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1.
PLoS Pathog ; 16(8): e1008776, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32845938

RESUMEN

Enteroaggregative Escherichia coli (EAEC) is a diarrheagenic pathotype associated with traveler's diarrhea, foodborne outbreaks and sporadic diarrhea in industrialized and developing countries. Regulation of virulence in EAEC is mediated by AggR and its negative regulator Aar. Together, they control the expression of at least 210 genes. On the other hand, we observed that about one third of Aar-regulated genes are related to metabolism and transport. In this study we show the AggR/Aar duo controls the metabolism of lipids. Accordingly, we show that AatD, encoded in the AggR-regulated aat operon (aatPABCD) is an N-acyltransferase structurally similar to the essential Apolipoprotein N-acyltransferase Lnt and is required for the acylation of Aap (anti-aggregation protein). Deletion of aatD impairs post-translational modification of Aap and causes its accumulation in the bacterial periplasm. trans-complementation of 042aatD mutant with the AatD homolog of ETEC or with the N-acyltransferase Lnt reestablished translocation of Aap. Site-directed mutagenesis of the E207 residue in the putative acyltransferase catalytic triad disrupted the activity of AatD and caused accumulation of Aap in the periplasm due to reduced translocation of Aap at the bacterial surface. Furthermore, Mass spectroscopy revealed that Aap is acylated in a putative lipobox at the N-terminal of the mature protein, implying that Aap is a lipoprotein. Lastly, deletion of aatD impairs bacterial colonization of the streptomycin-treated mouse model. Our findings unveiled a novel N-acyltransferase family associated with bacterial virulence, and that is tightly regulated by AraC/XylS regulators in the order Enterobacterales.


Asunto(s)
Acetiltransferasas/metabolismo , Factor de Transcripción de AraC/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Escherichia coli/patogenicidad , Regulación Bacteriana de la Expresión Génica , Acetiltransferasas/genética , Acilación , Animales , Factor de Transcripción de AraC/química , Factor de Transcripción de AraC/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Operón , Filogenia , Conformación Proteica , Virulencia
2.
Nat Commun ; 11(1): 4126, 2020 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-32807804

RESUMEN

Neisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association to identify loss-of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical relative to urethral isolates. This enrichment holds true for LOF mutations in another efflux pump, farAB, and in urogenitally-adapted versus typical N. meningitidis, providing evidence for a model in which expression of these pumps in the female urogenital tract incurs a fitness cost for pathogenic Neisseria. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.


Asunto(s)
Proteínas Bacterianas/metabolismo , Cuello del Útero/microbiología , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genética , Animales , Proteínas Bacterianas/genética , Farmacorresistencia Microbiana/genética , Femenino , Regulación Bacteriana de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Pruebas de Sensibilidad Microbiana , Mutación/genética , Neisseria gonorrhoeae/metabolismo , Operón/genética , Regiones Promotoras Genéticas/genética
3.
Proc Natl Acad Sci U S A ; 117(29): 17249-17259, 2020 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-32641516

RESUMEN

Control of infections caused by carbapenem-resistant Klebsiella pneumoniae continues to be challenging. The success of this pathogen is favored by its ability to acquire antimicrobial resistance and to spread and persist in both the environment and in humans. The emergence of clinically important clones, such as sequence types 11, 15, 101, and 258, has been reported worldwide. However, the mechanisms promoting the dissemination of such high-risk clones are unknown. Unraveling the factors that play a role in the pathobiology and epidemicity of K. pneumoniae is therefore important for managing infections. To address this issue, we studied a carbapenem-resistant ST-15 K. pneumoniae isolate (Kp3380) that displayed a remarkable adherent phenotype with abundant pilus-like structures. Genome sequencing enabled us to identify a chaperone-usher pili system (Kpi) in Kp3380. Analysis of a large K. pneumoniae population from 32 European countries showed that the Kpi system is associated with the ST-15 clone. Phylogenetic analysis of the operon revealed that Kpi belongs to the little-characterized γ2-fimbrial clade. We demonstrate that Kpi contributes positively to the ability of K. pneumoniae to form biofilms and adhere to different host tissues. Moreover, the in vivo intestinal colonizing capacity of the Kpi-defective mutant was significantly reduced, as was its ability to infect Galleria mellonella The findings provide information about the pathobiology and epidemicity of Kpi+ K. pneumoniae and indicate that the presence of Kpi may explain the success of the ST-15 clone. Disrupting bacterial adherence to the intestinal surface could potentially target gastrointestinal colonization.


Asunto(s)
Fimbrias Bacterianas/genética , Klebsiella pneumoniae/genética , Chaperonas Moleculares/genética , Células A549 , Animales , Antibacterianos , Adhesión Bacteriana/efectos de los fármacos , Adhesión Bacteriana/genética , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Carbapenémicos/farmacología , Línea Celular , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana Múltiple/genética , Células Epiteliales/microbiología , Europa (Continente) , Femenino , Eliminación de Gen , Genes Bacterianos/genética , Humanos , Infecciones por Klebsiella , Klebsiella pneumoniae/citología , Klebsiella pneumoniae/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Tipificación de Secuencias Multilocus , Operón , Filogenia
4.
PLoS Comput Biol ; 16(6): e1007997, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32598355

RESUMEN

Top-down proteomics has enabled the elucidation of heterogeneous protein complexes with different cofactors, post-translational modifications, and protein membership. This heterogeneity is believed to play a previously unknown role in cellular processes. The different molecular forms of a protein complex have come to be called "complex isoform" or "complexoform". Despite the elucidation of the complexoform, it remains unclear how and whether cellular circuits control the distribution of a complexoform. To help address this issue, we first simulate a generic three-protein complexoform to reveal the control of its distribution by the timing of gene transcription, mRNA translation, and protein transport. Overall, we ran 265 computational experiments: each averaged over 1,000 stochastic simulations. Based on the experiments, we show that genes arranged in a single operon, a cascade, or as two operons all give rise to the different protein composition of complexoform because of timing differences in protein-synthesis order. We also show that changes in the kinetics of expression, protein transport, or protein binding dramatically alter the distribution of the complexoform. Furthermore, both stochastic and transient kinetics control the assembly of the complexoform when the expression and assembly occur concurrently. We test our model against the biological cellulosome system. With biologically relevant rates, we find that the genetic circuitry controls the average final complexoform assembly and the variation in the assembly structure. Our results highlight the importance of both the genetic circuit architecture and kinetics in determining the distribution of a complexoform. Our work has a broad impact on our understanding of non-equilibrium processes in both living and synthetic biological systems.


Asunto(s)
Redes Reguladoras de Genes , Proteínas/metabolismo , Procesos Estocásticos , Simulación por Computador , Operón , Biosíntesis de Proteínas , ARN Mensajero/genética
5.
Nat Commun ; 11(1): 2794, 2020 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-32493973

RESUMEN

All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Here, we characterize a SAM-I riboswitch (SAM-IXcc) from the Xanthomonas campestris that regulates methionine synthesis via the met operon. In vitro and in vivo experiments show that SAM-IXcc controls the met operon primarily at the translational level in response to cellular S-adenosylmethionine (SAM) levels. Biochemical and genetic data demonstrate that SAM-IXcc expression platform not only can repress gene expression in response to SAM binding to SAM-IXcc aptamer but also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shine-Dalgarno (SD) sequence and freeing the SD for translation initiation. These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNA-sensing RNA element. This SAM-I riboswitch appears to be highly conserved in Xanthomonas species.


Asunto(s)
ARN de Transferencia de Metionina/metabolismo , Riboswitch , S-Adenosilmetionina/metabolismo , Secuencia de Bases , Sitios Genéticos , Modelos Biológicos , Conformación de Ácido Nucleico , Operón/genética , Biosíntesis de Proteínas , ARN de Transferencia de Metionina/química , ARN de Transferencia de Metionina/genética
6.
Nucleic Acids Res ; 48(12): 6547-6562, 2020 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-32453397

RESUMEN

Heme is a multifaceted molecule. While serving as a prosthetic group for many important proteins, elevated levels are toxic to cells. The complexity of this stimulus has shaped bacterial network evolution. However, only a small number of targets controlled by heme-responsive regulators have been described to date. Here, we performed chromatin affinity purification and sequencing to provide genome-wide insights into in vivo promoter occupancy of HrrA, the response regulator of the heme-regulated two-component system HrrSA of Corynebacterium glutamicum. Time-resolved profiling revealed dynamic binding of HrrA to more than 200 different genomic targets encoding proteins associated with heme biosynthesis, the respiratory chain, oxidative stress response and cell envelope remodeling. By repression of the extracytoplasmic function sigma factor sigC, which activates the cydABCD operon, HrrA prioritizes the expression of genes encoding the cytochrome bc1-aa3 supercomplex. This is also reflected by a significantly decreased activity of the cytochrome aa3 oxidase in the ΔhrrA mutant. Furthermore, our data reveal that HrrA also integrates the response to heme-induced oxidative stress by activating katA encoding the catalase. These data provide detailed insights in the systemic strategy that bacteria have evolved to respond to the versatile signaling molecule heme.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Regulación Bacteriana de la Expresión Génica , Hemo/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Bacterianas/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Operón , Regiones Promotoras Genéticas , Proteínas Quinasas/genética , Factor sigma/metabolismo
7.
BMC Bioinformatics ; 21(1): 162, 2020 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-32349661

RESUMEN

BACKGROUND: The reconstruction of metabolic networks and the three-dimensional coverage of protein structures have reached the genome-scale in the widely studied Escherichia coli K-12 MG1655 strain. The combination of the two leads to the formation of a structural systems biology framework, which we have used to analyze differences between the reactive oxygen species (ROS) sensitivity of the proteomes of sequenced strains of E. coli. As proteins are one of the main targets of oxidative damage, understanding how the genetic changes of different strains of a species relates to its oxidative environment can reveal hypotheses as to why these variations arise and suggest directions of future experimental work. RESULTS: Creating a reference structural proteome for E. coli allows us to comprehensively map genetic changes in 1764 different strains to their locations on 4118 3D protein structures. We use metabolic modeling to predict basal ROS production levels (ROStype) for 695 of these strains, finding that strains with both higher and lower basal levels tend to enrich their proteomes with antioxidative properties, and speculate as to why that is. We computationally assess a strain's sensitivity to an oxidative environment, based on known chemical mechanisms of oxidative damage to protein groups, defined by their localization and functionality. Two general groups - metalloproteins and periplasmic proteins - show enrichment of their antioxidative properties between the 695 strains with a predicted ROStype as well as 116 strains with an assigned pathotype. Specifically, proteins that a) utilize a molybdenum ion as a cofactor and b) are involved in the biogenesis of fimbriae show intriguing protective properties to resist oxidative damage. Overall, these findings indicate that a strain's sensitivity to oxidative damage can be elucidated from the structural proteome, though future experimental work is needed to validate our model assumptions and findings. CONCLUSION: We thus demonstrate that structural systems biology enables a proteome-wide, computational assessment of changes to atomic-level physicochemical properties and of oxidative damage mechanisms for multiple strains in a species. This integrative approach opens new avenues to study adaptation to a particular environment based on physiological properties predicted from sequence alone.


Asunto(s)
Adaptación Fisiológica , Escherichia coli K12/fisiología , Estrés Oxidativo , Proteoma/metabolismo , Antioxidantes/metabolismo , Proteínas de Escherichia coli/metabolismo , Fimbrias Bacterianas/metabolismo , Modelos Biológicos , Molibdeno/metabolismo , Operón/genética , Oxidación-Reducción , Periplasma/metabolismo , Fenotipo , Especies Reactivas de Oxígeno/metabolismo
8.
PLoS Pathog ; 16(5): e1008561, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32453788

RESUMEN

Several Xanthomonas species have a type IV secretion system (T4SS) that injects a cocktail of antibacterial proteins into neighbouring Gram-negative bacteria, often leading to rapid lysis upon cell contact. This capability represents an obvious fitness benefit since it can eliminate competition while the liberated contents of the lysed bacteria could provide an increase in the local availability of nutrients. However, the production of this Mega Dalton-sized molecular machine, with over a hundred subunits, also imposes a significant metabolic cost. Here we show that the chromosomal virB operon, which encodes the structural genes of this T4SS in X. citri, is regulated by the conserved global regulator CsrA. Relieving CsrA repression from the virB operon produced a greater number of T4SSs in the cell envelope and an increased efficiency in contact-dependent lysis of target cells. However, this was also accompanied by a physiological cost leading to reduced fitness when in co-culture with wild-type X. citri. We show that T4SS production is constitutive despite being downregulated by CsrA. Cells subjected to a wide range of rich and poor growth conditions maintain a constant density of T4SSs in the cell envelope and concomitant interbacterial competitiveness. These results show that CsrA provides a constant though partial repression on the virB operon, independent of the tested growth conditions, in this way controlling T4SS-related costs while at the same time maintaining X. citri's aggressive posture when confronted by competitors.


Asunto(s)
Proteínas Bacterianas/metabolismo , Homeostasis , Operón , Proteínas Represoras/metabolismo , Sistemas de Secreción Tipo IV/biosíntesis , Xanthomonas/metabolismo , Proteínas Bacterianas/genética , Proteínas Represoras/genética , Sistemas de Secreción Tipo IV/genética , Xanthomonas/genética
9.
PLoS One ; 15(5): e0232701, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32379807

RESUMEN

Bacterial live cell sensors are potentially powerful tools for the detection of environmental toxins. In this work, we have established and validated a flow cytometry readout for an existing bacterial arabinose sensor system with DNA methylation based memory function (Maier et al., 2017, Nat. Comm., 8:15336). Flow cytometry readout is convenient and enables a multiparameter analysis providing information about single-cell variability, which is beneficial for further development of sensor systems of this type in the future. We then designed a tetracycline sensor system, because of the importance of antibiotics pollution in the light of multi-resistant pathogens. To this end, a tetracycline trigger plasmid was constructed by replacing the araC repressor gene and the ara operator of the arabinose trigger plasmid with the tetR gene coding for the tetracycline repressor and the tet operon. After combination with the memory plasmid, the tetracycline sensor system was shown to be functional in E. coli allowing to detect and memorize the presence of tetracycline. Due to a positive feedback between the trigger and memory systems, the combined whole-cell biosensor showed a very high sensitivity for tetracycline with a detection threshold at 0.1 ng/ml tetracycline, which may be a general property of sensors of this type. Moreover, acute presence of tetracycline and past exposure can be detected by this sensor using the dual readout of two reporter fluorophores.


Asunto(s)
Antibacterianos/análisis , Técnicas Biosensibles/métodos , Escherichia coli/genética , Tetraciclina/análisis , Factor de Transcripción de AraC/genética , Metilación de ADN , Epigenómica , Proteínas de Escherichia coli/genética , Citometría de Flujo/métodos , Operón , Plásmidos/genética , Proteínas Represoras/genética
10.
PLoS One ; 15(5): e0232251, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32407412

RESUMEN

Lipids represent an important source of nutrition for infecting mycobacteria, accumulating within the necrotic core of granulomas and present in foamy macrophages associated with mycobacterial infection. In order to better understand the timing, process and importance of lipid accumulation, we developed methods for direct in vivo visualization and quantification of this process using the zebrafish-M. marinum larval model of infection. We find that neutral lipids accumulate cell-autonomously in mycobacterium-infected macrophages in vivo during early infection, with detectable levels of accumulation by two days post-infection. Treatment with ezetimibe, an FDA-approved drug, resulted in decreased levels of free cholesterol and neutral lipids, and a reduction of bacterial growth in vivo. The effect of ezetimibe in reducing bacterial growth was dependent on the mce4 operon, a key bacterial determinant of lipid utilization. Thus, in vivo, lipid accumulation can occur cell-autonomously at early timepoints of mycobacterial infection, and limitation of this process results in decreased bacterial burden.


Asunto(s)
Metabolismo de los Lípidos , Mycobacterium marinum/crecimiento & desarrollo , Ezetimiba/farmacología , Macrófagos/metabolismo , Macrófagos/microbiología , Mutación , Mycobacterium marinum/efectos de los fármacos , Mycobacterium marinum/genética , Mycobacterium marinum/fisiología , Operón/genética
11.
Acta Crystallogr D Struct Biol ; 76(Pt 4): 375-384, 2020 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-32254062

RESUMEN

Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Š(spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC.


Asunto(s)
Proteínas de Transporte de Anión/química , Proteínas Bacterianas/química , Hemo/análogos & derivados , Pseudomonas aeruginosa/enzimología , Proteínas de Transporte de Anión/genética , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Hemo/química , Modelos Moleculares , Operón , Multimerización de Proteína , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/genética
12.
PLoS Pathog ; 16(4): e1008440, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32294143

RESUMEN

In flea-borne plague, blockage of the flea's foregut by Yersinia pestis hastens transmission to the mammalian host. Based on microscopy observations, we first suggest that flea blockage results from primary infection of the foregut and not from midgut colonization. In this model, flea infection is characterized by the recurrent production of a mass that fills the lumen of the proventriculus and encompasses a large number of Y. pestis. This recurrence phase ends when the proventricular cast is hard enough to block blood ingestion. We further showed that ymt (known to be essential for flea infection) is crucial for cast production, whereas the hmsHFRS operon (known to be essential for the formation of the biofilm that blocks the gut) is needed for cast consolidation. By screening a library of mutants (each lacking a locus previously known to be upregulated in the flea gut) for biofilm formation, we found that rpiA is important for flea blockage but not for colonization of the midgut. This locus may initially be required to resist toxic compounds within the proventricular cast. However, once the bacterium has adapted to the flea, rpiA helps to form the biofilm that consolidates the proventricular cast. Lastly, we used genetic techniques to demonstrate that ribose-5-phosphate isomerase activity (due to the recent gain of a second copy of rpiA (y2892)) accentuated blockage but not midgut colonization. It is noteworthy that rpiA is an ancestral gene, hmsHFRS and rpiA2 were acquired by the recent ancestor of Y. pestis, and ymt was acquired by Y. pestis itself. Our present results (i) highlight the physiopathological and molecular mechanisms leading to flea blockage, (ii) show that the role of a gene like rpiA changes in space and in time during an infection, and (iii) emphasize that evolution is a gradual process punctuated by sudden jumps.


Asunto(s)
Insectos Vectores/microbiología , Peste/transmisión , Siphonaptera/microbiología , Yersinia pestis/fisiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , Sistema Digestivo/microbiología , Femenino , Humanos , Insectos Vectores/fisiología , Masculino , Ratones , Operón , Peste/microbiología , Siphonaptera/fisiología , Yersinia pestis/genética
13.
PLoS Pathog ; 16(4): e1008281, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32236137

RESUMEN

Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems. Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of bacilli, clostridia, streptococci, and actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.


Asunto(s)
Bacillus cereus/metabolismo , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Familia de Multigenes , Operón , Polisacáridos/metabolismo , Bacillus cereus/genética , Bacillus cereus/crecimiento & desarrollo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Filogenia , Conformación Proteica
14.
PLoS Comput Biol ; 16(4): e1007721, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32236097

RESUMEN

In bacteria functionally related genes comprising metabolic pathways and protein complexes are frequently encoded in operons and are widely conserved across phylogenetically diverse species. The evolution of these operon-encoded processes is affected by diverse mechanisms such as gene duplication, loss, rearrangement, and horizontal transfer. These mechanisms can result in functional diversification, increasing the potential evolution of novel biological pathways, and enabling pre-existing pathways to adapt to the requirements of particular environments. Despite the fundamental importance that these mechanisms play in bacterial environmental adaptation, a systematic approach for studying the evolution of operon organization is lacking. Herein, we present a novel method to study the evolution of operons based on phylogenetic clustering of operon-encoded protein families and genomic-proximity network visualizations of operon architectures. We applied this approach to study the evolution of the synthase dependent exopolysaccharide (EPS) biosynthetic systems: cellulose, acetylated cellulose, poly-ß-1,6-N-acetyl-D-glucosamine (PNAG), Pel, and alginate. These polymers have important roles in biofilm formation, antibiotic tolerance, and as virulence factors in opportunistic pathogens. Our approach revealed the complex evolutionary landscape of EPS machineries, and enabled operons to be classified into evolutionarily distinct lineages. Cellulose operons show phyla-specific operon lineages resulting from gene loss, rearrangement, and the acquisition of accessory loci, and the occurrence of whole-operon duplications arising through horizonal gene transfer. Our evolution-based classification also distinguishes between PNAG production from Gram-negative and Gram-positive bacteria on the basis of structural and functional evolution of the acetylation modification domains shared by PgaB and IcaB loci, respectively. We also predict several pel-like operon lineages in Gram-positive bacteria and demonstrate in our companion paper (Whitfield et al PLoS Pathogens, in press) that Bacillus cereus produces a Pel-dependent biofilm that is regulated by cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP).


Asunto(s)
Biología Computacional/métodos , Operón/genética , Operón/fisiología , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Evolución Biológica , Evolución Molecular , Duplicación de Gen , Filogenia , Factores de Virulencia
15.
Nat Commun ; 11(1): 1865, 2020 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-32313027

RESUMEN

Bacterial Rhs proteins containing toxic domains are often secreted by type VI secretion systems (T6SSs) through unclear mechanisms. Here, we show that the T6SS Rhs-family effector TseI of Aeromonas dhakensis is subject to self-cleavage at both the N- and the C-terminus, releasing the middle Rhs core and two VgrG-interacting domains (which we name VIRN and VIRC). VIRC is an endonuclease, and the immunity protein TsiI protects against VIRC toxicity through direct interaction. Proteolytic release of VIRC and VIRN is mediated, respectively, by an internal aspartic protease activity and by two conserved glutamic residues in the Rhs core. Mutations abolishing self-cleavage do not block secretion, but reduce TseI toxicity. Deletion of VIRN or the Rhs core abolishes secretion. TseI homologs from Pseudomonas syringae, P. aeruginosa, and Vibrio parahaemolyticus are also self-cleaved. VIRN and VIRC interact with protein VgrG1, while the Rhs core interacts with protein TecI. We propose that VIRN and the Rhs core act as T6SS intramolecular chaperones to facilitate toxin secretion and function.


Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/metabolismo , Toxinas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , Sistemas de Secreción Tipo VI/metabolismo , Aeromonas/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Genes Bacterianos , Mutación , Operón , Péptido Hidrolasas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo
16.
Nat Commun ; 11(1): 1203, 2020 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-32139702

RESUMEN

Auxotrophy, the inability to produce an organic compound essential for growth, is widespread among bacteria. Auxotrophic bacteria rely on transporters to acquire these compounds from their environment. Here, we study the expression of both low- and high-affinity transporters of the costly amino acid methionine in an auxotrophic lactic acid bacterium, Lactococcus lactis. We show that the high-affinity transporter (Met-transporter) is heterogeneously expressed at low methionine concentrations, resulting in two isogenic subpopulations that sequester methionine in different ways: one subpopulation primarily relies on the high-affinity transporter (high expression of the Met-transporter) and the other subpopulation primarily relies on the low-affinity transporter (low expression of the Met-transporter). The phenotypic heterogeneity is remarkably stable, inherited for tens of generations, and apparent at the colony level. This heterogeneity results from a T-box riboswitch in the promoter region of the met operon encoding the high-affinity Met-transporter. We hypothesize that T-box riboswitches, which are commonly found in the Lactobacillales, may play as-yet unexplored roles in the predominantly auxotrophic lifestyle of these bacteria.


Asunto(s)
Lactococcus lactis/genética , Riboswitch/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Lactococcus lactis/citología , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Operón/genética , Fenotipo , Análisis de la Célula Individual , Transcripción Genética
17.
Science ; 367(6482): 1140-1146, 2020 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-32139545

RESUMEN

Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.


Asunto(s)
Sistemas de Lectura Abierta , Péptidos/genética , Biosíntesis de Proteínas/genética , ARN Mensajero , Sistemas CRISPR-Cas , Humanos , Operón , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Transcriptoma
18.
Arch Microbiol ; 202(6): 1301-1315, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32130434

RESUMEN

Antimicrobial resistance is a serious public health threat worldwide today. Escherichia coli is known to resist low doses of antibiotics in the presence of sodium salicylate and related compounds by mounting non-heritable transient phenotypic antibiotic resistance (PAR). In the present study, we demonstrate that Bgl+ bacterial strains harboring a functional copy of the ß-glucoside (bgl) operon and are actively hydrolyzing plant-derived aromatic ß-glucosides such as salicin show PAR to low doses of antibiotics. The aglycone released during metabolism of aromatic ß-glucosides is responsible for conferring this phenotype by de-repressing the multiple antibiotics resistance (mar) operon. We also show that prolonged exposure of Bgl+ bacteria to aromatic ß-glucosides in the presence of sub-lethal doses of antibiotics can lead to a significant increase in the frequency of mutants that show heritable resistance to higher doses of antibiotics. Although heritable drug resistance in many cases is known to reduce the fitness of the carrier strain, we did not see a cost associated with resistance in the mutants, most of which carry clinically relevant mutations. These findings indicate that the presence of the activated form of the bgl operon in the genome facilitates the survival of bacteria in environments in which both aromatic ß-glucosides and antibiotics are present.


Asunto(s)
Farmacorresistencia Microbiana/genética , Glucósidos/metabolismo , Operón/genética , Antibacterianos/metabolismo , Antibacterianos/farmacología , Alcoholes Bencílicos/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Mutación , Fenotipo
19.
Nat Commun ; 11(1): 1587, 2020 03 27.
Artículo en Inglés | MEDLINE | ID: mdl-32221293

RESUMEN

RNA degradation is an essential process that allows bacteria to control gene expression and adapt to various environmental conditions. It is usually initiated by endoribonucleases (endoRNases), which produce intermediate fragments that are subsequently degraded by exoribonucleases (exoRNases). However, global studies of the coordinated action of these enzymes are lacking. Here, we compare the targetome of endoRNase Y with the targetomes of 3'-to-5' exoRNases from Streptococcus pyogenes, namely, PNPase, YhaM, and RNase R. We observe that RNase Y preferentially cleaves after guanosine, generating substrate RNAs for the 3'-to-5' exoRNases. We demonstrate that RNase Y processing is followed by trimming of the newly generated 3' ends by PNPase and YhaM. Conversely, the RNA 5' ends produced by RNase Y are rarely further trimmed. Our strategy enables the identification of processing events that are otherwise undetectable. Importantly, this approach allows investigation of the intricate interplay between endo- and exoRNases on a genome-wide scale.


Asunto(s)
Exorribonucleasas/metabolismo , RNA-Seq , Transcriptoma/genética , Regiones no Traducidas 5'/genética , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Guanosina/metabolismo , Operón/genética , Estabilidad del ARN/genética , Streptococcus pyogenes/genética
20.
Chemosphere ; 248: 126002, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32032872

RESUMEN

A mercury-resistant bacterial strain has been isolated from a rock of the Idrija mercury mine in Slovenia. The rock had 19 g carbon and 2952 mg mercury (Hg) per kg. Mass spectrometry and DNA sequencing showed that the bacterium belongs to the Pseudomonas genus. It is called Pseudomonas idrijaensis. This bacterial strain is sensitive to methylmercury (MeHg) like the reference P. aeruginosa strain PAO1, and is resistant to divalent mercury (Hg(II)) in contrast to PAO1. This difference could be attributed to the presence of the mer operon yet deprived of the merB gene encoding the organomercurial lyase, on the basis of whole genome sequencing. The P. idrijaensis mer operon displays the RTPCADE organization and is contained in the Tn5041 transposon. This transposon identified here occurs in other Gram-negative Hg-resistant strains isolated from mercury ores, aquatic systems and soils, including Pseudomonas strains from 15,000 to 40,000 years old Siberian permafrost. When P. idrijaensis was exposed to mercury chloride, two intracellular Hg species were identified by high energy-resolution XANES spectroscopy, a dithiolate Hg(SR)2 and a tetrathiolate Hg(SR)4 complex. P. idrijaensis had a much higher [Hg(SR)2]/[Hg(SR)4] molar ratio than bacteria lacking the mer operon when exposed to 4 µg Hg2+/L - resulting in an intracellular accumulation of 4.3 µg Hg/g dw. A higher amount of the Hg(SR)2 complex provides a chemical signature for the expression of the dicysteinate Mer proteins in response to mercury toxicity.


Asunto(s)
Mercurio/metabolismo , Pseudomonas/metabolismo , Contaminantes del Suelo/metabolismo , Bacterias/metabolismo , Secuencia de Bases , Mercurio/análisis , Compuestos de Metilmercurio/análisis , Minería , Operón , Eslovenia , Suelo , Espectroscopía de Absorción de Rayos X
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