ligands including substrates, reaction products, and inhibitors provided the first comparative structure-activity relationships for AAAEs. The fluorescence polarization assay was then miniaturized to a 384-well plate format, and high-throughput screening was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB) against BasE, an AAAE from Acinetobacter baumannii involved in production of the siderophore acinetobactin. Several small molecule inhibitors with new chemotypes were identified, and compound 23 containing a pyrazolo[5,4- a]pyridine scaffold emerged as the most promising ligand with a K D of 78 nM, which was independently confirmed by isothermal calorimetry, and inhibition was also verified in an ATP-[ (32)P]-pyrophosphate exchange steady-state kinetic assay.
Asunto(s)
Proteínas Bacterianas/metabolismo , Polarización de Fluorescencia/métodos , Ligasas/metabolismo , Sideróforos/biosíntesis , Adenosina/análogos & derivados , Adenosina/química , Coenzima A Ligasas/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Proteínas de Escherichia coli/metabolismo , Hidroxibenzoatos/metabolismo , Cinética , Ligandos , Estructura Molecular , Ácido Salicílico/metabolismo , Sideróforos/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The pikromycin polyketide synthase (PKS) is unique in its ability to generate both 12 and 14 membered ring macrolactones. As such, dissection of the molecular basis for controlling metabolic diversity in this system remains an important objective for understanding modular PKS function and expanding chemical diversity. Here, we describe a series of experiments designed to probe the importance of the protein-protein interaction that occurs between the final two monomodules, PikAIII (module 5) and PikAIV (module 6), for the production of the 12 membered ring macrolactone 10-deoxymethynolide. The results obtained from these in vitro studies demonstrate that PikAIII and PikAIV generate the 12 membered ring macrocycle most efficiently when engaged in their native protein-protein interaction. Accordingly, the data are consistent with PikAIV adopting an alternative conformation that enables the terminal thioesterase domain to directly off-load the PikAIII-bound hexaketide intermediate for macrocyclization.
Asunto(s)
Lactonas/química , Macrólidos/metabolismo , Sintasas Poliquetidas/metabolismo , Secuencia de Bases , Dominio Catalítico , Ciclización , Cartilla de ADN , Esterasas/metabolismo , Mutagénesis Sitio-Dirigida , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genéticaRESUMEN
Tuberculosis is the leading cause of infectious disease mortality in the world by a bacterial pathogen. We previously demonstrated that a bisubstrate inhibitor of the adenylation enzyme MbtA, which is responsible for the second step of mycobactin biosynthesis, exhibited potent antitubercular activity. Here we systematically investigate the structure-activity relationships of the bisubstrate inhibitor glycosyl domain resulting in the identification of a carbocyclic analogue that possesses a KIapp value of 2.3 nM and MIC99 values of 1.56 microM against M. tuberculosis H37Rv. The SAR data suggest the intriguing possibility that the bisubstrate inhibitors utilize a transporter for entry across the mycobacterial cell envelope. Additionally, we report improved conditions for the expression of MbtA and biochemical analysis, demonstrating that MbtA follows a random sequential enzyme mechanism for the adenylation half-reaction.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Nucleósidos/farmacología , Sideróforos/biosíntesis , Tuberculosis/tratamiento farmacológico , Adenosina Trifosfato/metabolismo , Antituberculosos/síntesis química , Antituberculosos/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Cinética , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Mycobacterium tuberculosis/enzimología , Nucleósidos/síntesis química , Nucleósidos/química , Relación Estructura-Actividad , Ácidos Sulfónicos/químicaRESUMEN
The pikromycin (Pik) polyketide synthase (PKS) from Streptomyces venezuelae comprises four multifunctional polypeptides (PikAI, PikAII, PikAIII, and PikAIV). This PKS can generate 12- and 14-membered ring macrolactones (10-deoxymethynolide and narbonolide, respectively) through the activity of its terminal modules (PikAIII and PikAIV). We performed a series of experiments involving the functional replacement of PikAIV in mutant strains with homodimeric and heterodimeric PikAIV modules to investigate the details of macrolactone ring size determination. The results suggest a new and surprising mechanism by which the penultimate hexaketide chain elongation intermediate is transferred from PikAIII ACP5 to PikAIV ACP6 before release by the terminal thioesterase domain. Elucidation of this chain transfer mechanism provides important new details about alternative macrolactone ring size formation in modular PKSs and contributes to the potential for rational design of structural diversity by combinatorial biosynthesis.
Asunto(s)
Antibacterianos/biosíntesis , Lactonas/metabolismo , Macrólidos/síntesis química , Complejos Multienzimáticos/metabolismo , Dominio Catalítico , Dimerización , Activación Enzimática , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Lactonas/síntesis química , Macrólidos/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutagénesis Sitio-Dirigida , Plásmidos/biosíntesis , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Streptomyces/enzimología , Streptomyces/genética , Streptomyces/metabolismo , Relación Estructura-ActividadRESUMEN
The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase, leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products.
Asunto(s)
Antibacterianos/biosíntesis , Complejos Multienzimáticos/genética , Streptomyces/enzimología , Secuencia de Aminoácidos , Antibacterianos/química , Eliminación de Gen , Prueba de Complementación Genética , Ingeniería Genética , Macrólidos , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Plásmidos/genética , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project was launched by the JGI in 2007 as a pilot project to sequence about 250 bacterial and archaeal genomes of elevated phylogenetic diversity. Herein, we propose to extend this approach to type strains of prokaryotes associated with soil or plants and their close relatives as well as type strains from newly described species. Understanding the microbiology of soil and plants is critical to many DOE mission areas, such as biofuel production from biomass, biogeochemistry, and carbon cycling. We are also targeting type strains of novel species while they are being described. Since 2006, about 630 new species have been described per year, many of which are closely aligned to DOE areas of interest in soil, agriculture, degradation of pollutants, biofuel production, biogeochemical transformation, and biodiversity.
RESUMEN
The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project was launched by the JGI in 2007 as a pilot project with the objective of sequencing 250 bacterial and archaeal genomes. The two major goals of that project were (a) to test the hypothesis that there are many benefits to the use the phylogenetic diversity of organisms in the tree of life as a primary criterion for generating their genome sequence and (b) to develop the necessary framework, technology and organization for large-scale sequencing of microbial isolate genomes. While the GEBA pilot project has not yet been entirely completed, both of the original goals have already been successfully accomplished, leading the way for the next phase of the project. Here we propose taking the GEBA project to the next level, by generating high quality draft genomes for 1,000 bacterial and archaeal strains. This represents a combined 16-fold increase in both scale and speed as compared to the GEBA pilot project (250 isolate genomes in 4+ years). We will follow a similar approach for organism selection and sequencing prioritization as was done for the GEBA pilot project (i.e. phylogenetic novelty, availability and growth of cultures of type strains and DNA extraction capability), focusing on type strains as this ensures reproducibility of our results and provides the strongest linkage between genome sequences and other knowledge about each strain. In turn, this project will constitute a pilot phase of a larger effort that will target the genome sequences of all available type strains of the Bacteria and Archaea.
RESUMEN
The unique ability of the pikromycin (Pik) polyketide synthase to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes underlying polyketide synthesis, specifically the mechanistic details of chain extension, keto group processing, acyl chain release, and macrocyclization. We have synthesized the natural pentaketide and hexaketide chain elongation intermediates as N-acetyl cysteamine (NAC) thioesters and have used them as substrates for in vitro conversions with engineered PikAIII+TE and in combination with native PikAIII (module 5) and PikAIV (module 6) multifunctional proteins. This investigation demonstrates directly the remarkable ability of these monomodules to catalyze one or two chain extension reactions, keto group processing steps, acyl-ACP release, and cyclization to generate 10-deoxymethynolide and narbonolide. The results reveal the enormous preference of Pik monomodules for their natural polyketide substrates and provide an important comparative analysis with previous studies using unnatural diketide NAC thioester substrates.
Asunto(s)
Macrólidos/metabolismo , Sintasas Poliquetidas/metabolismo , Secuencia de Aminoácidos , Cisteamina/análogos & derivados , Cisteamina/metabolismo , Cinética , Datos de Secuencia Molecular , Sintasas Poliquetidas/químicaRESUMEN
In Salmonella enterica serovar Typhimurium, gmk encodes guanylate kinase, an essential enzyme involved in the synthesis and salvage of guanine nucleotides. Here we report the isolation of a mutation in gmk that results in a nutritional requirement for adenine at low temperature. Comparisons of kinetic parameters from the wild-type and mutant Gmk enzymes revealed that the mutant enzyme had a more than 20-fold-higher Km for ATP than the wild-type enzyme. The growth dependence of the mutant on temperature and/or adenine could not be explained as a direct result of this kinetic difference. We propose a model in which previously described regulatory effects of GMP are responsible for these phenotypes.
Asunto(s)
Adenina/metabolismo , Frío , Regulación Bacteriana de la Expresión Génica , Mutación , Nucleósido-Fosfato Quinasa/genética , Salmonella typhimurium/enzimología , Genes Bacterianos , Genes Esenciales , Guanilato-Quinasas , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , Nucleósido-Fosfato Quinasa/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrollo , Análisis de Secuencia de ADNRESUMEN
The unique ability of the pikromycin (Pik) polyketide synthase to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes underlying polyketide synthesis, specifically the mechanistic details of the chain extension process. We have overexpressed and purified PikAIII (module 5) and PikAIV (module 6) and assessed the ability of these proteins to generate tri- and tetraketide lactone products using N-acetylcysteamine-activated diketides and (14)C-methylmalonyl-CoA as substrates. Comparison of the stereochemical specificities for PikAIII and PikAIV and the reported values for the DEBS modules reveals significant differences between these systems.
Asunto(s)
Macrólidos/metabolismo , Complejos Multienzimáticos/metabolismo , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Escherichia coli/enzimología , Escherichia coli/genética , Cinética , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/aislamiento & purificación , Streptomyces/enzimologíaRESUMEN
The unique ability of the pikromycin polyketide synthase (Pik PKS) to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes of polyketide synthesis, specifically, the mechanistic details of the chain extension process. We have overexpressed and purified PikAIII and PikAIV and demonstrated the ability of these proteins to generate triketide lactone products using (14)C-methylmalonyl-CoA as the sole substrate. Monomodular PikAIII generates TKL (1) when reacted alone, and synthesizes TKL (2) upon reaction in combination with PikAIV. Product formation remains dependent on the enzymatic decarboxylation of methylmalonyl-CoA and transfer of the acyl chain within the enzyme rather than acylation by propionyl-CoA from spontaneous decarboxylation. We propose that synthesis of TKL (1) by PikAIII involves iterative assembly of the triketide chain within a PikAIII homodimer analogous to the nonmodular type I PKS systems.
Asunto(s)
Antibacterianos/biosíntesis , Macrólidos , Complejos Multienzimáticos/metabolismo , Lactonas/metabolismo , Complejos Multienzimáticos/química , Streptomyces/enzimologíaRESUMEN
The pikromycin biosynthetic gene cluster contains the pikAV gene encoding a type II thioesterase (TEII). TEII is not responsible for polyketide termination and cyclization, and its biosynthetic role has been unclear. During polyketide biosynthesis, extender units such as methylmalonyl acyl carrier protein (ACP) may prematurely decarboxylate to generate the corresponding acyl-ACP, which cannot be used as a substrate in the condensing reaction by the corresponding ketosynthase domain, rendering the polyketide synthase module inactive. It has been proposed that TEII may serve as an "editing" enzyme and reactivate these modules by removing acyl moieties attached to ACP domains. Using a purified recombinant TEII we have tested this hypothesis by using in vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and methylmalonyl-ACP substrates derived from either PikAIII or the loading didomain of DEBS1 (6-deoxyerythronolide B synthase; AT(L)-ACP(L)). The pikromycin TEII exhibited high K(m) values (>100 microm) with all substrates and no apparent ACP specificity, catalyzing cleavage of methylmalonyl-ACP from both AT(L)-ACP(L) (k(cat)/K(m) 3.3 +/- 1.1 m(-1) s(-1)) and PikAIII (k(cat)/K(m) 2.9 +/- 0.9 m(-1) s(-1)). The TEII exhibited some acyl-group specificity, catalyzing hydrolysis of propionyl (k(cat)/K(m) 15.8 +/- 1.8 m(-1) s(-1)) and butyryl (k(cat)/K(m) 17.5 +/- 2.1 m(-1) s(-1)) derivatives of AT(L)-ACP(L) faster than acetyl (k(cat)/K(m) 4.9 +/- 0.7 m(-1) s(-1)), malonyl (k(cat)/K(m) 3.9 +/- 0.5 m(-1) s(-1)), or methylmalonyl derivatives. PikAIV containing a TEI domain catalyzed cleavage of propionyl derivative of AT(L)-ACP(L) at a dramatically lower rate than TEII. These results provide the first unequivocal in vitro evidence that TEII can hydrolyze acyl-ACP thioesters and a model for the action of TEII in which the enzyme remains primarily dissociated from the polyketide synthase, preferentially removing aberrant acyl-ACP species with long half-lives. The lack of rigorous substrate specificity for TEII may explain the surprising observation that high level expression of the protein in Streptomyces venezuelae leads to significant (>50%) titer decreases.
Asunto(s)
Antibacterianos/biosíntesis , Ácido Graso Sintasas/metabolismo , Macrólidos , Tioléster Hidrolasas/metabolismo , Secuencia de Bases , Catálisis , Cartilla de ADN , Ácido Graso Sintasas/genética , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimología , Tioléster Hidrolasas/genéticaRESUMEN
Evolutionary links between cytochrome P450 monooxygenases, a superfamily of extraordinarily divergent heme-thiolate proteins catalyzing a wide array of NADPH/NADH- and O(2)-dependent reactions, are becoming better understood because of availability of an increasing number of fully sequenced genomes. Among other reactions, P450s catalyze the site-specific oxidation of the precursors to macrolide antibiotics in the genus Streptomyces introducing regiochemical diversity into the macrolide ring system, thereby significantly increasing antibiotic activity. Developing effective uses for Streptomyces enzymes in biosynthetic processes and bioremediation requires identification and engineering of additional monooxygenases with activities toward a diverse array of small molecules. To elucidate the molecular basis for substrate specificity of oxidative enzymes toward macrolide antibiotics, the x-ray structure of CYP154C1 from Streptomyces coelicolor A3(2) was determined (Protein Data Bank code ). Relocation of certain common P450 secondary structure elements, along with a novel structural feature involving an additional beta-strand transforming the five-stranded beta-sheet into a six-stranded variant, creates an open cleft-shaped substrate-binding site between the two P450 domains. High sequence similarity to macrolide monooxygenases from other microbial species translates into catalytic activity of CYP154C1 toward both 12- and 14-membered ring macrolactones in vitro.