RESUMEN
Acinetobacter baumannii is an opportunistic Gram-negative pathogen that infects critically ill patients. The emergence of antimicrobial resistant A. baumannii has exacerbated the need to characterize environmental adaptation, antibiotic resistance and pathogenicity and their genetic regulators to inform intervention strategies. Critical to adaptation to changing environments in bacteria are small regulatory RNAs (sRNAs), however, the role that sRNAs play in the biology of A. baumannii is poorly understood. To assess the regulatory function of sRNAs and to uncover their RNA interaction partners, we employed an RNA proximity ligation and sequencing method (Hi-GRIL-seq) in three different environmental conditions. Forty sRNAs were ligated to sRNA-RNA chimeric sequencing reads, suggesting that sRNA-mediated gene regulation is pervasive in A. baumannii. In-depth characterization uncovered the sRNA Aar to be a post-transcriptional regulator of four mRNA targets including the transcript encoding outer membrane protein CarO. Aar initiates base-pairing with these mRNAs using a conserved seed region of nine nucleotides, sequestering the ribosome binding sites and inhibiting translation. Aar is differentially expressed in multiple stress conditions suggesting a role in fine-tuning translation of the Aar-target molecules. Our study provides mechanistic insights into sRNA-mediated gene regulation in A. baumannii and represents a valuable resource for future RNA-centric research endeavours.
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Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.
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Infecciones por Salmonella/genética , Salmonella typhimurium/genética , Animales , Gastroenteritis/microbiología , Perfilación de la Expresión Génica/métodos , Variación Genética/genética , Humanos , Macrófagos , Ratones , Infecciones por Salmonella/microbiología , VirulenciaRESUMEN
Salmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of Salmonella genomes has revealed that ST313 is closely related to the ST19 group of S Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by only â¼1,000 SNPs. We hypothesized that the phenotypic differences that distinguish African Salmonella from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes. Here we identified 3,597 transcriptional start sites of the ST313 strain D23580, and searched for a gene-expression signature linked to pathogenesis of Salmonella We identified a SNP in the promoter of the pgtE gene that caused high expression of the PgtE virulence factor in African S. Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance, and modulated virulence in the chicken infection model. We propose that high levels of PgtE expression by African S Typhimurium ST313 promote bacterial survival and dissemination during human infection. Our finding of a functional role for an extragenic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on noncoding regions of the genome.
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Evolución Molecular , Genoma Bacteriano/genética , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , ADN Bacteriano/genética , Epidemias , Humanos , Filogenia , Polimorfismo de Nucleótido Simple/genética , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
We present the first high-resolution determination of transcriptome architecture in the priority pathogen Acinetobacter baumannii. Pooled RNA from 16 laboratory conditions was used for differential RNA-seq (dRNA-seq) to identify 3731 transcriptional start sites (TSS) and 110 small RNAs, including the first identification in A. baumannii of sRNAs encoded at the 3' end of coding genes. Most sRNAs were conserved among sequenced A. baumannii genomes, but were only weakly conserved or absent in other Acinetobacter species. Single nucleotide mapping of TSS enabled prediction of -10 and -35 RNA polymerase binding sites and revealed an unprecedented base preference at position +2 that hints at an unrecognized transcriptional regulatory mechanism. To apply functional genomics to the problem of antimicrobial resistance, we dissected the transcriptional regulation of the drug efflux pump responsible for chloramphenicol resistance, craA. The two craA promoters were both down-regulated >1000-fold when cells were shifted to nutrient limited medium. This conditional down-regulation of craA expression renders cells sensitive to chloramphenicol, a highly effective antibiotic for the treatment of multidrug resistant infections. An online interface that facilitates open data access and visualization is provided as 'AcinetoCom' (http://bioinf.gen.tcd.ie/acinetocom/).
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Acinetobacter baumannii/genética , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , ARN Bacteriano/genética , Transcriptoma/genética , Acinetobacter baumannii/efectos de los fármacos , Mapeo Cromosómico , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ARN/métodosRESUMEN
We know a great deal about the genes used by the model pathogen Salmonella enterica serovar Typhimurium to cause disease, but less about global gene regulation. New tools for studying transcripts at the single nucleotide level now offer an unparalleled opportunity to understand the bacterial transcriptome, and expression of the small RNAs (sRNA) and coding genes responsible for the establishment of infection. Here, we define the transcriptomes of 18 mutants lacking virulence-related global regulatory systems that modulate the expression of the SPI1 and SPI2 Type 3 secretion systems of S. Typhimurium strain 4/74. Using infection-relevant growth conditions, we identified a total of 1257 coding genes that are controlled by one or more regulatory system, including a sub-class of genes that reflect a new level of cross-talk between SPI1 and SPI2. We directly compared the roles played by the major transcriptional regulators in the expression of sRNAs, and discovered that the RpoS (σ38) sigma factor modulates the expression of 23% of sRNAs, many more than other regulatory systems. The impact of the RNA chaperone Hfq upon the steady state levels of 280 sRNA transcripts is described, and we found 13 sRNAs that are co-regulated with SPI1 and SPI2 virulence genes. We report the first example of an sRNA, STnc1480, that is subject to silencing by H-NS and subsequent counter-silencing by PhoP and SlyA. The data for these 18 regulatory systems is now available to the bacterial research community in a user-friendly online resource, SalComRegulon.
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Proteínas Bacterianas/biosíntesis , Proteínas de la Membrana/biosíntesis , ARN Pequeño no Traducido/genética , Salmonella typhimurium/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Proteína de Factor 1 del Huésped/biosíntesis , Proteína de Factor 1 del Huésped/genética , Proteínas de la Membrana/genética , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/patogenicidad , Serogrupo , Factor sigma/biosíntesis , Factor sigma/genética , Transcriptoma/genética , VirulenciaRESUMEN
Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection.
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Regulación Bacteriana de la Expresión Génica/genética , Macrófagos/metabolismo , ARN Bacteriano/genética , Salmonella typhimurium/genética , Transcriptoma/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Genes Bacterianos/genética , Islas Genómicas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Vacunas contra la Salmonella/genética , Virulencia/genéticaRESUMEN
Pathogenic bacteria often need to survive in the host and the environment, and it is not well understood how cells transition between these equally challenging situations. For the human and animal pathogen Salmonella enterica serovar Typhimurium, biofilm formation is correlated with persistence outside a host, but the connection to virulence is unknown. In this study, we analyzed multicellular-aggregate and planktonic-cell subpopulations that coexist when S. Typhimurium is grown under biofilm-inducing conditions. These cell types arise due to bistable expression of CsgD, the central biofilm regulator. Despite being exposed to the same stresses, the two cell subpopulations had 1,856 genes that were differentially expressed, as determined by transcriptome sequencing (RNA-seq). Aggregated cells displayed the characteristic gene expression of biofilms, whereas planktonic cells had enhanced expression of numerous virulence genes. Increased type three secretion synthesis in planktonic cells correlated with enhanced invasion of a human intestinal cell line and significantly increased virulence in mice compared to the aggregates. However, when the same groups of cells were exposed to desiccation, the aggregates survived better, and the competitive advantage of planktonic cells was lost. We hypothesize that CsgD-based differentiation is a form of bet hedging, with single cells primed for host cell invasion and aggregated cells adapted for persistence in the environment. This allows S. Typhimurium to spread the risks of transmission and ensures a smooth transition between the host and the environment.
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Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidad , Transactivadores/metabolismo , Animales , Proteínas Bacterianas/genética , Células CACO-2 , GMP Cíclico/análogos & derivados , Humanos , Ratones , Transporte de Proteínas , Salmonella typhimurium/genética , Transcripción Genética , VirulenciaRESUMEN
In Salmonella enterica serovar Typhimurium (S. Typhimurium), the genomic island GEI4417/4436 is responsible for the utilization of myo-inositol (MI) as carbon and energy source. Here, we report the characterization of a novel, island-encoded positive autoregulator termed ReiD (STM4423) that is specific to certain S. enterica strains and Escherichia coli strain ED1a able to use MI. ReiD was essential for growth with this polyol and also contributed to S. Typhimurium proliferation in swine caecum content. Providing higher copy numbers of ReiD reduced the long lag phase of 2 days during growth of S. Typhimurium in MI medium by 50%. In a heterologous host, expression of ReiD activated the transcription from the promoter of iolE/iolG, whose products catalyse the initial two steps in MI degradation. Episomal expression of iolE/iolG1 rescued the otherwise zero growth phenotype of a reiD deletion mutant in MI medium. Gel mobility shift assays with purified ReiD demonstrated directed interaction of ReiD with its own promoter and that of iolE. The repressor IolR bound the reiD promoter, implying that reiD is part of the IolR regulon. Taken together, the regulator ReiD is a trigger to accelerate the switch from more easily accessible nutrients to MI utilization by S. Typhimurium.
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Regulación Bacteriana de la Expresión Génica , Inositol/metabolismo , Salmonella typhimurium/metabolismo , Factores de Transcripción/metabolismo , Animales , Carbono/metabolismo , Ciego/microbiología , Medios de Cultivo/química , ADN Bacteriano/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Metabolismo Energético , Regiones Promotoras Genéticas , Unión Proteica , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrollo , Porcinos , VirulenciaRESUMEN
More than 50 y of research have provided great insight into the physiology, metabolism, and molecular biology of Salmonella enterica serovar Typhimurium (S. Typhimurium), but important gaps in our knowledge remain. It is clear that a precise choreography of gene expression is required for Salmonella infection, but basic genetic information such as the global locations of transcription start sites (TSSs) has been lacking. We combined three RNA-sequencing techniques and two sequencing platforms to generate a robust picture of transcription in S. Typhimurium. Differential RNA sequencing identified 1,873 TSSs on the chromosome of S. Typhimurium SL1344 and 13% of these TSSs initiated antisense transcripts. Unique findings include the TSSs of the virulence regulators phoP, slyA, and invF. Chromatin immunoprecipitation revealed that RNA polymerase was bound to 70% of the TSSs, and two-thirds of these TSSs were associated with σ(70) (including phoP, slyA, and invF) from which we identified the -10 and -35 motifs of σ(70)-dependent S. Typhimurium gene promoters. Overall, we corrected the location of important genes and discovered 18 times more promoters than identified previously. S. Typhimurium expresses 140 small regulatory RNAs (sRNAs) at early stationary phase, including 60 newly identified sRNAs. Almost half of the experimentally verified sRNAs were found to be unique to the Salmonella genus, and <20% were found throughout the Enterobacteriaceae. This description of the transcriptional map of SL1344 advances our understanding of S. Typhimurium, arguably the most important bacterial infection model.
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Regulación Bacteriana de la Expresión Génica/genética , ARN Pequeño no Traducido/genética , Secuencias Reguladoras de Ácido Ribonucleico/genética , Salmonella typhimurium/genética , Transcripción Genética/genética , Secuencia de Bases , Northern Blotting , Inmunoprecipitación de Cromatina , Biblioteca de Genes , Análisis por Micromatrices , Datos de Secuencia Molecular , Oligonucleótidos/genética , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ARN/métodos , Sitio de Iniciación de la TranscripciónRESUMEN
IMPORTANCE: The capacity to utilize myo-inositol (MI) as sole carbon and energy source is widespread among bacteria, among them the intestinal pathogen S. Typhimurium. This study elucidates the complex and hierarchical regulation that underlies the utilization of MI by S. Typhimurium under substrate limitation. A total of seven regulatory factors have been identified so far, allowing the pathogen an environment-dependent, efficient, and fine-tuned regulation of a metabolic property that provides growth advantages in different environments.
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Salmonella enterica , Salmonella enterica/metabolismo , Salmonella typhimurium/genética , Regiones Promotoras Genéticas , Proteínas Bacterianas/genética , Inositol/metabolismo , Redes y Vías Metabólicas , Regulación Bacteriana de la Expresión GénicaRESUMEN
Invasive non-typhoidal Salmonella (iNTS) disease is a serious bloodstream infection that targets immune-compromised individuals, and causes significant mortality in sub-Saharan Africa. Salmonella enterica serovar Typhimurium ST313 causes the majority of iNTS in Malawi. We performed an intensive comparative genomic analysis of 608 S. Typhimurium ST313 isolates dating between 1996 and 2018 from Blantyre, Malawi. We discovered that following the arrival of the well-characterized S. Typhimurium ST313 lineage 2 in 1999, two multidrug-resistant variants emerged in Malawi in 2006 and 2008, designated sublineages 2.2 and 2.3, respectively. The majority of S. Typhimurium isolates from human bloodstream infections in Malawi now belong to sublineages 2.2 or 2.3. To understand the emergence of the prevalent ST313 sublineage 2.2, we studied two representative strains, D23580 (lineage 2) and D37712 (sublineage 2.2). The chromosome of ST313 lineage 2 and sublineage 2.2 only differed by 29 SNPs/small indels and a 3 kb deletion of a Gifsy-2 prophage region including the sseI pseudogene. Lineage 2 and sublineage 2.2 had distinctive plasmid profiles. The transcriptome was investigated in 15 infection-relevant in vitro conditions and within macrophages. During growth in physiological conditions that do not usually trigger S. Typhimurium SPI2 gene expression, the SPI2 genes of D37712 were transcriptionally active. We identified down-regulation of flagellar genes in D37712 compared with D23580. Following phenotypic confirmation of transcriptomic differences, we discovered that sublineage 2.2 had increased fitness compared with lineage 2 during mixed growth in minimal media. We speculate that this competitive advantage is contributing to the emergence of sublineage 2.2 in Malawi.
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Horizontal gene transfer by plasmids can confer metabolic capabilities that expand a host cell's niche. Yet, it is less understood whether the coalescence of specialized catabolic functions, antibiotic resistances and metal resistances on plasmids provides synergistic benefits. In this study, we report whole-genome assembly and phenotypic analysis of five Salmonella enterica strains isolated in the 1980s from milk powder in Munich, Germany. All strains exhibited the unusual phenotype of lactose-fermentation and encoded either of two variants of the lac operon. Surprisingly, all strains encoded the mobilized colistin resistance gene 9 (mcr-9), long before the first report of this gene in the literature. In two cases, the mcr-9 gene and the lac locus were linked within a large gene island that formed an IncHI2A-type plasmid in one strain but was chromosomally integrated in the other strain. In two other strains, the mcr-9 gene was found on a large IncHI1B/IncP-type plasmid, whereas the lac locus was encoded on a separate chromosomally integrated plasmidic island. The mcr-9 sequences were identical and genomic contexts could not explain the wide range of colistin resistances exhibited by the Salmonella strains. Nucleotide variants did explain phenotypic differences in motility and exopolysaccharide production. The observed linkage of mcr-9 to lactose metabolism, an array of heavy-metal detoxification systems, and other antibiotic resistance genes may reflect a coalescence of specialized phenotypes that improve the spread of colistin resistance in dairy facilities, much earlier than previously suspected.
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Colistina , Salmonella enterica , Colistina/farmacología , Salmonella enterica/genética , Lactosa , Serogrupo , Farmacorresistencia Bacteriana/genética , Plásmidos/genéticaRESUMEN
The combination of genomics and high-throughput cDNA sequencing technologies has facilitated the identification of many small RNAs (sRNAs) that play a central role in the post-transcriptional gene regulation of Salmonella enterica serovar Typhimurium. To date, most of the functionally characterized sRNAs have been involved in the regulation of processes which are not directly linked to virulence. Just five sRNAs have been found to affect the ability of Salmonella to replicate within mammalian cells, but the precise regulatory mechanisms that are used by sRNAs to control Salmonella pathogenicity at the post-transcriptional level remain to be identified. It is anticipated that an improved understanding of sRNA biology will shed new light on the virulence of Salmonella.
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Infecciones por Salmonella/microbiología , Animales , Secuencia de Bases , Secuencia Conservada , Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Genes Bacterianos , Interacciones Huésped-Parásitos/genética , Humanos , Interferencia de ARN , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/fisiología , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Virulencia/genéticaRESUMEN
The capability of Salmonella enterica serovar Typhimurium strain 14028 (S. Typhimurium 14028) to utilize myo-inositol (MI) is determined by the genomic island GEI4417/4436 carrying the iol genes that encode enzymes, transporters, and a repressor responsible for the MI catabolic pathway. In contrast to all bacteria investigated thus far, S. Typhimurium 14028 growing on MI as the sole carbon source is characterized by a remarkable long lag phase of 40 to 60 h. We report here that on solid medium with MI as the sole carbon source, this human pathogen exhibits a bistable phenotype characterized by a dissection into large colonies and a slow-growing bacterial background. This heterogeneity is reversible and therefore not caused by mutation, and it is not observed in the absence of the iol gene repressor IolR nor in the presence of at least 0.55% CO(2). Bistability is correlated with the activity of the iolE promoter (P(iolE)), but not of P(iolC) or P(iolD), as shown by promoter-gfp fusions. On the single-cell level, fluorescence microscopy and flow cytometry analysis revealed a gradual switch of P(iolE) from the "off" to the "on" status during the late lag phase and the transition to the log phase. Deletion of iolR or the addition of 0.1% NaHCO(3) induced an early growth start of S. Typhimurium 14028 in minimal medium with MI. The addition of ethoxyzolamide, an inhibitor of carboanhydrases, elongated the lag phase in the presence of bicarbonate. The positive-feedback loop via repressor release and positive induction by bicarbonate-CO(2) might allow S. Typhimurium 14028 to adapt to rapidly changing environments. The phenomenon described here is a novel example of bistability in substrate degradation, and, to our knowledge, is the first demonstration of gene regulation by bicarbonate-CO(2) in Salmonella.
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Regulación Bacteriana de la Expresión Génica , Inositol/metabolismo , Salmonella typhimurium/metabolismo , Fusión Artificial Génica , Carbono/metabolismo , Medios de Cultivo/química , Citometría de Flujo , Eliminación de Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente , Fenotipo , Regiones Promotoras Genéticas , Salmonella typhimurium/crecimiento & desarrollo , Activación TranscripcionalRESUMEN
We report the draft genome sequence of Acinetobacter soli AS15, which was isolated in 2018 from a rectal screen of a patient at St. Vincent's University Hospital (Dublin, Ireland). The draft genome sequence is 3,589,002 bp and was assembled into 82 contigs.
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Alzheimer's disease (AD) is a common neurodegenerative disease that is accompanied by pronounced neuroinflammatory responses mainly characterized by marked microgliosis and astrogliosis. However, it remains open as to how different aspects of astrocytic and microglial activation affect disease progression. Previously, we found that microglia expansion in the spinal cord, initiated by IKK2/NF-κB activation in astrocytes, exhibits stage-dependent beneficial effects on the progression of amyotrophic lateral sclerosis. Here, we investigated the impact of NF-κB-initiated neuroinflammation on AD pathogenesis using the APP23 mouse model of AD in combination with conditional activation of IKK2/NF-κB signaling in astrocytes. We show that NF-κB activation in astrocytes triggers a distinct neuroinflammatory response characterized by striking astrogliosis as well as prominent microglial reactivity. Immunohistochemistry and Congo red staining revealed an overall reduction in the size and number of amyloid plaques in the cerebral cortex and hippocampus. Interestingly, isolated primary astrocytes and microglia cells exhibit specific marker gene profiles which, in the case of microglia, point to an enhanced plaque clearance capacity. In contrast, direct IKK2/NF-κB activation in microglia results in a pro-inflammatory polarization program. Our findings suggest that IKK2/NF-κB signaling in astrocytes may activate paracrine mechanisms acting on microglia function but also on APP processing in neurons.
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Péptidos beta-Amiloides/metabolismo , Astrocitos/metabolismo , Polaridad Celular , Quinasa I-kappa B/metabolismo , Microglía/patología , FN-kappa B/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Inflamación/patología , Ratones Transgénicos , Microglía/metabolismo , Modelos Biológicos , Fagocitosis , Fenotipo , Placa Amiloide/genética , Placa Amiloide/patología , Proteolisis , Transducción de SeñalRESUMEN
In Salmonella enterica serovar Typhimurium, the genomic island GEI4417/4436 has recently been identified to be responsible for myo-inositol (MI) utilization. Here, two of the four island-encoded permeases are identified as the MI transporters of this pathogen. In-frame deletion of iolT1 (STM4418) led to a severe growth defect, and deletion of iolT1 (STM4419) to a slight growth defect in the presence of MI. These phenotypes could be complemented by providing the putative transporter genes in trans. Bioluminescence-based reporter assays demonstrated a strong induction of their promoters P(iolT1) and P(iolT2) in the presence of MI but not of glucose. Deletion of iolR, which encodes the negative regulator of most genes involved in MI degradation, resulted in upregulation of P(iolT1) and P(iolT2), indicating that the expression of IolT1 and IolT2 is repressed by IolR. This finding was supported by bandshift assays using purified IolR. Both transporters are located in the membrane when expressed in Escherichia coli. Heterologously expressed IolT1 had its optimal activity at pH 5.5. Together with the strongly reduced MI uptake in the presence of protonophores, this indicates that IolT1 operates as a proton symporter. Using myo-[1,2-[(3)H](N)]inositol, a saturable uptake activity of IolT1 with a K(m) value between 0.49 and 0.79 mM was determined in DH5alpha expressing IolT1, in S. enterica serovar Typhimurium strain 14028, and in mutant 14028 DeltaiolT2. Phylogenetic analysis of IolT1 identified putative MI transporters in Gram-negative bacteria also able to utilize MI.
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Proteínas Bacterianas/metabolismo , Islas Genómicas , Inositol/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Salmonella typhimurium/metabolismo , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Proteínas de Transporte de Membrana/genética , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrolloRESUMEN
Prophages are integrated phage elements that are a pervasive feature of bacterial genomes. The fitness of bacteria is enhanced by prophages that confer beneficial functions such as virulence, stress tolerance or phage resistance, and these functions are encoded by 'accessory' or 'moron' loci. Whilst the majority of phage-encoded genes are repressed during lysogeny, accessory loci are often highly expressed. However, it is challenging to identify novel prophage accessory loci from DNA sequence data alone. Here, we use bacterial RNA-seq data to examine the transcriptional landscapes of five Salmonella prophages. We show that transcriptomic data can be used to heuristically enrich for prophage features that are highly expressed within bacterial cells and represent functionally important accessory loci. Using this approach, we identify a novel antisense RNA species in prophage BTP1, STnc6030, which mediates superinfection exclusion of phage BTP1. Bacterial transcriptomic datasets are a powerful tool to explore the molecular biology of temperate phages.
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Bacteriófagos/fisiología , Lisogenia , Transcriptoma , Bacteriófagos/genética , Profagos/genética , Profagos/fisiología , Salmonella/virología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Knockout mutation of STM4432 resulted in a growth-deficient phenotype of Salmonella enterica serovar Typhimurium in the presence of myo-inositol (MI) as the sole carbon source. STM4432 is part of a 22.6-kb genomic island which spans STM4417 to STM4436 (genomic island 4417/4436) and is responsible for MI degradation. Genome comparison revealed the presence of this island in only six Salmonella strains and a high variability of the iol gene organization in gram-negative bacteria. Upon nonpolar deletion of 11 island loci, the genes involved in six enzymatic steps of the MI pathway were identified. The generation time of S. enterica serovar Typhimurium in minimal medium with MI decreases with higher concentrations of this polyol. Reverse transcriptase PCR showed five separate transcriptional units encompassing the genes iolA-iolB, iolE-iolG1, iolC1-iolC2, iolD1-iolD2-iolG2, and iolI2-iolH. Luciferase reporter assays revealed a strong induction of their promoters in the presence of MI but not glucose. The main regulator, IolR, was identified due to a reduced lag phase of a strain mutated in STM4417 (iolR). Deletion of iolR resulted in stimulation of the iol operons, indicating its negative effect on the iol genes of S. enterica serovar Typhimurium in rich medium at a transcriptional level. Bandshift assays demonstrated the binding of this putative repressor to promoter sequences of iolA, iolC1, and iolD1. Binding of IolR to its own promoter and induced iolR expression in an IolR-negative background demonstrate that its transcription is autoregulated. This is the first characterization of MI degradation in a gram-negative bacterium, revealing a complex transcriptional organization and regulation of the S. enterica serovar Typhimurium iol genes.
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Islas Genómicas , Inositol/metabolismo , Salmonella typhimurium/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Mutación , Regiones Promotoras Genéticas , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
We report here the draft genome sequence of Acinetobacter junii MHI21018, isolated in 2009 from bovine colostrum. The draft genome sequence is composed of 3,267,995 bp, has a GC content of 38.54%, and was assembled into 114 contigs (contig size, >500 bp) with an N 50 value of 72,566 bp.