Response of Burkholderia cenocepacia H111 to micro-oxia.
ABSTRACT: B. cenocepacia is an opportunistic human pathogen that is particularly problematic for patients suffering from cystic fibrosis (CF). In the CF lung bacteria grow to high densities within the viscous mucus that is limited in oxygen. Pseudomonas aeruginosa, the dominant pathogen in CF patients, is known to grow and survive under oxygen-limited to anaerobic conditions by using micro-oxic respiration, denitrification and fermentative pathways. In contrast, inspection of the genome sequences of available B. cenocepacia strains suggested that B. cenocepacia is an obligate aerobic and non-fermenting bacterium. In accordance with the bioinformatics analysis we observed that B. cenocepacia H111 is able to grow with as little as 0.1% O2 but not under strictly anoxic conditions. Phenotypic analyses revealed that H111 produced larger amounts of biofilm, pellicle and proteases under micro-oxic conditions (0.5%-5% O2, i.e. conditions that mimic those encountered in CF lung infection), and was more resistant to several antibiotics. RNA-Seq and shotgun proteomics analyses of cultures of B. cenocepacia H111 grown under micro-oxic and aerobic conditions showed up-regulation of genes involved in the synthesis of the exopolysaccharide (EPS) cepacian as well as several proteases, two isocitrate lyases and other genes potentially important for life in micro-oxia.RNA-Seq raw data files are accessible through the GEO Series accession number GSE48585. MS data have been deposited in the ProteomeXchange database (PXD000270).
Project description:B. cenocepacia is an opportunistic human pathogen that is particularly problematic for patients suffering from cystic fibrosis (CF). In the CF lung, bacteria grow to high densities within the viscous mucus that is limited in oxygen. Pseudomonas aeruginosa, the dominant pathogen in CF patients, is known to grow and survive under oxygen-limited to anaerobic conditions by using micro-oxic respiration, denitrification and fermentative pathways. In contrast, inspection of the genome sequences of available B. cenocepacia strains suggested that B. cenocepacia is an obligate aerobic and non-fermenting bacterium. In accordance with the bioinformatics analysis, we observed that B. cenocepacia H111 is able to grow with as little as 0.1% O2 but not under strictly anoxic conditions. Phenotypic analyses revealed that H111 produced larger amounts of biofilm, pellicle and proteases under micro-oxic conditions (0.5% - 5% O2, i.e. conditions that mimic those encountered in CF lung infection), and was more resistant to several antibiotics. RNA-Seq and shotgun proteomics analyses of cultures of B. cenocepacia H111 grown under micro-oxic and aerobic conditions showed up-regulation of genes involved in the synthesis of the exopolysaccharide (EPS) cepacian as well as several proteases, two isocitrate lyases and other genes potentially important for life in micro-oxia. Oxygen regulation in Burkholderia cenocepacia was investigated using RNA-Seq of cells grown under aerobic or micro-oxic conditions.
Project description:Characterization of prophages in sequenced bacterial genomes is important for virulence assessment, evolutionary analysis, and phage application development. The objective of this study was to identify complete, inducible prophages in the cystic fibrosis (CF) clinical isolate Burkholderia cenocepacia H111. Using the prophage-finding program PHAge Search Tool (PHAST), we identified three putative intact prophages in the H111 sequence. Virions were readily isolated from H111 culture supernatants following extended incubation. Using shotgun cloning and sequencing, one of these virions (designated ?H111-1 [vB_BceM_?H111-1]) was identified as the infective particle of a PHAST-detected intact prophage. ?H111-1 has an extremely broad host range with respect to B. cenocepacia strains and is predicted to use lipopolysaccharide (LPS) as a receptor. Bioinformatics analysis indicates that the prophage is 42,972 base pairs in length, encodes 54 proteins, and shows relatedness to the virion morphogenesis modules of AcaML1 and "Vhmllikevirus" myoviruses. As ?H111-1 is active against a broad panel of clinical strains and encodes no putative virulence factors, it may be therapeutically effective for Burkholderia infections.
Project description:The study of the minimum set of genes required to sustain life is a fundamental question in biological research. Recent studies on bacterial essential genes suggested that between 350 and 700 genes are essential to support autonomous bacterial cell growth. Essential genes are of interest as potential new antimicrobial drug targets; hence, our aim was to identify the essential genome of the cystic fibrosis (CF) isolate Burkholderia cenocepacia H111. Using a transposon sequencing (Tn-Seq) approach, we identified essential genes required for growth in rich medium under aerobic and microoxic conditions as well as in a defined minimal medium with citrate as a sole carbon source. Our analysis suggests that 398 genes are required for autonomous growth in rich medium, a number that represents only around 5% of the predicted genes of this bacterium. Five hundred twenty-six genes were required to support growth in minimal medium, and 434 genes were essential under microoxic conditions (0.5% O2). A comparison of these data sets identified 339 genes that represent the minimal set of essential genes required for growth under all conditions tested and can be considered the core essential genome of B. cenocepacia H111. The majority of essential genes were found to be located on chromosome 1, and few such genes were located on chromosome 2, where most of them were clustered in one region. This gene cluster is fully conserved in all Burkholderia species but is present on chromosome 1 in members of the closely related genus Ralstonia, suggesting that the transfer of these essential genes to chromosome 2 in a common ancestor contributed toward the separation of the two genera.IMPORTANCE Transposon sequencing (Tn-Seq) is a powerful method used to identify genes that are essential for autonomous growth under various conditions. In this study, we have identified a set of "core essential genes" that are required for growth under multiple conditions, and these genes represent potential antimicrobial targets. We also identified genes specifically required for growth under low-oxygen and nutrient-limited environments. We generated conditional mutants to verify the results of our Tn-Seq analysis and demonstrate that one of the identified genes was not essential per se but was an artifact of the construction of the mutant library. We also present verified examples of genes that were not truly essential but, when inactivated, showed a growth defect. These examples have identified so-far-underestimated shortcomings of this powerful method.
Project description:Burkholderia cenocepacia H111, a strain isolated from a cystic fibrosis patient, has been shown to effectively kill the nematode Caenorhabditis elegans. We used the C. elegans model of infection to screen a mini-Tn5 mutant library of B. cenocepacia H111 for attenuated virulence. Of the approximately 5,500 B. cenocepacia H111 random mini-Tn5 insertion mutants that were screened, 22 showed attenuated virulence in C. elegans. Except for the quorum-sensing regulator cepR, none of the mutated genes coded for the biosynthesis of classical virulence factors such as extracellular proteases or siderophores. Instead, the mutants contained insertions in metabolic and regulatory genes. Mutants attenuated in virulence in the C. elegans infection model were also tested in the Drosophila melanogaster pricking model, and those also attenuated in this model were further tested in Galleria mellonella. Six of the 22 mutants were attenuated in D. melanogaster, and five of these were less pathogenic in the G. mellonella model. We show that genes encoding enzymes of the purine, pyrimidine, and shikimate biosynthesis pathways are critical for virulence in multiple host models of infection.
Project description:Burkholderia cenocepacia H111 is an opportunistic pathogen associated with chronic lung infections in cystic fibrosis patients. Biofilm formation, motility and virulence of B. cenocepacia are regulated by the second messenger cyclic di-guanosine monophosphate (c-di-GMP). In the present study, we analyzed the role of all 25 putative c-di-GMP metabolizing proteins of B. cenocepacia H111 with respect to motility, colony morphology, pellicle formation, biofilm formation, and virulence. We found that RpfR is a key regulator of c-di-GMP signaling in B. cenocepacia, affecting a broad spectrum of phenotypes under various environmental conditions. In addition, we identified Bcal2449 as a regulator of B. cenocepacia virulence in Galleria mellonella larvae. While Bcal2449 consists of protein domains that may catalyze both c-di-GMP synthesis and degradation, only the latter was essential for larvae killing, suggesting that a decreased c-di-GMP level mediated by the Bcal2449 protein is required for virulence of B. cenocepacia. Finally, our work suggests that some individual proteins play a role in regulating exclusively motility (CdpA), biofilm formation (Bcam1160) or both (Bcam2836).
Project description:BACKGROUND:Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. RESULTS:Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential [Formula: see text]-dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. CONCLUSIONS:We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.
Project description:Burkholderia cenocepacia belongs to the Burkholderia cepacia complex (Bcc), a group of at least 18 distinct species that establish chronic infections in the lung of people with the genetic disease cystic fibrosis (CF). The sputum of CF patients is rich in amino acids and was previously shown to increase flagellar gene expression in B. cenocepacia. We examined flagellin expression and flagellar morphology of B. cenocepacia grown in synthetic cystic fibrosis sputum medium (SCFM) compared to minimal medium. We found that CF nutritional conditions induce increased motility and flagellin expression. Individual amino acids added at the same concentrations as found in SCFM also increased motility but not flagellin expression, suggesting a chemotactic effect of amino acids. Electron microscopy and flagella staining demonstrated that the increase in flagellin corresponds to a change in the number of flagella per cell. In minimal medium, the ratio of multiple: single: aflagellated cells was 2:3.5:4.5; while under SCFM conditions, the ratio was 7:2:1. We created a deletion mutant, ?flhF, to study whether this putative GTPase regulates the flagellation pattern of B. cenocepacia K56-2 during growth in CF conditions. The ?flhF mutant exhibited 80% aflagellated, 14% single and 6% multiple flagellated bacterial subpopulations. Moreover, the ratio of multiple to single flagella in WT and ?flhF was 3.5 and 0.43, respectively in CF conditions. The observed differences suggest that FlhF positively regulates flagellin expression and the flagellation pattern in B. cenocepacia K56-2 during CF nutritional conditions.
Project description:Using a discovery shotgun proteomics approach, the expressed proteome of Burkholderia cenocepacia (strain H111) under aerobic (21% oxygen) and micro-oxic (0.5% oxygen) conditions was analyzed. B.cenocepacia is an opportunistic human pathogen that is particularly problematic for patients suffering from cystic fibrosis. Cells were sub-cellular fractionated (Cyt=cytoplasmic, TM=total membrane, EC=extracellular) and proteins were extracted and separated by 1D SDS-PAGE. Gels were cut into ten slices, digested using trypsin and resulting peptides were analyzed using LC-MS/MS once with a discovery run followed by a subsequent exclusion list run (precursor ions identified in the discovery run were excluded from fragmentation in the exclusion run). Mass spectra were searched against a protein sequence database containing 7258 annotated B. cenocepacia H111 proteins (accession CAFQ00000000.1) and protein sequences of 259 common contaminants. Spectra were searched against this database with MS-GF+ (MS-GFDB v7747, kindly provided by Dr. Sangtae Kim, UCSD, USA) using the following parameters: Carbamidomethylation was set as a fixed modification on all Cysteines, oxidation of Methionines, deamidation of Asparagines and Glutamines, as well as cyclization of N-terminal Glutamines were considered as optional modifications. Spectra were searched for a match to fully-tryptic and semi-tryptic peptides and the automatic decoy search option was enabled. Search results were filtered and summarized using in-house tools. A total of 2,128 proteins was identified at an estimated FDR of less than 1%. Additional RNA-seq data are held at GEO under the accession: GSE48585.
Project description:The opportunistic pathogen Burkholderia cenocepacia is particularly life-threatening for cystic fibrosis (CF) patients. Chronic lung infections with these bacteria can rapidly develop into fatal pulmonary necrosis and septicaemia. We have recently shown that macrophages are a critical site for replication of B. cenocepacia K56-2 and the induction of fatal pro-inflammatory responses using a zebrafish infection model. Here, we show that ShvR, a LysR-type transcriptional regulator that is important for biofilm formation, rough colony morphotype and inflammation in a rat lung infection model, is also required for the induction of fatal pro-inflammatory responses in zebrafish larvae. ShvR was not essential, however, for bacterial survival and replication in macrophages. Temporal, rhamnose-induced restoration of shvR expression in the shvR mutant during intramacrophage stages unequivocally demonstrated a key role for ShvR in transition from intracellular persistence to acute fatal pro-inflammatory disease. ShvR has been previously shown to tightly control the expression of the adjacent afc gene cluster, which specifies the synthesis of a lipopeptide with antifungal activity. Mutation of afcE, encoding an acyl-CoA dehydrogenase, has been shown to give similar phenotypes as the shvR mutant. We found that, like shvR, afcE is also critical for the switch from intracellular persistence to fatal infection in zebrafish. The closely related B. cenocepacia H111 has been shown to be less virulent than K56-2 in several infection models, including Galleria mellonella and rats. Interestingly, constitutive expression of shvR in H111 increased virulence in zebrafish larvae to almost K56-2 levels in a manner that absolutely required afc. These data confirm a critical role for afc in acute virulence caused by B. cenocepacia that depends on strain-specific regulatory control by ShvR. We propose that ShvR and AFC are important virulence factors of the more virulent Bcc species, either through pro-inflammatory effects of the lipopeptide AFC, or through AFC-dependent membrane properties.
Project description:In Burkholderia cenocepacia H111, the large surface protein BapA plays a crucial role in the formation of highly structured communities, known as biofilms. We have recently demonstrated that quorum sensing (QS) is necessary for the maximal expression of bapA. In this study we identify BapR, a protein from the IclR family of transcriptional regulators that, in conjunction with QS, controls biofilm formation by affecting the expression of bapA. We present evidence that, in addition to bapA, BapR influences the expression of extracellular proteases, swimming motility and has a profound impact in the incidence of persister cells, making this regulator an interesting target for persister cells and biofilm eradication.