ABSTRACT
Xenorhabdus nematophila is a Gram-negative bacterium, mutualistically associated with the soil nematode Steinernema carpocapsae, and this nemato-bacterial complex is parasitic for a broad spectrum of insects. The transcriptional regulator OxyR is widely conserved in bacteria and activates the transcription of a set of genes that influence cellular defence against oxidative stress. It is also involved in the virulence of several bacterial pathogens. The aim of this study was to identify the X. nematophila OxyR regulon and investigate its role in the bacterial life cycle. An oxyR mutant was constructed in X. nematophila and phenotypically characterized in vitro and in vivo after reassociation with its nematode partner. OxyR plays a major role during the X. nematophila resistance to oxidative stress in vitro. Transcriptome analysis allowed the identification of 59 genes differentially regulated in the oxyR mutant compared to the parental strain. In vivo, the oxyR mutant was able to reassociate with the nematode as efficiently as the control strain. These nemato-bacterial complexes harbouring the oxyR mutant symbiont were able to rapidly kill the insect larvae in less than 48 h after infestation, suggesting that factors other than OxyR could also allow X. nematophila to cope with oxidative stress encountered during this phase of infection in insect. The significantly increased number of offspring of the nemato-bacterial complex when reassociated with the X. nematophila oxyR mutant compared to the control strain revealed a potential role of OxyR during this symbiotic stage of the bacterial life cycle.
Subject(s)
Bacterial Proteins , Gene Expression Regulation, Bacterial , Oxidative Stress , Symbiosis , Xenorhabdus , Xenorhabdus/genetics , Xenorhabdus/metabolism , Xenorhabdus/physiology , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Rhabditida/microbiology , Rhabditida/genetics , Rhabditida/physiology , Larva/microbiology , Virulence , Regulon , Gene Expression Profiling , MutationABSTRACT
Bacteria have evolved sophisticated mechanisms to deliver potent toxins into bacterial competitors or into eukaryotic cells in order to destroy rivals and gain access to a specific niche or to hijack essential metabolic or signaling pathways in the host. Delivered effectors carry various activities such as nucleases, phospholipases, peptidoglycan hydrolases, enzymes that deplete the pools of NADH or ATP, compromise the cell division machinery, or the host cell cytoskeleton. Effectors categorized in the family of polymorphic toxins have a modular structure, in which the toxin domain is fused to additional elements acting as cargo to adapt the effector to a specific secretion machinery. Here we show that Photorhabdus laumondii, an entomopathogen species, delivers a polymorphic antibacterial toxin via a type VI secretion system. This toxin inhibits protein synthesis in a NAD+-dependent manner. Using a biotinylated derivative of NAD, we demonstrate that translation is inhibited through ADP-ribosylation of the ribosomal 23S RNA. Mapping of the modification further showed that the adduct locates on helix 44 of the thiostrepton loop located in the GTPase-associated center and decreases the GTPase activity of the EF-G elongation factor.
Subject(s)
Bacterial Toxins/pharmacology , GTP Phosphohydrolases/genetics , RNA, Ribosomal, 23S/genetics , Type VI Secretion Systems/drug effects , ADP-Ribosylation/drug effects , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , NAD/genetics , Peptide Elongation Factor G/genetics , Photorhabdus/chemistry , Photorhabdus/genetics , Protein Biosynthesis/drug effects , RNA, Ribosomal, 23S/drug effects , Thiostrepton/chemistry , Thiostrepton/pharmacologyABSTRACT
In bacteria, DNA-methyltransferase are responsible for DNA methylation of specific motifs in the genome. This methylation usually occurs at a very high rate. In the present study, we studied the MTases encoding genes found in the entomopathogenic bacteria Xenorhabdus. Only one persistent MTase was identified in the various species of this genus. This MTase, also broadly conserved in numerous Gram-negative bacteria, is called Dam: DNA-adenine MTase. Methylome analysis confirmed that the GATC motifs recognized by Dam were methylated at a rate of >99% in the studied strains. The observed enrichment of unmethylated motifs in putative promoter regions of the X. nematophila F1 strain suggests the possibility of epigenetic regulations. The overexpression of the Dam MTase responsible for additional motifs to be methylated was associated with impairment of two major phenotypes: motility, caused by a downregulation of flagellar genes, and hemolysis. However, our results suggest that dam overexpression did not modify the virulence properties of X. nematophila. This study increases the knowledge on the diverse roles played by MTases in bacteria.
Subject(s)
Site-Specific DNA-Methyltransferase (Adenine-Specific) , Xenorhabdus , Adenine , DNA , DNA Methylation , DNA Modification Methylases/genetics , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Xenorhabdus/geneticsABSTRACT
The different strains of Bacillus cereus can grow at temperatures covering a very diverse range. Some B. cereus strains can grow in chilled food and consequently cause food poisoning. We have identified a new sensor/regulator mechanism involved in low-temperature B. cereus growth. Construction of a mutant of this two-component system enabled us to show that this system, called CasKR, is required for growth at the minimal temperature (Tmin). CasKR was also involved in optimal cold growth above Tmin and in cell survival below Tmin. Microscopic observation showed that CasKR plays a key role in cell shape during cold growth. Introducing the casKR genes in a ΔcasKR mutant restored its ability to grow at Tmin. Although it was first identified in the ATCC 14579 model strain, this mechanism has been conserved in most strains of the B. cereus group. We show that the role of CasKR in cold growth is similar in other B. cereus sensu lato strains with different growth temperature ranges, including psychrotolerant strains.
Subject(s)
Bacillus cereus/growth & development , Bacillus cereus/radiation effects , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Protein Kinases/metabolism , Stress, Physiological , Transcription Factors/metabolism , Bacillus cereus/genetics , Bacterial Proteins/genetics , Cold Temperature , Gene Deletion , Genetic Complementation Test , Protein Kinases/genetics , Transcription Factors/geneticsABSTRACT
Using a previously developed Bacillus cereus in vivo expression technology (IVET) promoter trap system, we showed that spsA, a gene of unknown function, was specifically expressed in the larval gut during infection. Search for gut-related compounds inducing spsA transcription identified glucose-6-phosphate (G6P) as an activation signal. Analysis of the spsA-related 5-gene cluster indicated that SpsA is part of a new sugar phosphate sensor system composed of a 2-component system (TCS) encoded by spsR and spsK, and 2 additional downstream genes, spsB and spsC. In B. cereus, American Type Culture Collection (ATCC) 14579, spsRK, and spsABC are separate transcriptional units, of which only spsABC was activated by extracellular G6P. lacZ transcriptional fusions tested in mutant and complemented strains showed that SpsRK, SpsA, and SpsB are essential for the transcription of spsABC. Deletion mutant analysis showed that SpsC is essential for the G6P uptake. gfp-transcriptional fusions showed that these genes are required for host-activated expression, as well. This sugar phosphate sensor and transport system is found in pathogenic Bacillus group and Clostridia bacteria and may be important for host adaptation. Our findings provide new insights into the function of 2-component sensor systems in host-pathogen interactions, specifically in the gut.
Subject(s)
Bacillus cereus/genetics , Bacterial Proteins/genetics , Glucose-6-Phosphate/pharmacology , Sugar Phosphates/analysis , Animals , Biosensing Techniques/methods , Gastrointestinal Tract/microbiology , Gene Expression Regulation, Bacterial , Genes, Bacterial , Glucose-6-Phosphate/metabolism , Host-Pathogen Interactions , Larva/microbiology , Moths/genetics , Moths/microbiology , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/physiology , Sugar Phosphates/metabolismABSTRACT
In this study, growth rates and lag times of the five RNA helicase-deleted mutants of Bacillus cereus ATCC 14579 were compared to those of the wild-type strain under thermal, oxidative, and pH stresses. Deletion of cshD and cshE had no impact under any of the tested conditions. Deletion of cshA, cshB, and cshC abolished growth at 12°C, confirming previous results. In addition, we found that each RNA helicase had a role in a specific temperature range: deletion of cshA reduced growth at all the tested temperatures up to 45°C, deletion of cshB had impact below 30°C and over 37°C, and deletion of cshC led mainly to a cold-sensitive phenotype. Under oxidative conditions, deletion of cshB and cshC reduced growth rate and increased lag time, while deletion of cshA increased lag time only with H(2)O(2) and reduced growth rate at a high diamide concentration. Growth of the ΔcshA strain was affected at a basic pH independently of the temperature, while these conditions had a limited effect on ΔcshB and ΔcshC strain growth. The RNA helicases CshA, CshB, and CshC could participate in a general adaptation pathway to stressful conditions, with a stronger impact at low temperature and a wider role of CshA.
Subject(s)
Adaptation, Physiological , Bacillus cereus/enzymology , Oxidative Stress , RNA Helicases/metabolism , Temperature , Bacillus cereus/drug effects , Bacillus cereus/genetics , Bacillus cereus/growth & development , Diamide/pharmacology , Gene Deletion , Genes, Bacterial , Hydrogen Peroxide/pharmacology , Hydrogen-Ion Concentration , RNA Helicases/geneticsABSTRACT
DNA methylation can be part of epigenetic mechanisms, leading to cellular subpopulations with heterogeneous phenotypes. While prokaryotic phenotypic heterogeneity is of critical importance for a successful infection by several major pathogens, the exact mechanisms involved in this phenomenon remain unknown in many cases. Powerful sequencing tools have been developed to allow the detection of the DNA methylated bases at the genome level, and they have recently been extensively applied on numerous bacterial species. Some of these tools are increasingly used for metagenomics analysis but only a limited amount of the available methylomic data is currently being exploited. Because newly developed tools now allow the detection of subpopulations differing in their genome methylation patterns, it is time to emphasize future strategies based on a more extensive use of methylomic data. This will ultimately help to discover new epigenetic gene regulations involved in bacterial phenotypic heterogeneity, including during host-pathogen interactions.
ABSTRACT
Global pollinators, like honeybees, are declining in abundance and diversity, which can adversely affect natural ecosystems and agriculture. Therefore, we tested the current hypotheses describing honeybee losses as a multifactorial syndrome, by investigating integrative effects of an infectious organism and an insecticide on honeybee health. We demonstrated that the interaction between the microsporidia Nosema and a neonicotinoid (imidacloprid) significantly weakened honeybees. In the short term, the combination of both agents caused the highest individual mortality rates and energetic stress. By quantifying the strength of immunity at both the individual and social levels, we showed that neither the haemocyte number nor the phenoloxidase activity of individuals was affected by the different treatments. However, the activity of glucose oxidase, enabling bees to sterilize colony and brood food, was significantly decreased only by the combination of both factors compared with control, Nosema or imidacloprid groups, suggesting a synergistic interaction and in the long term a higher susceptibility of the colony to pathogens. This provides the first evidences that interaction between an infectious organism and a chemical can also threaten pollinators, interactions that are widely used to eliminate insect pests in integrative pest management.
Subject(s)
Bees , Imidazoles/toxicity , Insecticides/toxicity , Microsporidiosis/veterinary , Nitro Compounds/toxicity , Nosema , Agriculture , Animals , Bees/drug effects , Bees/microbiology , Bees/physiology , Humans , Immunity/drug effects , Microsporidiosis/mortality , Neonicotinoids , Nosema/pathogenicity , Nosema/physiology , Social BehaviorABSTRACT
Bacillus cereus ATCC 14579 possesses five RNA helicase-encoding genes overexpressed under cold growth conditions. Out of the five corresponding mutants, only the ΔcshA, ΔcshB, and ΔcshC strains were cold sensitive. Growth of the ΔcshA strain was also reduced at 30°C but not at 37°C. The cold phenotype was restored with the cshA gene for the ΔcshA strain and partially for the ΔcshB strain but not for the ΔcshC strain, suggesting different functions at low temperature.
Subject(s)
Adaptation, Physiological , Bacillus cereus/enzymology , Bacillus cereus/physiology , Bacterial Proteins/metabolism , Cold Temperature , RNA Helicases/metabolism , Bacillus cereus/growth & development , Bacterial Proteins/genetics , Gene Deletion , Genetic Complementation Test , RNA Helicases/deficiencyABSTRACT
The mechanisms involved in the ability of Bacillus cereus to multiply at low temperatures were investigated. It was assumed that many genes involved in cold acclimation would be upregulated at low temperatures. Recombinase-based in vivo expression technology (IVET) was adapted to the detection of the transient activation of B. cereus promoters during growth at 10 degrees C. Four independent screenings of a promoter library from type strain ATCC 14579 were performed, and 17 clones were isolated. They corresponded to 17 promoter regions that displayed reproducibly elevated expression at 10 degrees C relative to expression at 30 degrees C. This analysis revealed several genes that may be important for B. cereus to grow successfully under the restrictive conditions of cold habitats. Among them, a locus corresponding to open reading frames BC5402 to BC5398, harboring a lipase-encoding gene and a putative transcriptional regulator, was identified three times. While a mutation in the putative regulator-encoding gene did not cause any particular phenotype, a mutant deficient in the lipase-encoding gene showed reduced growth abilities at low temperatures compared with the parental strain. The mutant did not change its fatty acid profiles in the same way as the wild type when grown at 12 degrees C instead of 37 degrees C. This study demonstrates the feasibility of a promoter trap strategy for identifying cold-induced genes. It outlines a first picture of the different processes involved in B. cereus cold acclimation.
Subject(s)
Bacillus cereus/physiology , Cold Temperature , Gene Expression Profiling , Genes, Bacterial , Stress, Physiological , Bacillus cereus/chemistry , Bacillus cereus/growth & development , Bacterial Proteins/genetics , Fatty Acids/analysis , Gene Deletion , Lipase/genetics , Promoter Regions, GeneticABSTRACT
Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens during the whole bacterial life cycle including during symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that during the life cycle of P. luminescens, Dam is not involved the bacterial symbiosis with the nematode H. bacteriophora, but it contributes to the pathogenicity of the nemato-bacterial complex.
Subject(s)
Bacterial Proteins/metabolism , Insecta/microbiology , Nematoda/microbiology , Photorhabdus/enzymology , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Symbiosis/physiology , AnimalsABSTRACT
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.
Subject(s)
Bacillus cereus/genetics , Bacterial Proteins/metabolism , Trans-Activators/metabolism , Virulence Factors/metabolism , Amino Acid Sequence , Animals , Bacillaceae Infections/microbiology , Bacillus cereus/metabolism , Bacillus cereus/pathogenicity , Bacterial Proteins/genetics , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Genes, Bacterial , Genes, Reporter , Lepidoptera/microbiology , Molecular Sequence Data , Mutation , Plasmids , Proteomics , RNA, Bacterial/genetics , Regulon , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, DNA , Trans-Activators/genetics , Transcription, Genetic , Virulence , Virulence Factors/geneticsABSTRACT
The Bacillus cereus group represents sporulating soil bacteria containing pathogenic strains which may cause diarrheic or emetic food poisoning outbreaks. Multiple locus sequence typing revealed a presence in natural samples of these bacteria of about 30 clonal complexes. Application of genomic methods to this group was however biased due to the major interest for representatives closely related to Bacillus anthracis. Albeit the most important food-borne pathogens were not yet defined, existing data indicate that they are scattered all over the phylogenetic tree. The preliminary analysis of the sequences of three genomes discussed in this paper narrows down the gaps in our knowledge of the B. cereus group. The strain NVH391-98 is a rare but particularly severe food-borne pathogen. Sequencing revealed that the strain should be a representative of a novel bacterial species, for which the name Bacillus cytotoxis or Bacillus cytotoxicus is proposed. This strain has a reduced genome size compared to other B. cereus group strains. Genome analysis revealed absence of sigma B factor and the presence of genes encoding diarrheic Nhe toxin, not detected earlier. The strain B. cereus F837/76 represents a clonal complex close to that of B. anthracis. Including F837/76, three such B. cereus strains had been sequenced. Alignment of genomes suggests that B. anthracis is their common ancestor. Since such strains often emerge from clinical cases, they merit a special attention. The third strain, KBAB4, is a typical facultative psychrophile generally found in soil. Phylogenic studies show that in nature it is the most active group in terms of gene exchange. Genomic sequence revealed high presence of extra-chromosomal genetic material (about 530kb) that may account for this phenomenon. Genes coding Nhe-like toxin were found on a big plasmid in this strain. This may indicate a potential mechanism of toxicity spread from the psychrophile strain community. The results of this genomic work and ecological compartments of different strains incite to consider a necessity of creating prophylactic vaccines against bacteria closely related to NVH391-98 and F837/76. Presumably developing of such vaccines can be based on the properties of non-pathogenic strains such as KBAB4 or ATCC14579 reported here or earlier. By comparing the protein coding genes of strains being sequenced in this project to others we estimate the shared proteome, or core genome, in the B. cereus group to be 3000+/-200 genes and the total proteome, or pan-genome, to be 20-25,000 genes.
Subject(s)
Bacillus cereus/genetics , Food Microbiology , Foodborne Diseases/microbiology , Genome, Bacterial , HumansABSTRACT
DNA methylation can serve to control diverse phenomena in eukaryotes and prokaryotes, including gene regulation leading to cell differentiation. In bacteria, DNA methylomes (i.e., methylation state of each base of the whole genome) have been described for several species, but methylome profile variation during the lifecycle has rarely been studied, and only in a few model organisms. Moreover, major phenotypic changes have been reported in several bacterial strains with a deregulated methyltransferase, but the corresponding methylome has rarely been described. Here we report the first methylome description of an entomopathogenic bacterium, Photorhabdus luminescens. Eight motifs displaying a high rate of methylation (>94%) were identified. The methylome was strikingly stable over course of growth, but also in a subpopulation responsible for a critical step in the bacterium's lifecycle: successful survival and proliferation in insects. The rare unmethylated GATC motifs were preferentially located in putative promoter regions, and most of them were methylated after Dam methyltransferase overexpression, suggesting that DNA methylation is involved in gene regulation. Our findings bring key insight into bacterial methylomes and encourage further research to decipher the role of loci protected from DNA methylation in gene regulation.
Subject(s)
Adenine/metabolism , DNA Methylation , Gene Expression Regulation, Bacterial , Insecta/microbiology , Photorhabdus/genetics , Animals , DNA, Bacterial/genetics , Genetic Loci/genetics , Genome, Bacterial/genetics , Nucleotide Motifs/genetics , Photorhabdus/metabolism , Promoter Regions, Genetic/genetics , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Whole Genome SequencingABSTRACT
BACKGROUND: In the human pathogen Bacillus cereus, the expression of most extracellular virulence factors is controlled by the transcriptional activator PlcR. Among these virulence factors, cereolysin O (Clo) is an haemolysin belonging to the cholesterol-dependant cytolysins, a protein family extensively studied in Gram-positive bacteria. RESULTS: In the genomes of bacteria belonging to the B. cereus group, including Bacillus anthracis and Bacillus thuringiensis, a small gene encoding a 26-amino acid peptide was present in multicopy. One copy was always found upstream from the gene encoding Clo. In B. cereus ATCC 14579, the small gene and the clo gene are co-transcribed. Transcriptional fusions showed that the three paralogues identified in this strain were expressed in a PlcR-dependent manner. We propose to name these peptides Spp for small PlcR-regulated peptides. We show that a synthetic peptide corresponding to the deduced product of the spp genes displayed antibacterial activity. CONCLUSION: The co-expression of spp, a small PlcR-regulated multicopy gene with clo suggests a yet unidentified relationship between Spp and the cholesterol-dependent cytolysin in bacteria belonging to the B.cereus group.
Subject(s)
Bacillus cereus/genetics , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Gene Expression Regulation, Bacterial , Trans-Activators/metabolism , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/pharmacology , Artificial Gene Fusion , Bacillus anthracis/genetics , Bacillus thuringiensis/genetics , Bacterial Proteins/metabolism , Gene Dosage , Genes, Bacterial , Genes, Reporter , Peptides/genetics , beta-Galactosidase/analysis , beta-Galactosidase/geneticsABSTRACT
BACKGROUND: Three enterotoxins are implicated in diarrhoeal food poisoning due to Bacillus cereus: Haemolysin BL (Hbl), Non-haemolytic enterotoxin (Nhe), and Cytotoxin K (CytK). Toxin gene profiling and assays for detection of toxin-producing stains have been used in attempts to evaluate the enterotoxic potential of B. cereus group strains. B. cereus strain NVH 391/98, isolated from a case of fatal enteritis, was genetically remote from other B. cereus group strains. This strain lacked the genes encoding Hbl and Nhe, but contains CytK-1. The high virulence of this strain is thought to be due to the greater cytotoxic activity of CytK-1 compared to CytK-2, and to a high level of cytK expression. To date, only three strains containing cytK-1 have been identified; B. cereus strains NVH 391/98, NVH 883/00, and INRA AF2. RESULTS: A novel gene variant encoding Nhe was identified in these three strains, which had an average of 80% identity in protein sequence with previously identified Nhe toxins. While culture supernatants containing CytK and Nhe from NVH 391/98 and INRA AF2 were highly cytotoxic, NVH 883/00 expressed little or no CytK and Nhe and was non-cytotoxic. Comparative sequence and expression studies indicated that neither the PlcR/PapR quorum sensing system, nor theYvrGH and YvfTU two-component systems, were responsible for the observed difference in toxin production. Additionally, phylogenetic analysis of 13 genes showed that NVH 391/98, NVH 883/00, and INRA AF2 comprise a novel cluster of strains genetically distant from other B. cereus group strains. CONCLUSION: Due to its divergent sequence, the novel nhe operon had previously not been detected in NVH 391/98 using PCR and several monoclonal antibodies. Thus, toxigenic profiling based on the original nhe sequence will fail to detect the toxin in this group of strains. The observation that strain NVH 883/00 carries cytK-1 but is non-cytotoxic indicates that the detection of this gene variant is not a sufficient criterion for identification of highly cytotoxic strains. The presence of the novel nhe operon and the cytK-1 gene variant in this cluster of strains reflect their phylogenetically remote relationship towards other B. cereus group strains.
Subject(s)
Bacillus cereus/metabolism , Bacterial Toxins/metabolism , Enterotoxins/metabolism , Phylogeny , Animals , Bacillus cereus/classification , Bacillus cereus/genetics , Bacterial Toxins/genetics , Bacterial Toxins/pharmacology , Blotting, Western , Cell Survival/drug effects , Chlorocebus aethiops , Electrophoresis, Polyacrylamide Gel , Enterotoxins/genetics , Enterotoxins/pharmacology , Molecular Sequence Data , Protein Biosynthesis/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Vero Cells , Virulence/geneticsABSTRACT
Dam, the most described bacterial DNA-methyltransferase, is widespread in gamma-proteobacteria. Dam DNA methylation can play a role in various genes expression and is involved in pathogenicity of several bacterial species. The purpose of this study was to determine the role played by the dam ortholog identified in the entomopathogenic bacterium Photorhabdus luminescens. Complementation assays of an Escherichia coli dam mutant showed the restoration of the DNA methylation state of the parental strain. Overexpression of dam in P. luminescens did not impair growth ability in vitro. In contrast, compared to a control strain harboring an empty plasmid, a significant decrease in motility was observed in the dam-overexpressing strain. A transcriptome analysis revealed the differential expression of 208 genes between the two strains. In particular, the downregulation of flagellar genes was observed in the dam-overexpressing strain. In the closely related bacterium Xenorhabdus nematophila, dam overexpression also impaired motility. In addition, the dam-overexpressing P. luminescens strain showed a delayed virulence compared to that of the control strain after injection in larvae of the lepidopteran Spodoptera littoralis. These results reveal that Dam plays a major role during P. luminescens insect infection.
ABSTRACT
Some of the bacterial cells in isogenic populations behave differently from others. We describe here how a new type of phenotypic heterogeneity relating to resistance to cationic antimicrobial peptides (CAMPs) is determinant for the pathogenic infection process of the entomopathogenic bacterium Photorhabdus luminescens. We demonstrate that the resistant subpopulation, which accounts for only 0.5% of the wild-type population, causes septicemia in insects. Bacterial heterogeneity is driven by the PhoPQ two-component regulatory system and expression of pbgPE, an operon encoding proteins involved in lipopolysaccharide (LPS) modifications. We also report the characterization of a core regulon controlled by the DNA-binding PhoP protein, which governs virulence in P. luminescens. Comparative RNAseq analysis revealed an upregulation of marker genes for resistance, virulence and bacterial antagonism in the pre-existing resistant subpopulation, suggesting a greater ability to infect insect prey and to survive in cadavers. Finally, we suggest that the infection process of P. luminescens is based on a bet-hedging strategy to cope with the diverse environmental conditions experienced during the lifecycle.
Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Drug Resistance, Bacterial , Enterobacteriaceae Infections/microbiology , Photorhabdus/drug effects , Photorhabdus/genetics , Animals , Disk Diffusion Antimicrobial Tests , Gene Expression Profiling , Gene Expression Regulation , Gene Order , Genes, Bacterial , Insecta/microbiology , Mutation , Operon , Photorhabdus/pathogenicity , Virulence/geneticsABSTRACT
The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species. Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs. Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs. The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.
Subject(s)
Bacillus cereus/chemistry , Chromatography , Fatty Acids, Unsaturated/analysis , Fatty Acids/analysis , Adaptation, PhysiologicalABSTRACT
Two-component systems (TCS) allow a cell to elaborate a variety of adaptive responses to environment changes. The recently discovered CasK/R TCS plays a role in the optimal unsaturation of fatty acids necessary for cold adaptation of the foodborne-pathogen Bacillus cereus Here, we showed that the promoter activity of the operon encoding this TCS was repressed during growth at low temperature in the stationary phase in the parental strain when compared to the casK/R mutant, suggesting that CasR negatively regulates the activity of its own promoter in these conditions. The promoter activity of the desA gene encoding the Δ5 fatty acid desaturase, providing unsaturated fatty acids (UFAs) required for low temperature adaptation, was repressed in the casK/R mutant grown at 12°C versus 37°C. This result suggests that CasK/R activates desA expression during B. cereus growth at low temperature, allowing an optimal unsaturation of the fatty acids. In contrast, desA expression was repressed during the lag phase at low temperature in presence of UFAs, in a CasK/R-independent manner. Our findings confirm that the involvement of this major TCS in B. cereus cold adaptation is linked to the upregulation of a fatty acid desaturase.