RESUMEN
The extracellular biofilm matrix often contains a network of amyloid fibers which, in the human opportunistic pathogen Bacillus cereus, includes the two homologous proteins TasA and CalY. We show here, in the closely related entomopathogenic species Bacillus thuringiensis, that CalY also displays a second function. In the early stationary phase of planktonic cultures, CalY was located at the bacterial cell-surface, as shown by immunodetection. Deletion of calY revealed that this protein plays a major role in adhesion to HeLa epithelial cells, to the insect Galleria mellonella hemocytes and in the bacterial virulence against larvae of this insect, suggesting that CalY is a cell-surface adhesin. In mid-stationary phase and in biofilms, the location of CalY shifted from the cell surface to the extracellular medium, where it was found as fibers. The transcription study and the deletion of sipW suggested that CalY change of location is due to a delayed activity of the SipW signal peptidase. Using purified CalY, we found that the protein polymerization occurred only in the presence of cell-surface components. CalY is, therefore, a bifunctional protein, which switches from a cell-surface adhesin activity in early stationary phase, to the production of fibers in mid-stationary phase and in biofilms.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Bacillus thuringiensis/genética , Biopelículas/crecimiento & desarrollo , Metaloproteasas/metabolismo , Factores de Virulencia/metabolismo , Adhesinas Bacterianas/genética , Animales , Bacillus thuringiensis/enzimología , Adhesión Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Matriz Extracelular de Sustancias Poliméricas/genética , Matriz Extracelular de Sustancias Poliméricas/metabolismo , Células HeLa , Hemocitos/microbiología , Humanos , Larva/microbiología , Metaloproteasas/genética , Mariposas Nocturnas/microbiología , Factores de Virulencia/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1002629.].
RESUMEN
IStrons are chimeric genetic elements composed of a group I intron associated with an insertion sequence (IS). The group I intron is a catalytic RNA providing the IStron with self-splicing ability, which renders IStron insertions harmless to the host genome. The IS element is a DNA transposon conferring mobility, and thus allowing the IStron to spread in genomes. IStrons are therefore a striking example of a molecular symbiosis between unrelated genetic elements endowed with different functions. In this study, we have conducted the first comprehensive survey of IStrons in sequenced genomes that provides insights into the distribution, diversity, origin and evolution of IStrons. We show that IStrons have a restricted phylogenetic distribution limited to two bacterial phyla, the Firmicutes and the Fusobacteria. Nevertheless, diverse IStrons representing two major groups targeting different insertion site motifs were identified. This taken with the finding that while the intron components of all IStrons belong to the same structural class, they are fused to different IS families, indicates that multiple intron-IS symbioses have occurred during evolution. In addition, introns and IS elements related to those that were at the origin of IStrons were also identified.
Asunto(s)
Elementos Transponibles de ADN , Genoma Bacteriano , Intrones , ARN Catalítico , Evolución Molecular , Empalme del ARNRESUMEN
How pathogenic bacteria infect and kill their host is currently widely investigated. In comparison, the fate of pathogens after the death of their host receives less attention. We studied Bacillus thuringiensis (Bt) infection of an insect host, and show that NprR, a quorum sensor, is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes, including many encoding degradative enzymes or proteins involved in the synthesis of a nonribosomal peptide named kurstakin. These degradative enzymes are essential in vitro to degrade several substrates and are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We show that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. We report that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to spore spreading.
Asunto(s)
Bacillus thuringiensis/fisiología , Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno/fisiología , Insectos/microbiología , Percepción de Quorum/fisiología , Animales , Proteínas Bacterianas/genética , MutaciónRESUMEN
Bacillus anthracis is the cause of anthrax, and two large plasmids are essential for toxicity: pXO1, which contains the toxin genes, and pXO2, which encodes a capsule. B. anthracis forms a highly monomorphic lineage within the B. cereus group, but strains of Bacillus thuringiensis and B. cereus exist that are genetically closely related to the B. anthracis cluster. During the past five years B. cereus strains that contain the pXO1 virulence plasmid were discovered, and strains with both pXO1 and pXO2 have been isolated from great apes in Africa. Therefore, the presence of pXO1 and pXO2 no longer principally separates B. anthracis from other Bacilli. The B. anthracis lineage carries a specific mutation in the global regulator PlcR, which controls the transcription of secreted virulence factors in B. cereus and B. thuringiensis. Coevolution of the B. anthracis chromosome with its plasmids may be the basis for the successful development and uniqueness of the B. anthracis lineage.
Asunto(s)
Bacillus anthracis/patogenicidad , Bacillus cereus/patogenicidad , Bacillus thuringiensis/patogenicidad , Plásmidos , Factores de Virulencia/biosíntesis , Animales , Bacillus anthracis/genética , Bacillus cereus/genética , Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Transactivadores/genética , Factores de Virulencia/genéticaRESUMEN
The recently discovered HEAT-like repeat (HLR) DNA glycosylase superfamily is widely distributed in all domains of life. The present bioinformatics and phylogenetic analysis shows that HLR DNA glycosylase superfamily members in the genus Bacillus form three subfamilies: AlkC, AlkD and AlkF/AlkG. The crystal structure of AlkF shows structural similarity with the DNA glycosylases AlkC and AlkD, however neither AlkF nor AlkG display any DNA glycosylase activity. Instead, both proteins have affinity to branched DNA structures such as three-way and Holliday junctions. A unique ß-hairpin in the AlkF/AlkG subfamily is most likely inserted into the DNA major groove, and could be a structural determinant regulating DNA substrate affinity. We conclude that AlkF and AlkG represent a new family of HLR proteins with affinity for branched DNA structures.
Asunto(s)
Bacillus cereus/enzimología , Proteínas Bacterianas/química , ADN Glicosilasas/química , Sitios de Unión , Cromatografía Líquida de Alta Presión , Análisis por Conglomerados , Escherichia coli/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Estructura Terciaria de ProteínaRESUMEN
The Bacillus cereus type strain ATCC 14579 harbours pBClin15, a linear plasmid with similar genome organization to tectiviruses. Since phage morphogenesis is not known to occur it has been suggested that pBClin15 may be a defect relic of a tectiviral prophage without relevance for the bacterial physiology. However, in this paper, we demonstrate that a pBClin15-cured strain is more tolerant to antibiotics interfering with DNA integrity than the WT strain. Growth in the presence of crystal violet or the quinolones nalidixic acid, norfloxacin or ciprofloxacin resulted in aggregation and lysis of the WT strain, whereas the pBClin15-cured strain was unaffected. Microarray analysis comparing the gene expression in the WT and pBClin15-cured strains showed that pBClin15 gene expression was strongly upregulated in response to norfloxacin stress, and coincided with lysis and aggregation of the WT strain. The aggregating bacteria experienced a significant survival benefit compared with the planktonic counterparts in the presence of norfloxacin. There was no difference between the WT and pBClin15-cured strains during growth in the absence of norfloxacin, the pBClin15 genes were moderately expressed, and no effect was observed on chromosomal gene expression. These data demonstrate for the first time that although pBClin15 may be a remnant of a temperate phage, it negatively affects the DNA stress tolerance of B. cereus ATCC 14579. Furthermore, our results warrant a recommendation to always verify the presence of pBClin15 following genetic manipulation of B. cereus ATCC 14579.
Asunto(s)
Bacillus cereus/efectos de los fármacos , Bacteriólisis , Adhesión Celular , ADN Bacteriano/metabolismo , Expresión Génica/efectos de los fármacos , Plásmidos , Quinolonas/metabolismo , Antibacterianos/metabolismo , Bacillus cereus/genética , Bacillus cereus/fisiología , Perfilación de la Expresión Génica , Análisis por Micromatrices , Transcripción GenéticaRESUMEN
Core genome multilocus sequence typing (cgMLST) employs a strategy where the set of orthologous genes common to all members of a group of organisms are used for phylogenetic analysis of the group members. The Bacillus cereus group consists of species with pathogenicity towards insect species as well as warm-blooded animals including humans. While B. cereus is an opportunistic pathogen linked to a range of human disease conditions, including emesis and diarrhoea, Bacillus thuringiensis is an entomopathogenic species with toxicity toward insect larvae, and therefore used as a biological pesticide worldwide. Bacillus anthracis is a classical obligate pathogen causing anthrax, an acute lethal condition in herbivores as well as humans, and which is endemic in many parts of the world. The group also includes a range of additional species, and B. cereus group bacteria have been subject to analysis with a wide variety of phylogenetic typing systems. Here we present, based on analyses of 173 complete genomes from B. cereus group species available in public databases, the identification of a set of 1568 core genes which were used to create a core genome multilocus typing scheme for the group which is implemented in the PubMLST system as an open online database freely available to the community. The new cgMLST system provides unprecedented resolution over existing phylogenetic analysis schemes covering the B. cereus group.
Asunto(s)
Bacillus anthracis , Bacillus , Animales , Humanos , Tipificación de Secuencias Multilocus , Bacillus cereus/genética , Filogenia , Bacillus anthracis/genética , Genoma Bacteriano/genéticaRESUMEN
Many short (<400 bp) interspersed sequence repeats exist in bacteria, yet little is known about their origins, mode of generation, or possible function. Here, we present a comprehensive analysis of 18 different previously identified repeated DNA elements, bcr1-bcr18 (Økstad OA, Hegna I, Lindback T, Rishovd AL, Kolstø AB. 1999. Genome organization is not conserved between Bacillus cereus and Bacillus subtilis. Microbiology. 145:621-631.; Tourasse NJ, Helgason E, Økstad OA, Hegna IK, Kolstø AB. 2006. The Bacillus cereus group: novel aspects of population structure and genome dynamics. J Appl Microbiol. 101:579-593.), in 36 sequenced genomes from the Bacillus cereus group of bacteria. This group consists of genetically closely related species with variable pathogenic specificity toward different hosts and includes among others B. anthracis, B. cereus, and B. thuringiensis. The B. cereus group repeat elements could be classified into three categories with different properties: Group A elements (bcr1-bcr3) exhibited highly variable copy numbers ranging from 4 to 116 copies per strain, showed a nonconserved chromosomal distribution pattern between strains, and displayed several features characteristic of mobile elements. Group B repeats (bcr4-bcr6) were present in 0-10 copies per strain and were associated with strain-specific genes and disruptions of genome synteny, implying a possible contribution to genome rearrangements and/or horizontal gene transfer events. bcr5, in particular, was associated with large gene clusters showing resemblance to integrons. In agreement with their potentially mobile nature or involvement in horizontal transfers, the sequences of the repeats from Groups A and B (bcr1-bcr6) followed a phylogeny different from that of the host strains. Conversely, repeats from Group C (bcr7-bcr18) had a conserved chromosomal location and orthologous gene neighbors in the investigated B. cereus group genomes, and their phylogeny matched that of the host chromosome. Several of the group C repeats exhibited a conserved secondary structure or had parts of the structure conserved, possibly indicating functional RNAs. Accordingly, five of the repeats in group C overlapped regions encoding previously characterized riboswitches. Similarly, other group C repeats could represent novel riboswitches, encode small RNAs, and/or constitute other types of regulatory elements with specific biological functions. The current analysis suggests that the multitude of repeat elements identified in the B. cereus group promote genome dynamics and plasticity and could contribute to the flexible and adaptive life style of these bacteria.
Asunto(s)
Bacillus cereus/clasificación , Bacillus cereus/genética , ADN Bacteriano/genética , Bacillus cereus/fisiología , Filogenia , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Here we report the use of a multi-genome DNA microarray to investigate the genome diversity of Bacillus cereus group members and elucidate the events associated with the emergence of Bacillus anthracis the causative agent of anthrax-a lethal zoonotic disease. We initially performed directed genome sequencing of seven diverse B. cereus strains to identify novel sequences encoded in those genomes. The novel genes identified, combined with those publicly available, allowed the design of a "species" DNA microarray. Comparative genomic hybridization analyses of 41 strains indicate that substantial heterogeneity exists with respect to the genes comprising functional role categories. While the acquisition of the plasmid-encoded pathogenicity island (pXO1) and capsule genes (pXO2) represents a crucial landmark dictating the emergence of B. anthracis, the evolution of this species and its close relatives was associated with an overall shift in the fraction of genes devoted to energy metabolism, cellular processes, transport, as well as virulence.
Asunto(s)
Bacillus anthracis/genética , Evolución Molecular , Genoma Bacteriano , Bacillus anthracis/patogenicidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , VirulenciaRESUMEN
The Bacillus cereus group of bacteria is a group of closely related species that are of medical and economic relevance, including B. anthracis, B. cereus, and B. thuringiensis. Bacteria from the Bacillus cereus group encode three large, highly conserved genes of unknown function (named crdA, crdB, and crdC) that are composed of 16 to 35 copies of a repeated domain of 132 amino acids at the protein level. Bioinformatic analysis revealed that there is a phylogenetic bias in the genomic distribution of these genes and that strains harboring all three large genes mainly belong to cluster III of the B. cereus group phylogenetic tree. The evolutionary history of the three large genes implicates gain, loss, duplication, internal deletion, and lateral transfer. Furthermore, we show that the transcription of previously identified antisense open reading frames in crdB is simultaneously regulated with its host gene throughout the life cycle in vitro, with the highest expression being at the onset of sporulation. In B. anthracis, different combinations of double- and triple-knockout mutants of the three large genes displayed slower and less efficient sporulation processes than the parental strain. Altogether, the functional studies suggest an involvement of these three large genes in the sporulation process.
Asunto(s)
Bacillus cereus/genética , Proteínas Bacterianas/genética , Evolución Molecular , Esporas Bacterianas/genética , Bacillus cereus/fisiología , Proteínas Bacterianas/clasificación , Biología Computacional , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esporas Bacterianas/fisiologíaRESUMEN
Bacillus pseudofirmus OF4 is an extreme but facultative alkaliphile that grows non-fermentatively in a pH range from 7.5 to above 11.4 and can withstand large sudden increases in external pH. It is a model organism for studies of bioenergetics at high pH, at which energy demands are higher than at neutral pH because both cytoplasmic pH homeostasis and ATP synthesis require more energy. The alkaliphile also tolerates a cytoplasmic pH > 9.0 at external pH values at which the pH homeostasis capacity is exceeded, and manages other stresses that are exacerbated at alkaline pH, e.g. sodium, oxidative and cell wall stresses. The genome of B. pseudofirmus OF4 includes two plasmids that are lost from some mutants without viability loss. The plasmids may provide a reservoir of mobile elements that promote adaptive chromosomal rearrangements under particular environmental conditions. The genome also reveals a more acidic pI profile for proteins exposed on the outer surface than found in neutralophiles. A large array of transporters and regulatory genes are predicted to protect the alkaliphile from its overlapping stresses. In addition, unanticipated metabolic versatility was observed, which could ensure requisite energy for alkaliphily under diverse conditions.
Asunto(s)
Adaptación Fisiológica/genética , Bacillus/genética , Genoma Bacteriano , Concentración de Iones de Hidrógeno , Bacillus/fisiología , Proteínas Bacterianas/química , Pared Celular/fisiología , Citoplasma/química , Elementos Transponibles de ADN , ADN Bacteriano/genética , Metabolismo Energético , Intrones , Anotación de Secuencia Molecular , Estrés Oxidativo , Fosforilación , Plásmidos/genética , Origen de Réplica , Sodio/químicaRESUMEN
Bacillus cereus can use swarming to move over and colonize solid surfaces in different environments. This kind of motility is a collective behavior accompanied by the production of long and hyperflagellate swarm cells. In this study, the genome-wide transcriptional response of B. cereus ATCC 14579 during swarming was analyzed. Swarming was shown to trigger the differential expression (>2-fold change) of 118 genes. Downregulated genes included those required for basic cellular metabolism. In accordance with the hyperflagellate phenotype of the swarm cell, genes encoding flagellin were overexpressed. Some genes associated with K(+) transport, phBC6A51 phage genes, and the binding component of the enterotoxin hemolysin BL (HBL) were also induced. Quantitative reverse transcription-PCR (qRT-PCR) experiments indicated an almost 2-fold upregulation of the entire hbl operon during swarming. Finally, BC1435 and BC1436, orthologs of liaI-liaH that are known to be involved in the resistance of Bacillus subtilis to daptomycin, were upregulated under swarming conditions. Accordingly, phenotypic assays showed reduced susceptibility of swarming B. cereus cells to daptomycin, and P(spac)-induced hyper-expression of these genes in liquid medium highlighted the role of BC1435 and BC1436 in the response of B. cereus to daptomycin.
Asunto(s)
Bacillus cereus/citología , Flagelos/genética , Flagelina/biosíntesis , Flagelina/genética , Bacillus cereus/genética , Bacillus cereus/fisiología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Daptomicina/farmacología , Farmacorresistencia Bacteriana/genética , Enterotoxinas/biosíntesis , Expresión Génica , Perfilación de la Expresión Génica , Genotipo , Proteínas Hemolisinas/biosíntesis , Proteínas Hemolisinas/genética , Análisis por Micromatrices , Pruebas de Sensibilidad Microbiana , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Microbiología del SueloRESUMEN
The B.c.I4 group II intron from Bacillus cereus ATCC 10987 harbors an unusual 3' extension. Here, we report the discovery of four additional group II introns with a similar 3' extension in Bacillus thuringiensis kurstaki 4D1 that splice at analogous positions 53/56 nt downstream of domain VI in vivo. Phylogenetic analyses revealed that the introns are only 47-61% identical to each other. Strikingly, they do not form a single evolutionary lineage even though they belong to the same Bacterial B class. The extension of these introns is predicted to form a conserved two-stem-loop structure. Mutational analysis in vitro showed that the smaller stem S1 is not critical for self-splicing, whereas the larger stem S2 is important for efficient exon ligation and lariat release in presence of the extension. This study clearly demonstrates that previously reported B.c.I4 is not a single example of a specialized intron, but forms a new functional class with an unusual mode that ensures proper positioning of the 3' splice site.
Asunto(s)
Bacillus thuringiensis/genética , Intrones , Empalme del ARN , ARN Catalítico/química , Secuencia de Bases , Secuencia Conservada , Datos de Secuencia Molecular , Mutagénesis , Conformación de Ácido NucleicoRESUMEN
The Bacillus cereus group of bacteria includes species that can cause food-poisoning or spoilage, such as B. cereus, as well as Bacillus anthracis, the cause of anthrax. In the present report we have conducted a multi-datatype analysis using tools from the HyperCAT database (http://mlstoslo.uio.no/) that we recently developed, combining data from multilocus sequence typing (Tourasse et al., 2010), amplified fragment length polymorphism, and multilocus enzyme electrophoresis typing techniques. We provide a comprehensive snapshot of the B. cereus group population, incorporating 2213 isolates including 450 from food and dairy products, in the form of both phylogenetic supertrees and superclusters of genetically closely related isolates. Our main findings include the detection of phylogenetically separated groups of isolates possibly representing novel evolutionary lineages within the B. cereus group, a putative new branch of B. anthracis, as well as new groups of related strains containing both environmental and clinical isolates. In addition, the multi-datatype analysis revealed to a larger extent than previously recognized that food-borne isolates can share identical genotyping profiles with strains from various other origins. Altogether, the global analysis confirms and extends the results underlining the opportunistic nature of B. cereus group organisms, and the fact that isolates responsible for disease outbreaks and contamination of foodstuffs can originate from various genetic backgrounds.
Asunto(s)
Bacillus cereus/clasificación , Bacillus cereus/genética , Microbiología de Alimentos , Filogenia , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Técnicas de Tipificación Bacteriana , Bases de Datos Genéticas/estadística & datos numéricos , Tipificación de Secuencias Multilocus , Reacción en Cadena de la PolimerasaRESUMEN
Bacillus cereus is found in food, soil, and plants, and the ability to cause food-borne diseases and opportunistic infection presumably varies among strains. Therefore, measuring harmful toxin production, in addition to the detection of the bacterium itself, may be key for food and hospital safety purposes. All previous studies have focused on the main known virulence factors, cereulide, Hbl, Nhe, and CytK. We examined whether other virulence factors may be specific to pathogenic strains. InhA1, NprA, and HlyII have been described as possibly contributing to B. cereus pathogenicity. We report the prevalence and expression profiles of these three new virulence factor genes among 57 B. cereus strains isolated from various sources, including isolates associated with gastrointestinal and nongastrointestinal diseases. Using PCR, quantitative reverse transcriptase PCR, and virulence in vivo assays, we unraveled these factors as potential markers to differentiate pathogenic from nonpathogenic strains. We show that the hlyII gene is carried only by strains with a pathogenic potential and that the expression levels of inhA1 and nprA are higher in the pathogenic than in the nonpathogenic group of strains studied. These data deliver useful information about the pathogenicity of various B. cereus strains.
Asunto(s)
Bacillus cereus/clasificación , Bacillus cereus/genética , Toxinas Bacterianas/genética , Técnicas Bacteriológicas/métodos , Factores de Virulencia/genética , Animales , Línea Celular , Perfilación de la Expresión Génica , Humanos , Larva/microbiología , Lepidópteros/microbiología , Macrófagos/microbiología , Ratones , Reacción en Cadena de la Polimerasa , Prevalencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de SupervivenciaRESUMEN
The Bacillus cereus group of bacteria is an important group including mammalian and insect pathogens, such as B. anthracis, the anthrax bacterium, B. thuringiensis, used as a biological pesticide and B. cereus, often involved in food poisoning incidents. To characterize the population structure and epidemiology of these bacteria, five separate multilocus sequence typing (MLST) schemes have been developed, which makes results difficult to compare. Therefore, we have developed a database that compiles and integrates MLST data from all five schemes for the B. cereus group, accessible at http://mlstoslo.uio.no/. Supertree techniques were used to combine the phylogenetic information from analysis of all schemes and datasets, in order to produce an integrated view of the B. cereus group population. The database currently contains strain information and sequence data for 1029 isolates and 26 housekeeping gene fragments, which can be searched by keywords, MLST scheme, or sequence similarity. Supertrees can be browsed according to various criteria such as species, isolate source, or genetic distance, and subtrees containing strains of interest can be extracted. Besides analysis of the available data, the user has the possibility to enter her/his own sequences and compare them to the database and/or include them into the supertree reconstructions.
Asunto(s)
Bacillus anthracis/clasificación , Bacillus cereus/clasificación , Bacillus thuringiensis/clasificación , Bases de Datos de Ácidos Nucleicos , Filogenia , Bacillus anthracis/genética , Bacillus cereus/genética , Bacillus thuringiensis/genética , Técnicas de Tipificación Bacteriana , Internet , Análisis de Secuencia de ADN , Interfaz Usuario-ComputadorRESUMEN
Group I and group II introns are different catalytic self-splicing and mobile RNA elements that contribute to genome dynamics. In this study, we have analyzed their distribution and evolution in 29 sequenced genomes from the Bacillus cereus group of bacteria. Introns were of different structural classes and evolutionary origins, and a large number of nearly identical elements are shared between multiple strains of different sources, suggesting recent lateral transfers and/or that introns are under a strong selection pressure. Altogether, 73 group I introns were identified, inserted in essential genes from the chromosome or newly described prophages, including the first elements found within phages in bacterial plasmids. Notably, bacteriophages are an important source for spreading group I introns between strains. Furthermore, 77 group II introns were found within a diverse set of chromosomal and plasmidic genes. Unusual findings include elements located within conserved DNA metabolism and repair genes and one intron inserted within a novel retroelement. Group II introns are mainly disseminated via plasmids and can subsequently invade the host genome, in particular by coupling mobility with host cell replication. This study reveals a very high diversity and variability of mobile introns in B. cereus group strains.
Asunto(s)
Bacillus cereus/genética , Evolución Molecular , Genoma Bacteriano , Intrones , Retroelementos , Bacillus cereus/clasificación , Bacillus cereus/aislamiento & purificación , Bacillus thuringiensis/genética , Secuencia de Bases , Biología Computacional , ADN Intergénico/química , Genómica , Datos de Secuencia Molecular , Filogenia , Profagos/genética , ARN Catalítico/química , Alineación de SecuenciaRESUMEN
All group II introns known to date fold into six functional domains. However, we recently identified an intron in Bacillus cereus ATCC 10987, B.c.I4, that splices 56 nt downstream of the expected 3' splice site in vivo (Tourasse et al. 2005, J. Bacteriol., 187, 5437-5451). In this study, we confirmed by ribonuclease protection assay that the 56-bp segment is part of the intron RNA molecule, and computational prediction suggests that it might form a stable stem-loop structure downstream of domain VI. The splicing of B.c.I4 was further investigated both in vivo and in vitro. Lariat formation proceeded primarily by branching at the ordinary bulged adenosine in domain VI without affecting the fidelity of splicing. In addition, the splicing efficiency of the wild-type intron was better than that of a mutant construct deleted of the 56-bp 3' extension. These results indicate that the intron has apparently adapted to the extra segment, possibly through conformational adjustments. The extraordinary group II intron B.c.I4 harboring an unprecedented extra 3' segment constitutes a dramatic example of the flexibility and adaptability of group II introns.
Asunto(s)
Bacillus cereus/genética , Intrones , Sitios de Empalme de ARN , Empalme del ARN , ARN Bacteriano/química , Retroelementos , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN/química , ARN Bacteriano/metabolismo , ARN CircularRESUMEN
BACKGROUND: Most extracellular virulence factors produced by Bacillus cereus are regulated by the pleiotropic transcriptional activator PlcR. Among strains belonging to the B. cereus group, the plcR gene is always located in the vicinity of genes encoding the YvfTU two-component system. The putative role of YvfTU in the expression of the PlcR regulon was therefore investigated. RESULTS: Expression of the plcR gene was monitored using a transcriptional fusion with a lacZ reporter gene in a yvfTU mutant and in its B. cereus ATCC 14579 parental strain. Two hours after the onset of the stationary phase, a stage at which the PlcR regulon is highly expressed, the plcR expression in the yvfTU mutant was only 50% of that of its parental strain. In addition to the reduced plcR expression in the yvfTU mutant, a few members of the PlcR regulon showed a differential expression, as revealed by transcriptomic and proteomic analyses. The virulence of the yvfTU mutant in a Galleria mellonella insect model was slightly lower than that of the parental strain. CONCLUSION: The YvfTU two-component system is not required for the expression of most of the virulence factors belonging to the PlcR regulon. However, YvfTU is involved in expression of plcR, a major regulator of virulence in B. cereus.