RESUMEN
Predation of starter lactic acid bacteria such as Streptococcus thermophilus by bacteriophages is a persistent and costly problem in the dairy industry. CRISPR-mediated bacteriophage insensitive mutants (BIMs), while straightforward to generate and verify, can quickly be overcome by mutant phages. The aim of this study was to develop a tool allowing the generation of derivatives of commercial S. thermophilus strains which are resistant to phage attack through a non-CRISPR-mediated mechanism, with the objective of generating BIMs exhibiting stable resistance against a range of isolated lytic S. thermophilus phages. To achieve this, standard BIM generation was complemented by the use of the wild-type (WT) strain which had been transformed with an antisense mRNA-generating plasmid (targeting a crucial CRISPR-associated [cas] gene) in order to facilitate the generation of non-CRISPR-mediated BIMs. Phage sensitivity assays suggest that non-CRISPR-mediated BIMs exhibit some advantages compared to CRISPR-mediated BIMs derived from the same strain.IMPORTANCE The outlined approach reveals the presence of a powerful host-imposed barrier for phage infection in S. thermophilus Considering the detrimental economic consequences of phage infection in the dairy processing environment, the developed methodology has widespread applications, particularly where other methods may not be practical or effective in obtaining robust, phage-tolerant S. thermophilus starter strains.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Interferencia de ARN , Fagos de Streptococcus/genética , Streptococcus thermophilus/virología , ADN Bacteriano/genética , ADN Intergénico , Mutación , ARN sin Sentido , Streptococcus thermophilus/genéticaRESUMEN
In the current study, we characterized 137 Lactococcus lactis bacteriophages that had been isolated between 1997 and 2012 from whey samples obtained from industrial facilities located in 16 countries. Multiplex PCR grouping of these 137 phage isolates revealed that the majority (61.31%) belonged to the 936 group, with the remainder belonging to the P335 and c2 groups (23.36 and 15.33%, respectively). Restriction profile analysis of phage genomic DNA indicated a high degree of genetic diversity within this phage collection. Furthermore, based on a host-range survey of the phage collection using 113 dairy starter strains, we showed that the c2-group isolates exhibited a broader host range than isolates of the 936 and P335 groups.
Asunto(s)
Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiología , Biodiversidad , Lactococcus lactis/virología , Suero Lácteo/virología , Animales , Bacteriófagos/clasificación , Bacteriófagos/genética , Bovinos , Variación Genética , FilogeniaRESUMEN
BACKGROUND: Despite continuous research efforts, bacterio(phages) infecting Lactococcus lactis starter strains persist as a major threat to dairy fermentations. The lactococcal P335 phages, which are currently classified into four sub-groups (I-IV), are the second most frequently isolated phage group in an industrial dairy context. RESULTS: The current work describes the isolation and comparative genomic analysis of 17 novel P335 group phages. Detailed analysis of the genomic region of P335 phages encoding the so-called "baseplate", which includes the receptor binding protein (RBP) was combined with a functional characterization of the RBP of sub-group III and IV phages. Additionally, calcium-dependence assays revealed a specific requirement for calcium by sub-group IV phages while host range analysis highlighted a higher number of strains with CWPS type A (11 of 39 strains) are infected by the P335 phages assessed in this study than those with a C (five strains), B (three of 39 strains) or unknown (one of 39 strains) CWPS type. CONCLUSIONS: These analyses revealed significant divergence among RBP sequences, apparently reflecting their unique interactions with the host and particularly for strains with a type A CWPS. The implications of the genomic architecture of lactococcal P335 phages on serving as a general model for Siphoviridae phages are discussed.
Asunto(s)
Bacteriófagos/genética , Bacteriófagos/fisiología , Lactococcus lactis/virología , Bacteriófagos/metabolismo , Calcio/metabolismo , Variación Genética , Genómica , Especificidad del Huésped/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The Gram-positive bacterium Bacillus subtilis contains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion in B. subtilis is a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed in Escherichia coli, both B. subtilis Tat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of the E. coli AmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently in B. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate in B. subtilis. Several investigated GFP fusion proteins were indeed secreted in B. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from the E. coli AmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by the B. subtilis Tat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway.
Asunto(s)
Bacillus subtilis/metabolismo , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Salinidad , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Señales de Clasificación de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Staphylococcus aureus is an important Gram-positive bacterial pathogen producing many secreted and cell surface-localized virulence factors. Here we report that the staphylococcal thiol-disulfide oxidoreductase DsbA is essential for stable biogenesis of the ComGC pseudopilin. The signal peptidase ComC is indispensable for ComGC maturation and optimal cell surface exposure.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Serina Endopeptidasas/metabolismo , Staphylococcus aureus/enzimología , Staphylococcus aureus/metabolismo , Expresión GénicaRESUMEN
Thiol-disulfide oxidoreductases (TDORs) catalyze thiol-disulfide exchange reactions that are crucial for protein activity and stability. Specifically, they can function as thiol oxidases, disulfide reductases or disulfide isomerases. The generally established view is that particular TDORs act unidirectionally within a fixed cascade of specific, sequentially arranged reactions. However, recent studies on both Gram-negative and Gram-positive bacteria imply that this view needs to be expanded, at least for thiol-disulfide exchanges in proteins that are exported from the cytoplasm. Here, we present our opinion that various TDORs can function as interchangeable modules in different thiol-disulfide exchange pathways. Such TDOR modules, thus, fulfil important functions in generating the diversity in activity and specificity that is needed in productive extracytoplasmic thiol-disulfide exchange.
Asunto(s)
Bacillus subtilis/metabolismo , Disulfuros/metabolismo , Escherichia coli/metabolismo , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Disulfuros/química , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Oxidación-Reducción , Proteína Disulfuro Reductasa (Glutatión)/química , Proteína Disulfuro Reductasa (Glutatión)/genética , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Compuestos de Sulfhidrilo/químicaRESUMEN
Bacillus subtilis is a well-established cell factory for efficient secretion of many biotechnologically relevant enzymes that are naturally produced by it or related organisms. However, the use of B. subtilis as a host for production of heterologous secretory proteins can be complicated by problems related to inefficient translocation of the foreign proteins across the plasma membrane or to inefficient release of the exported proteins from the cell surface into the surrounding medium. Therefore, there is a clear need for tools that allow more efficient membrane targeting, translocation, and release during the production of these proteins. In the present study, we investigated the contributions of the pre (pre(lip)) and pro (pro(lip)) sequences of a Staphylococcus hyicus lipase to secretion of a heterologous protein, the alkaline phosphatase PhoA of Escherichia coli, by B. subtilis. The results indicate that the presence of the pro(lip)-peptide, in combination with the lipase signal peptide (pre(lip)), contributes significantly to the efficient secretion of PhoA by B. subtilis and that pre(lip) directs PhoA secretion more efficiently than the authentic signal peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses indicate that, under the conditions tested, no known secretion or membrane-cell wall stress responses were provoked by the production of PhoA with any of the pre- and pro-region sequences used. Our data underscore the view that the pre-pro signals of the S. hyicus lipase are very useful tools for secretion of heterologous proteins in B. subtilis.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipasa/química , Staphylococcus/enzimología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biotecnología/métodos , Membrana Celular/genética , Membrana Celular/metabolismo , Clonación Molecular , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Proteínas de Escherichia coli/genética , Lipasa/genética , Pliegue de Proteína , Señales de Clasificación de Proteína/genética , Transporte de Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Staphylococcus/genética , Staphylococcus/metabolismo , alfa-Amilasas/metabolismoRESUMEN
Bacteria secrete numerous proteins into their environment for growth and survival under complex and ever-changing conditions. The highly different characteristics of secreted proteins pose major challenges to the cellular protein export machinery and, accordingly, different pathways have evolved. While the main secretion (Sec) pathway transports proteins in an unfolded state, the twin-arginine translocation (Tat) pathway transports folded proteins. To date, these pathways were believed to act in strictly independent ways. Here, we have employed proteogenomics to investigate the secretion mechanism of the esterase LipA of Bacillus subtilis, using a serendipitously obtained hyper-producing strain. While LipA is secreted Sec-dependently under standard conditions, hyper-produced LipA is secreted predominantly Tat-dependently via an unprecedented overflow mechanism. Two previously identified B. subtilis Tat substrates, PhoD and YwbN, require each a distinct Tat translocase for secretion. In contrast, hyper-produced LipA is transported by both Tat translocases of B. subtilis, showing that they have distinct but overlapping specificities. The identified overflow secretion mechanism for LipA focuses interest on the possibility that secretion pathway choice can be determined by environmental and intracellular conditions. This may provide an explanation for the previous observation that many Sec-dependently transported proteins have potential twin-arginine signal peptides for export via the Tat pathway.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Vías Secretoras/fisiología , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Clonación Molecular , Electroforesis en Gel Bidimensional , Genómica/métodos , Proteínas de Transporte de Membrana/genética , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Proteómica/métodos , Vías Secretoras/genéticaRESUMEN
Bacillus subtilis serves as an excellent model to study protein secretion at a proteomic scale. Most of the extracellular proteins are exported from the cytoplasm via the secretory (Sec) pathway. Despite extensive studies, the secretion mechanisms of about 25% of the extracellular proteins are unknown. This suggests that B. subtilis makes use of alternative mechanisms to release proteins into its environment. In search for novel pathways, which contribute to biogenesis of the B. subtilis exoproteome, we investigated a possible role of the large conductance mechanosensitive channel protein MscL. We compared protein secretion by MscL deficient and proficient B. subtilis cells. MscL did not contribute to secretion under standard growth conditions. Unexpectedly, we discovered that under hypo-osmotic shock conditions specific, normally cytoplasmic proteins were released by mscL mutant cells. This protein release was selective since not all cytoplasmic proteins were equally well released. We established that this protein release by mscL mutant cells cannot be attributed to cell death or lysis. The presence of MscL, therefore, seems to prevent the specific release of cytoplasmic proteins by B. subtilis during hypo-osmotic shock. Our unprecedented findings imply that an unidentified system for selective release of cytoplasmic proteins is active in B. subtilis.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Canales Iónicos/metabolismo , Mecanotransducción Celular/fisiología , Apoptosis , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Electroforesis en Gel Bidimensional , Canales Iónicos/genética , Mecanotransducción Celular/genética , Viabilidad Microbiana , Microscopía Fluorescente , Mutación , Presión Osmótica/fisiología , Proteómica , Vías Secretoras/genética , Vías Secretoras/fisiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Bacillus subtilis strain 168 produces the extremely stable and broad-spectrum lantibiotic sublancin 168. Known sublancin 168-susceptible organisms include important pathogens, such as Staphylococcus aureus. Nevertheless, since its discovery, the mode of action of sublancin 168 has remained elusive. The present studies were, therefore, aimed at the identification of cellular determinants for bacterial susceptibility toward sublancin 168. Growth inhibition and competition assays on plates and in liquid cultures revealed that sublancin 168-mediated growth inhibition of susceptible B. subtilis and S. aureus cells is affected by the NaCl concentration in the growth medium. Added NaCl did not influence the production, activity, or stability of sublancin 168 but, instead, lowered the susceptibility of sensitive cells toward this lantibiotic. Importantly, the susceptibility of B. subtilis and S. aureus cells toward sublancin 168 was shown to depend on the presence of the large mechanosensitive channel of conductance MscL. In contrast, MscL was not involved in susceptibility toward the bacteriocin nisin or Pep5. Taken together, our unprecedented results demonstrate that MscL is a critical and specific determinant in bacterial sublancin 168 susceptibility that may serve either as a direct target for this lantibiotic or as a gate of entry to the cytoplasm.
Asunto(s)
Bacillus subtilis/efectos de los fármacos , Proteínas Bacterianas/fisiología , Bacteriocinas/farmacología , Mecanotransducción Celular/fisiología , Péptidos/farmacología , Staphylococcus aureus/efectos de los fármacos , Glicopéptidos , Pruebas de Sensibilidad Microbiana , Cloruro de Sodio/farmacologíaRESUMEN
Bacillus subtilis strain 168 produces the extremely stable lantibiotic sublancin 168, which has a broad spectrum of bactericidal activity. Both sublancin 168 production and producer immunity are determined by the SPbeta prophage. While the sunA and sunT genes for sublancin 168 production have been known for several years, the genetic basis for sublancin 168 producer immunity has remained elusive. Therefore, the present studies were aimed at identifying an SPbeta gene(s) for sublancin 168 immunity. By systematic deletion analysis, we were able to pinpoint one gene, named yolF, as the sublancin 168 producer immunity gene. Growth inhibition assays performed using plates and liquid cultures revealed that YolF is both required and sufficient for sublancin 168 immunity even when heterologously produced in the sublancin-sensitive bacterium Staphylococcus aureus. Accordingly, we propose to rename yolF to sunI (for sublancin immunity). Subcellular localization studies indicate that the SunI protein is anchored to the membrane with a single N-terminal membrane-spanning domain that has an N(out)-C(in) topology. Thus, the bulk of the protein faces the cytoplasm of B. subtilis. This topology has not yet been reported for known bacteriocin producer immunity proteins, which implies that SunI belongs to a novel class of bacteriocin antagonists.
Asunto(s)
Bacillus subtilis/genética , Bacteriocinas/farmacología , Farmacorresistencia Bacteriana/genética , Genes Bacterianos/genética , Genes Bacterianos/fisiología , Péptidos/farmacología , Bacillus subtilis/efectos de los fármacos , Western Blotting , Medios de Cultivo , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Glicopéptidos , Pruebas de Sensibilidad Microbiana , Plásmidos , Profagos/genética , Staphylococcus aureus/efectos de los fármacosRESUMEN
Bacillus subtilis is a well-established cellular factory for proteins and fine chemicals. In particular, the direct secretion of proteinaceous products into the growth medium greatly facilitates their downstream processing, which is an important advantage of B. subtilis over other biotechnological production hosts, such as Escherichia coli. The application spectrum of B. subtilis is, however, often confined to proteins from Bacillus or closely related species. One of the major reasons for this (current) limitation is the inefficient formation of disulfide bonds, which are found in many, especially eukaryotic, proteins. Future exploitation of B. subtilis to fulfill the ever-growing demand for pharmaceutical and other high-value proteins will therefore depend on overcoming this particular hurdle. Recently, promising advances in this area have been achieved, which focus attention on the need to modulate the cellular levels and activity of thiol-disulfide oxidoreductases (TDORs). These TDORs are enzymes that control the cleavage or formation of disulfide bonds. This review will discuss readily applicable approaches for TDOR modulation and aims to provide leads for further improvement of the Bacillus cell factory for production of disulfide bond-containing proteins.
Asunto(s)
Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteína Disulfuro Reductasa (Glutatión)/metabolismoRESUMEN
Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of disulfide bonds that is the greatest bottleneck. Degradation of inefficiently or incorrectly oxidized proteins and the requirement for costly and time-consuming reduction and oxidation steps in the downstream processing of the proteins still are major limitations for full exploitation of B. subtilis for biopharmaceutical production. Therefore, the present study was aimed at developing a novel in vivo strategy for improved production of secreted disulfide-bond-containing proteins. Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; introduction of the heterologous oxidase DsbA from Staphylococcus carnosus; and addition of redox-active compounds to the growth medium. As shown using the disulfide-bond-containing molecule Escherichia coli PhoA as a model protein, combined use of these three approaches resulted in secretion of amounts of active PhoA that were approximately 3.5-fold larger than the amounts secreted by the parental strain B. subtilis 168. Our findings indicate that Bacillus strains with improved oxidizing properties can be engineered for biotechnological production of heterologous high-value proteins containing disulfide bonds.
Asunto(s)
Bacillus subtilis/enzimología , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteína Disulfuro Reductasa (Glutatión)/genética , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Fosfatasa Alcalina/biosíntesis , Biotecnología/métodos , Medios de Cultivo/química , Proteínas de Escherichia coli/biosíntesis , Oxidación-Reducción , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus/enzimología , Staphylococcus/genéticaRESUMEN
Most analyzed Lactococcus lactis strains are predicted to harbor one or more prophage genomes within their chromosome; however, the true extent of the inducibility and functionality of such prophages cannot easily be deduced from sequence analysis alone. Chemical treatment of lysogenic strains with Mitomycin C is known to cause induction of temperate phages, though it is not always easy to clearly identify a lysogenic strain or to measure the number of released phage particles. Here, we report the application of flow cytometry as a reliable tool for the detection and enumeration of released lactococcal prophages using the green dye SYTO-9.
RESUMEN
Despite the persistent and costly problem caused by (bacterio)phage predation of Streptococcus thermophilus in dairy plants, DNA sequence information relating to these phages remains limited. Genome sequencing is necessary to better understand the diversity and proliferative strategies of virulent phages. In this report, whole genome sequences of 40 distinct bacteriophages infecting S. thermophilus were analyzed for general characteristics, genomic structure and novel features. The bacteriophage genomes display a high degree of conservation within defined groupings, particularly across the structural modules. Supporting this observation, four novel members of a recently discovered third group of S. thermophilus phages (termed the 5093 group) were found to be conserved relative to both phage 5093 and to each other. Replication modules of S. thermophilus phages generally fall within two main groups, while such phage genomes typically encode one putative transcriptional regulator. Such features are indicative of widespread functional synteny across genetically distinct phage groups. Phage genomes also display nucleotide divergence between groups, and between individual phages of the same group (within replication modules and at the 3' end of the lysis module)-through various insertions and/or deletions. A previously described multiplex PCR phage detection system was updated to reflect current knowledge on S. thermophilus phages. Furthermore, the structural protein complement as well as the antireceptor (responsible for the initial attachment of the phage to the host cell) of a representative of the 5093 group was defined. Our data more than triples the currently available genomic information on S. thermophilus phages, being of significant value to the dairy industry, where genetic knowledge of lytic phages is crucial for phage detection and monitoring purposes. In particular, the updated PCR detection methodology for S. thermophilus phages is highly useful in monitoring particular phage group(s) present in a given whey sample. Studies of this nature therefore not only provide information on the prevalence and associated threat of known S. thermophilus phages, but may also uncover newly emerging and genomically distinct phages infecting this dairy starter bacterium.
RESUMEN
The oxidative folding of proteins involves disulfide bond formation, which is usually catalyzed by thiol-disulfide oxidoreductases (TDORs). In bacteria, this process takes place in the cytoplasmic membrane and other extracytoplasmic compartments. While it is relatively easy to study oxidative folding of water-soluble proteins on a proteome-wide scale, this has remained a major challenge for membrane proteins due to their high hydrophobicity. Here, we have assessed whether proteomic techniques can be applied to probe the oxidative folding of membrane proteins using the Gram-positive bacterium Bacillus subtilis as a model organism. Specifically, we investigated the membrane proteome of a B. subtilis bdbCD mutant strain, which lacks the primary TDOR pair BdbC and BdbD, by gel-free mass spectrometry. In total, 18 membrane-associated proteins showed differing behavior in the bdbCD mutant and the parental strain. These included the ProA protein involved in osmoprotection. Consistent with the absence of ProA, the bdbCD mutant was found to be sensitive to osmotic shock. We hypothesize that membrane proteomics is a potentially effective approach to profile oxidative folding of bacterial membrane proteins.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteómica/métodos , Bacillus subtilis/metabolismo , Electroforesis en Gel de Poliacrilamida , Pliegue de ProteínaRESUMEN
In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of eukaryotic serine/threonine kinases exist in bacteria. Moreover, various pathogenic species, such as Yersinia and Mycobacterium, require serine/threonine kinases for successful invasion of human host cells. The substrates targeted by bacterial serine/threonine kinases have remained largely unknown. Here we report that the serine/threonine kinase PknB from the important pathogen Staphylococcus aureus is released into the external milieu, which opens up the possibility that PknB does not only phosphorylate bacterial proteins but also proteins of the human host. To identify possible human targets of purified PknB, we studied in vitro phosphorylation of peptide microarrays and detected 68 possible human targets for phosphorylation. These results show that PknB is a proline-directed kinase with MAP kinase-like enzymatic activity. As the potential cellular targets for PknB are involved in apoptosis, immune responses, transport, and metabolism, PknB secretion may help the bacterium to evade intracellular killing and facilitate its growth. In apparent agreement with this notion, phosphorylation of the host-cell response coordinating transcription factor ATF-2 by PknB was confirmed by mass spectrometry. Taken together, our results identify PknB as the first prokaryotic representative of the proline-directed kinase/MAP kinase family of enzymes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Prolina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Staphylococcus aureus/enzimología , Factor de Transcripción Activador 2/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Western Blotting , Humanos , MAP Quinasa Quinasa 4/metabolismo , Espectrometría de Masas , Mutación , Péptidos/metabolismo , Fosforilación , Análisis por Matrices de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Staphylococcus aureus/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Rhodomyrtone [6,8-dihydroxy-2,2,4,4-tetramethyl-7-(3-methyl-1-oxobutyl)-9-(2-methylpropyl)-4,9-dihydro-1H-xanthene-1,3(2H)-di-one] from Rhodomyrtus tomentosa (Aiton) Hassk. displayed significant antibacterial activities against gram-positive bacteria including Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Streptococcus gordonii, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus salivarius. Especially noteworthy was the activity against MRSA with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) ranging from 0.39 to 0.78 microg/ml. As shown for S. pyogenes, no surviving cells were detected within 5 and 6h after treatment with the compound at 8MBC and 4MBC concentrations, respectively. Rhodomyrtone displays no bacteriolytic activity, as determined by measurement of the optical density at 620 nm. A rhodomyrtone killing test with S. mutans using phase contrast microscopy showed that this compound caused a few morphological changes as the treated cells were slightly changed in color and bigger than the control when they were killed. Taken together, the results support the view that rhodomyrtone has a strong bactericidal activity on gram-positive bacteria, including major pathogens.
Asunto(s)
Antibacterianos/farmacología , Myrtaceae/química , Xantonas/farmacología , Antibacterianos/aislamiento & purificación , Bacterias Grampositivas/clasificación , Bacterias Grampositivas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Xantonas/aislamiento & purificaciónRESUMEN
Thioredoxin functions in nearly all organisms as the major thiol-disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as "mixed disulfide fishing" in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme-substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5A) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Disulfuros/química , Mutación , Estructura Cuaternaria de Proteína , Tiorredoxinas/química , Tiorredoxinas/genética , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Cisteína/química , Dimerización , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Tiorredoxinas/metabolismoRESUMEN
Disulphide bond formation catalysed by thiol-disulphide oxidoreductases (TDORs) is a universally conserved mechanism for stabilizing extracytoplasmic proteins. In Escherichia coli, disulphide bond formation requires a concerted action of distinct TDORs in thiol oxidation and subsequent quinone reduction. TDOR function in other bacteria has remained largely unexplored. Here we focus on TDORs of low-GC Gram-positive bacteria, in particular DsbA of Staphylococcus aureus and BdbA-D of Bacillus subtilis. Phylogenetic analyses reveal that the homologues DsbA and BdbD cluster in distinct groups typical for Staphylococcus and Bacillus species respectively. To compare the function of these TDORs, DsbA was produced in various bdb mutants of B. subtilis. Next, we assessed the ability of DsbA to sustain different TDOR-dependent processes, including heterologous secretion of E. coli PhoA, competence development and bacteriocin (sublancin 168) production. The results show that DsbA can function in all three processes. While BdbD needs a quinone oxidoreductase for activity, DsbA activity appears to depend on redox-active medium components. Unexpectedly, both quinone oxidoreductases of B. subtilis are sufficient to sustain production of sublancin. Moreover, DsbA can functionally replace these quinone oxidoreductases in sublancin production. Taken together, our unprecedented findings imply that TDOR systems of low-GC Gram-positive bacteria have a modular composition.