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Bacterial infections often involve more than one pathogen. While it is well established that polymicrobial infections can impact disease outcomes, we know little about how pathogens interact and affect each other's behaviour and fitness. Here, we used a microscopy approach to explore interactions between Pseudomonas aeruginosa and six human opportunistic pathogens that often co-occur in polymicrobial infections: Acinetobacter baumannii, Burkholderia cenocepacia, Escherichia coli, Enterococcus faecium, Klebsiella pneumoniae, and Staphylococcus aureus. When following growing microcolonies on agarose pads over time, we observed a broad spectrum of species-specific ecological interactions, ranging from mutualism to antagonism. For example, P. aeruginosa engaged in a mutually beneficial interaction with E. faecium but suffered from antagonism by E. coli. While we found little evidence for active directional growth towards or away from cohabitants, we observed that some pathogens increased growth in double layers in response to competition and that physical forces due to fast colony expansion had a major impact on fitness. Overall, our work provides an atlas of pathogen interactions, highlighting the diversity of potential species dynamics that may occur in polymicrobial infections. We discuss possible mechanisms driving pathogen interactions and offer predictions of how the different ecological interactions could affect virulence., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Across the Burkholderia genus O -linked protein glycosylation is highly conserved. While the inhibition of glycosylation has been shown to be detrimental for virulence in Burkholderia cepacia complex species, such as Burkholderia cenocepacia , little is known about how specific glycosylation sites impact protein functionality. Within this study, we sought to improve our understanding of the breadth, dynamics, and requirement for glycosylation across the B. cenocepacia O- glycoproteome. Assessing the B. cenocepacia glycoproteome across different culture media using complementary glycoproteomic approaches, we increase the known glycoproteome to 141 glycoproteins. Leveraging this repertoire of glycoproteins, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) revealing the B. cenocepacia glycoproteome is largely stable across conditions with most glycoproteins constitutively expressed. Examination of how the absence of glycosylation impacts the glycoproteome reveals that the protein abundance of only five glycoproteins (BCAL1086, BCAL2974, BCAL0525, BCAM0505, and BCAL0127) are altered by the loss of glycosylation. Assessing Δ fliF (ΔBCAL0525), Δ motB (ΔBCAL0127), and ΔBCAM0505 strains, we demonstrate the loss of FliF, and to a lesser extent MotB, mirror the proteomic effects observed in the absence of glycosylation in Δ pglL . While both MotB and FliF are essential for motility, we find loss of glycosylation sites in MotB or FliF does not impact motility supporting these sites are dispensable for function. Combined this work broadens our understanding of the B. cenocepacia glycoproteome supporting that the loss of glycoproteins in the absence of glycosylation is not an indicator of the requirement for glycosylation for protein function., Importance: Burkholderia cenocepacia is an opportunistic pathogen of concern within the Cystic Fibrosis community. Despite a greater appreciation of the unique physiology of B. cenocepacia gained over the last 20 years a complete understanding of the proteome and especially the O-glycoproteome, is lacking. In this study, we utilize systems biology approaches to expand the known B. cenocepacia glycoproteome as well as track the dynamics of glycoproteins across growth phases, culturing media and in response to the loss of glycosylation. We show that the glycoproteome of B. cenocepacia is largely stable across conditions and that the loss of glycosylation only impacts five glycoproteins including the motility associated proteins FliF and MotB. Examination of MotB and FliF shows, while these proteins are essential for motility, glycosylation is dispensable. Combined this work supports that B. cenocepacia glycosylation can be dispensable for protein function and may influence protein properties beyond stability., Competing Interests: The authors declare no conflict of interest.

A first clue to gene function can be obtained by examining whether a gene is required for life in certain standard conditions, that is, whether a gene is essential. In bacteria, essential genes are usually identified by high-density transposon mutagenesis followed by sequencing of insertion sites (Tn-seq). These studies assign the term "essential" to whole genes rather than the protein domain sequences that encode the essential functions. However, genes can code for multiple protein domains that evolve their functions independently. Therefore, when essential genes code for more than one protein domain, only one of them could be essential. In this study, we defined this subset of genes as "essential domain-containing" (EDC) genes. Using a Tn-seq data set built-in Burkholderia cenocepacia K56-2, we developed an in silico pipeline to identify EDC genes and the essential protein domains they encode. We found forty candidate EDC genes and demonstrated growth defect phenotypes using CRISPR interference (CRISPRi). This analysis included two knockdowns of genes encoding the protein domains of unknown function DUF2213 and DUF4148. These putative essential domains are conserved in more than two hundred bacterial species, including human and plant pathogens. Together, our study suggests that essentiality should be assigned to individual protein domains rather than genes, contributing to a first functional characterization of protein domains of unknown function., (© 2022. The Author(s).)

Many bacteria cycle between sessile and motile forms in which they must sense and respond to internal and external signals to coordinate appropriate physiology. Maintaining fitness requires genetic networks that have been honed in variable environments to integrate these signals. The identity of the major regulators and how their control mechanisms evolved remain largely unknown in most organisms. During four different evolution experiments with the opportunist betaproteobacterium Burkholderia cenocepacia in a biofilm model, mutations were most frequently selected in the conserved gene rpfR RpfR uniquely integrates two major signaling systems-quorum sensing and the motile-sessile switch mediated by cyclic-di-GMP-by two domains that sense, respond to, and control the synthesis of the autoinducer cis-2-dodecenoic acid (BDSF). The BDSF response in turn regulates the activity of diguanylate cyclase and phosphodiesterase domains acting on cyclic-di-GMP. Parallel adaptive substitutions evolved in each of these domains to produce unique life history strategies by regulating cyclic-di-GMP levels, global transcriptional responses, biofilm production, and polysaccharide composition. These phenotypes translated into distinct ecology and biofilm structures that enabled mutants to coexist and produce more biomass than expected from their constituents grown alone. This study shows that when bacterial populations are selected in environments challenging the limits of their plasticity, the evolved mutations not only alter genes at the nexus of signaling networks but also reveal the scope of their regulatory functions., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The use of antagonistic microorganisms and their volatile organic compounds (VOCs) to control plant fungal pathogens is an eco-friendly and promising substitute for chemical fungicides. In this work, endophytic bacterium ETR-B22, isolated from the root of Sophora tonkinensis Gagnep., was found to exhibit strong antagonistic activity against 12 fungal pathogens found in agriculture. Strain ETR-B22 was identified as Burkholderia cenocepacia based on 16S rRNA and recA sequences. We evaluated the antifungal activity of VOCs emitted by ETR-B22. The VOCs from strain ETR-B22 also showed broad-spectrum antifungal activity against 12 fungal pathogens. The composition of the volatile profiles was analyzed based on headspace solid phase microextraction (HS-SPME) gas chromatography coupled to mass spectrometry (GC-MS). Different extraction strategies for the SPME process significantly affected the extraction efficiency of the VOCs. Thirty-two different VOCs were identified. Among the VOC of ETR-B22, dimethyl trisulfide, indole, methyl anthranilate, methyl salicylate, methyl benzoate, benzyl propionate, benzyl acetate, 3,5-di-tert-butylphenol, allyl benzyl ether and nonanoic acid showed broad-spectrum antifungal activity, and are key inhibitory compounds produced by strain ETR-B22 against various fungal pathogens. Our results suggest that the endophytic strain ETR-B22 and its VOCs have high potential for use as biological controls of plant fungal pathogens.

Natural environments are rarely static; rather selection can fluctuate on timescales ranging from hours to centuries. However, it is unclear how adaptation to fluctuating environments differs from adaptation to constant environments at the genetic level. For bacteria, one key axis of environmental variation is selection for planktonic or biofilm modes of growth. We conducted an evolution experiment with Burkholderia cenocepacia, comparing the evolutionary dynamics of populations evolving under constant selection for either biofilm formation or planktonic growth with populations in which selection fluctuated between the two environments on a weekly basis. Populations evolved in the fluctuating environment shared many of the same genetic targets of selection as those evolved in constant biofilm selection, but were genetically distinct from the constant planktonic populations. In the fluctuating environment, mutations in the biofilm-regulating genes wspA and rpfR rose to high frequency in all replicate populations. A mutation in wspA first rose rapidly and nearly fixed during the initial biofilm phase but was subsequently displaced by a collection of rpfR mutants upon the shift to the planktonic phase. The wspA and rpfR genotypes coexisted via negative frequency-dependent selection around an equilibrium frequency that shifted between the environments. The maintenance of coexisting genotypes in the fluctuating environment was unexpected. Under temporally fluctuating environments, coexistence of two genotypes is only predicted under a narrow range of conditions, but the frequency-dependent interactions we observed provide a mechanism that can increase the likelihood of coexistence in fluctuating environments., (© 2019 John Wiley & Sons Ltd.)

C109 is a potent but poorly soluble FtsZ inhibitor displaying promising activity against Burkholderia cenocepacia, a high-risk pathogen for cystic fibrosis (CF) sufferers. To harness C109 for inhalation, we developed nanocrystal-embedded dry powders for inhalation suspension consisting in C109 nanocrystals stabilized with D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) embedded in hydroxypropyl-β-cyclodextrin (CD). The powders could be safely re-dispersed in water for in vitro aerosolization. Owing to the presence of a PEG shell, the rod shape and the peculiar aspect ratio, C109 nanocrystals were able to diffuse through artificial CF mucus. The promising technological features were completed by encouraging in vitro/in vivo effects. The formulations displayed no toxicity towards human bronchial epithelial cells and were active against planktonic and sessile B. cenocepacia strains. The efficacy of C109 nanosuspensions in combination with piperacillin was confirmed in a Galleria mellonella infection model, strengthening their potential for combined therapy of B. cenocepacia lung infections., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

O -linked protein glycosylation is a conserved feature of the Burkholderia genus. The addition of the trisaccharide β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc to membrane exported proteins in Burkholderia cenocepacia is required for bacterial fitness and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. Using proteomics, luciferase reporter assays, and DNA cross-linking, we demonstrate the loss of glycosylation leads to changes in transcriptional regulation of multiple proteins, including the repression of the master quorum CepR/I. These proteomic and transcriptional alterations lead to the abolition of biofilm formation and defects in siderophore activity. Surprisingly, the abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants, except for BCAL1086 and BCAL2974, which were found in reduced amounts, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, biofilm formation, or siderophore activity. Together, our results show that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the transcriptional regulatory systems, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins. IMPORTANCE Protein glycosylation is increasingly recognized as a common posttranslational protein modification in bacterial species. Despite this commonality, our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption of O -linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular, and phenotypic assays. We find that in contrast to recent findings on the N -linked glycosylation systems of Campylobacter jejuni , O -linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that loss of glycosylation in B. cenocepacia strains leads to global proteome and transcriptional changes, including the repression of the quorum-sensing regulator cepR ( BCAM1868 ) gene. These alterations lead to dramatic phenotypic changes in glycosylation-null strains, which are paralleled by both global proteomic and transcriptional alterations, which do not appear to directly result from the loss of glycosylation per se. This research unravels the pleiotropic effects of O -linked glycosylation in B. cenocepacia , demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome., (Copyright © 2019 Oppy et al.)

Small non-coding sRNAs have versatile roles in regulating bacterial metabolism. Four short homologous Burkholderia cenocepacia sRNAs strongly expressed under conditions of growth arrest were recently identified. Here we report the detailed investigation of one of these, NcS27. sRNA NcS27 contains a short putative target recognition sequence, which is conserved throughout the order Burkholderiales . This sequence is the reverse complement of the Shine-Dalgarno sequence of a large number of genes involved in transport and metabolism of amino acids and carbohydrates. Overexpression of NcS27 sRNA had a distinct impact on growth, attenuating growth on a variety of substrates such as phenylalanine, tyrosine, glycerol and galactose, while having no effect on growth on other substrates. Transcriptomics and proteomics of NcS27 overexpression and silencing mutants revealed numerous predicted targets changing expression, notably of genes involved in degradation of aromatic amino acids phenylalanine and tyrosine, and in transport of carbohydrates. The conserved target recognition sequence was essential for growth phenotypes and gene expression changes. Cumulatively, our data point to a role of NcS27 in regulating the shutdown of metabolism upon nutrient deprivation in B. cenocepacia . We propose Burkholderia double-hairpin sRNA regulator bdhR1 as designation for ncS27 .

Bacterial strain 71-2 with phosphate-solubilizing activity was isolated from tobacco rhizosphere and classified as Burkholderia cenocepacia based on sequence analyses of 16S rRNA and recA genes. To learn phosphate-solubilizing mechanisms of 71-2, mutants showing reduced solubilizing phosphate activity were obtained using the EZ-Tn5 transposon. Mutant 71-2-MT51 was reduced in the solubilizing phosphate content to 34.36% as compared with the wild-type strain 71-2. The disrupted gene in 71-2-MT51 was cloned and sequenced, and the putative protein from the gene shared 65.26% identity to protein sequences of enolase from Escherichia coli, which suggests the gene encodes an enzyme of enolase. Complementation analyzing showed that Eno was responsible for phosphate solubilizing for B. cenocepacia strain 71-2. To our knowledge, this is the first report of Eno involved in phosphate solubilizing in B. cenocepacia as well as in other bacteria.

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant bacteria. As potentiators do not interfere with essential processes, it has been hypothesized that they are less likely to induce resistance. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop reduced susceptibility toward one such adjuvant, baicalin hydrate (BH). Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to TOB and TOB+BH compared to the start population, and the potentiating effect of BH toward TOB was lost. Whole-genome sequencing was performed to probe which changes were involved in the reduced effect of BH, and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter). No changes in the MIC or MBC of TOB or changes in the number of persister cells were observed. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that B. cenocepacia J2315 biofilms rapidly lose susceptibility toward the antibiotic-potentiating activity of BH and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as mechanisms., (Copyright © 2019 American Society for Microbiology.)

Reduced antimicrobial susceptibility due to resistance and tolerance has become a serious threat to human health. An approach to overcome this reduced susceptibility is the use of antibiotic adjuvants, also known as potentiators. These are compounds that have little or no antibacterial effect on their own but increase the susceptibility of bacterial cells towards antimicrobial agents. Baicalin hydrate, previously described as a quorum sensing inhibitor, is such a potentiator that increases the susceptibility of Burkholderia cenocepacia J2315 biofilms towards tobramycin. The goal of the present study is to elucidate the molecular mechanisms behind the potentiating activity of baicalin hydrate and related flavonoids. We first determined the effect of multiple flavonoids on susceptibility of B. cenocepacia J2315 towards tobramycin. Increased antibiotic susceptibility was most pronounced in combination with apigenin 7-O-glucoside and baicalin hydrate. For baicalin hydrate, also other B. cepacia complex strains and other antibiotics were tested. The potentiating effect was only observed for aminoglycosides and was both strain- and aminoglycoside-dependent. Subsequently, gene expression was compared between baicalin hydrate treated and untreated cells, in the presence and absence of tobramycin. This revealed that baicalin hydrate affected cellular respiration, resulting in increased reactive oxygen species production in the presence of tobramycin. We subsequently showed that baicalin hydrate has an impact on oxidative stress via several pathways including oxidative phosphorylation, glucarate metabolism and by modulating biosynthesis of putrescine. Furthermore, our data strongly suggest that the influence of baicalin hydrate on oxidative stress is unrelated to quorum sensing. Our data indicate that the potentiating effect of baicalin hydrate is due to modulating the oxidative stress response, which in turn leads to increased tobramycin-mediated killing.

Quorum sensing (QS) signals are used by bacteria to regulate biological functions in response to cell population densities. Cyclic diguanosine monophosphate (c-di-GMP) regulates cell functions in response to diverse environmental chemical and physical signals that bacteria perceive. In Burkholderia cenocepacia , the QS signal receptor RpfR degrades intracellular c-di-GMP when it senses the QS signal cis -2-dodecenoic acid, also called Burkholderia diffusible signal factor (BDSF), as a proxy for high cell density. However, it was unclear how this resulted in control of BDSF-regulated phenotypes. Here, we found that RpfR forms a complex with a regulator named GtrR (BCAL1536) to enhance its binding to target gene promoters under circumstances where the BDSF signal binds to RpfR to stimulate its c-di-GMP phosphodiesterase activity. In the absence of BDSF, c-di-GMP binds to the RpfR-GtrR complex and inhibits its ability to control gene expression. Mutations in rpfR and gtrR had overlapping effects on both the B. cenocepacia transcriptome and BDSF-regulated phenotypes, including motility, biofilm formation, and virulence. These results show that RpfR is a QS signal receptor that also functions as a c-di-GMP sensor. This protein thus allows B. cenocepacia to integrate information about its physical and chemical surroundings as well as its population density to control diverse biological functions including virulence. This type of QS system appears to be widely distributed in beta and gamma proteobacteria., Competing Interests: The authors declare no conflict of interest.

Small RNAs play a regulatory role in many central metabolic processes of bacteria, as well as in developmental processes such as biofilm formation. Small RNAs of Burkholderia cenocepacia, an opportunistic pathogenic beta-proteobacterium, are to date not well characterised. To address that, we performed genome-wide transcriptome structure analysis of biofilm grown B. cenocepacia J2315. 41 unannotated short transcripts were identified in intergenic regions of the B. cenocepacia genome. 15 of these short transcripts, highly abundant in biofilms, widely conserved in Burkholderia sp. and without known function, were selected for in-depth analysis. Expression profiling showed that most of these sRNAs are more abundant in biofilms than in planktonic cultures. Many are also highly abundant in cells grown in minimal media, suggesting they are involved in adaptation to nutrient limitation and growth arrest. Their computationally predicted targets include a high proportion of genes involved in carbon metabolism. Expression and target genes of one sRNA suggest a potential role in regulating iron homoeostasis. The strategy used for this study to detect sRNAs expressed in B. cenocepacia biofilms has successfully identified sRNAs with a regulatory function.

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% O 2 ). 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., (Copyright © 2017 American Society for Microbiology.)

Interactions between microbes are central to the dynamics of microbial communities. Understanding these interactions is essential for the characterization of communities, yet challenging to accomplish in practice. There are limited available tools for characterizing diffusion-mediated, contact-independent microbial interactions. A practical and widely implemented technique in such characterization involves the simultaneous co-culture of distinct bacterial species and subsequent analysis of relative abundance in the total population. However, distinguishing between species can be logistically challenging. In this paper, we present a low-cost, vertical membrane, co-culture plate to quantify contact-independent interactions between distinct bacterial populations in co-culture via real-time optical density measurements. These measurements can be used to facilitate the analysis of the interaction between microbes that are physically separated by a semipermeable membrane yet able to exchange diffusible molecules. We show that diffusion across the membrane occurs at a sufficient rate to enable effective interaction between physically separate cultures. Two bacterial species commonly found in the cystic fibrotic lung, Pseudomonas aeruginosa and Burkholderia cenocepacia, were co-cultured to demonstrate how this plate may be implemented to study microbial interactions. We have demonstrated that this novel co-culture device is able to reliably generate real-time measurements of optical density data that can be used to characterize interactions between microbial species.

Knowledge about the molecular mechanisms that are involved in the regulation of biofilm formation is essential for the development of biofilm-control measures. It is well established that the nucleotide second messenger cyclic diguanosine monophosphate (c-di-GMP) is a positive regulator of biofilm formation in many bacteria, but more knowledge about c-di-GMP effectors is needed. We provide evidence that c-di-GMP, the alternative sigma factor RpoN (σ54), and the enhancer-binding protein BerB play a role in biofilm formation of Burkholderia cenocepacia by regulating the production of a biofilm-stabilizing exopolysaccharide. Our findings suggest that BerB binds c-di-GMP, and activates RpoN-dependent transcription of the berA gene coding for a c-di-GMP-responsive transcriptional regulator. An increased level of the BerA protein in turn induces the production of biofilm-stabilizing exopolysaccharide in response to high c-di-GMP levels. Our findings imply that the production of biofilm exopolysaccharide in B. cenocepacia is regulated through a cascade involving two consecutive transcription events that are both activated by c-di-GMP. This type of regulation may allow tight control of the expenditure of cellular resources., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Explaining the enormous biodiversity observed in bacterial communities is challenging because ecological theory predicts that competition between species occupying the same niche should lead to the exclusion of less competitive community members. Competitive exclusion should be particularly strong when species compete for a single limiting resource or live in unstructured habitats that offer no refuge for weaker competitors. Here, we describe the 'cheating effect', a form of intra-specific competition that can counterbalance between-species competition, thereby fostering biodiversity in unstructured habitats. Using experimental communities consisting of the strong competitor Pseudomonas aeruginosa (PA) and its weaker counterpart Burkholderia cenocepacia (BC), we show that co-existence is impossible when the two species compete for a single limiting resource, iron. However, when introducing a PA cheating mutant, which specifically exploits the iron-scavenging siderophores produced by the PA wild type, we found that biodiversity was preserved under well-mixed conditions where PA cheats could outcompete the PA wild type. Cheating fosters biodiversity in our system because it creates strong intra-specific competition, which equalizes fitness differences between PA and BC. Our study identifies cheating - typically considered a destructive element - as a constructive force in shaping biodiversity.

Lipid A anchors the lipopolysaccharide (LPS) to the outer membrane and is usually composed of a hexa-acylated diglucosamine backbone. Burkholderia cenocepacia, an opportunistic pathogen, produces a mixture of tetra- and penta-acylated lipid A. "Late" acyltransferases add secondary acyl chains to lipid A after the incorporation of four primary acyl chains to the diglucosamine backbone. Here, we report that B. cenocepacia has only one late acyltransferase, LpxL (BCAL0508), which adds a myristoyl chain to the 2' position of lipid A resulting in penta-acylated lipid A. We also identified PagL (BCAL0788), which acts as an outer membrane lipase by removing the primary β-hydroxymyristate (3-OH-C14:0) chain at the 3 position, leading to tetra-acylated lipid A. Unlike PagL, LpxL depletion caused reduced cell growth and defects in cell morphology, both of which were suppressed by overexpressing the LPS flippase MsbA (BCAL2408), suggesting that lipid A molecules lacking the fifth acyl chain contributed by LpxL are not good substrates for the flippase. We also show that intracellular B. cenocepacia within macrophages produced more penta-acylated lipid A, suggesting lipid A penta-acylation in B. cenocepacia is required not only for bacterial growth and morphology but also for adaptation to intracellular lifestyle., (© 2017 John Wiley & Sons Ltd.)

Background: Genomic islands (GIs) are genomic regions that reveal evidence of horizontal DNA transfer. They can code for many functions and may augment a bacterium's adaptation to its host or environment. GIs have been identified in strain J2315 of Burkholderia cenocepacia, whereas in strain AU 1054 there has been no published works on such regions according to our text mining and keyword search in Medline., Results: In this study, we identified 21 GIs in AU 1054 by combining two computational tools. Feature analyses suggested that the predictions are highly reliable and hence illustrated the advantage of joint predictions by two independent methods. Based on putative virulence factors, four GIs were further identified as pathogenicity islands (PAIs). Through experiments of gene deletion mutants in live bacteria, two putative PAIs were confirmed, and the virulence factors involved were identified as lipA and copR. The importance of the genes lipA (from PAI 1) and copR (from PAI 2) for bacterial invasion and replication indicates that they are required for the invasive properties of B. cenocepacia and may function as virulence determinants for bacterial pathogenesis and host infection., Conclusions: This approach of in silico prediction of GIs and subsequent identification of potential virulence factors in the putative island regions with final validation using wet experiments could be used as an effective strategy to rapidly discover novel virulence factors in other bacterial species and strains.

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, ΔflhF, to study whether this putative GTPase regulates the flagellation pattern of B. cenocepacia K56-2 during growth in CF conditions. The ΔflhF mutant exhibited 80% aflagellated, 14% single and 6% multiple flagellated bacterial subpopulations. Moreover, the ratio of multiple to single flagella in WT and ΔflhF 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.

Bacteria usually grow forming biofilms, which are communities of cells embedded in a self-produced dynamic polymeric matrix, characterized by a complex three-dimensional structure. The matrix holds cells together and above a surface, and eventually releases them, resulting in colonization of other surfaces. Although exopolysaccharides (EPOLs) are important components of the matrix, determination of their structure is usually performed on samples produced in non-biofilm conditions, or indirectly through genetic studies. Among the Burkholderia cepacia complex species, Burkholderia cenocepacia is an important pathogen in cystic fibrosis (CF) patients and is generally more aggressive than other species. In the present investigation, B. cenocepacia strain BTS2, a CF isolate, was grown in biofilm mode on glass slides and cellulose membranes, using five growth media, one of which mimics the nutritional content of CF sputum. The structure of the matrix EPOLs was determined by 1H-NMR spectroscopy, while visualization of the biofilms on glass slides was obtained by means of confocal laser microscopy, phase-contrast microscopy and atomic force microscopy. The results confirmed that the type of EPOLs biosynthesized depends both on the medium used and on the type of support, and showed that mucoid conditions do not always lead to significant biofilm production, while bacteria in a non-mucoid state can still form biofilm containing EPOLs.

Respiratory infection with Burkholderia cenocepacia is associated with accelerated decline in lung function and increased mortality in cystic fibrosis (CF) patients (A. M. Jones, M. E. Dodd, J. R. W. Govan, V. Barcus, C. J. Doherty, J. Morris, and A. K. Webb, Thorax 59:948-951, 2004, http://dx.doi.org/10.1136/thx.2003.017210). B. cenocepacia often possesses innate resistance to multiple antimicrobial classes, making eradication uncommon in established infection (P. B. Davis, Am J Respir Crit Care Med 173:475-482, 2006, http://dx.doi.org/10.1164/rccm.200505-840OE). We report the use of clinafloxacin in a CF patient with advanced B. cenocepacia infection, present pharmacokinetic (PK) data, and discuss the potential therapeutic role of clinafloxacin in patients with this condition., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The Gram-negative bacterial type VI secretion system (T6SS) delivers toxins to kill or inhibit the growth of susceptible bacteria, while other secretion systems target eukaryotic cells. Deletion of atsR, a negative regulator of virulence factors in B. cenocepacia K56-2, increases T6SS activity. Macrophages infected with a K56-2 ΔatsR mutant display dramatic alterations in their actin cytoskeleton architecture that rely on the T6SS, which is responsible for the inactivation of multiple Rho-family GTPases by an unknown mechanism. We employed a strategy to standardize the bacterial infection of macrophages and densitometrically quantify the T6SS-associated cellular phenotype, which allowed us to characterize the phenotype of systematic deletions of each gene within the T6SS cluster and ten vgrG genes in K56-2 ΔatsR. None of the genes from the T6SS core cluster nor the individual vgrG genes were directly responsible for the cytoskeletal changes in infected cells. However, a mutant strain with all vgrG genes deleted was unable to cause macrophage alterations. Despite not being able to identify a specific effector protein responsible for the cytoskeletal defects in macrophages, our strategy resulted in the identification of the critical core components and accessory proteins of the T6SS assembly machinery and provides a screening method to detect T6SS effectors targeting the actin cytoskeleton in macrophages by random mutagenesis.

Essential gene studies often reveal novel essential functions for genes with dispensable homologues in other species. This is the case with the widespread family of electron transfer flavoproteins (ETFs), which are required for the metabolism of specific substrates or for symbiotic nitrogen fixation in some bacteria. Despite these non-essential functions high-throughput screens have identified ETFs as putatively essential in several species. In this study, we constructed a conditional expression mutant of one of the ETFs in Burkholderia cenocepacia, and demonstrated that its expression is essential for growth on both complex media and a variety of single-carbon sources. We further demonstrated that the two subunits EtfA and EtfB interact with each other, and that cells depleted of ETF are non-viable and lack redox potential. These cells also transition from the short rods characteristic of Burkholderia cenocepacia to small spheres independently of MreB. The putative membrane partner ETF dehydrogenase also induced the same rod-to-sphere change. We propose that the ETF of Burkholderia cenocepacia is a novel antibacterial target.

To study the role of Quorum Sensing (QS) regulation in the control of the cellular processes of Burkholderia cenocepacia 370, plasmid pME6863 was transferred into its cells. The plasmid contains a heterologous gene encoding for AiiA N-acyl-homoserine lactonase, which degrades the signaling molecules of the QS system of N-acyl-homoserine lactones (AHL). An absence or reduction of AHL in the culture was revealed with the biosensors Chromobacterium violaceum CV026 and Agrobacterium tumifaciens NT1/pZLR4, respectively. The presence of the aiiA gene, which was cloned from Bacillus sp. A24 in the cells of B. cenocepacia 370, resulted in a lack of hemolytic activity, which reduced the extracellular proteolytic activity and decreased the cells' ability to migration in swarms on the surface of the agar medium. The introduction of the aiiA gene did not affect lipase activity, fatty acids synthesis, HCN synthesis, or biofilm formation. Hydrogen peroxide was shown to stimulate biofilm formation by B. cenocepacia 370 in concentrations that inhibited or weakly suppressed bacterial growth. The introduction of the aiiA gene into the cells did not eliminate this effect but it did reduce it.

Members of the genus Burkholderia are versatile bacteria capable of colonizing highly diverse environmental niches. In this study, we investigated the global response of the opportunistic pathogen Burkholderia cenocepacia H111 to nitrogen limitation at the transcript and protein expression levels. In addition to a classical response to nitrogen starvation, including the activation of glutamine synthetase, PII proteins, and the two-component regulatory system NtrBC, B. cenocepacia H111 also upregulated polyhydroxybutyrate (PHB) accumulation and exopolysaccharide (EPS) production in response to nitrogen shortage. A search for consensus sequences in promoter regions of nitrogen-responsive genes identified a σ(54) consensus sequence. The mapping of the σ(54) regulon as well as the characterization of a σ(54) mutant suggests an important role of σ(54) not only in control of nitrogen metabolism but also in the virulence of this organism., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Infections with the bacteria Burkholderia cepacia complex (Bcc) are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, B. cenocepacia K56-2. The primary screen used bacterial growth in 96-well plate format and identified 206 primary actives among 30,259 compounds. From 100 compounds with no previous record of antibacterial activity secondary screening and data mining selected a total of Bce bioactives that were further analyzed. An experimental pipeline, evaluating in vitro antibacterial and antibiofilm activity, toxicity and in vivo antibacterial activity using C. elegans was used for prioritizing compounds with better chances to be further investigated as potential Bcc antibacterial drugs. This high throughput screen, along with the in vitro and in vivo analysis highlights the utility of this experimental method to quickly identify bioactives as a starting point of antibacterial drug discovery.

Two-component systems are important regulatory systems that allow bacteria to adjust to environmental conditions, and in some bacteria are used in pathogenesis. We identified a novel two-component system in Burkholderia cenocepacia, an opportunistic pathogen that causes pneumonia in cystic fibrosis (CF) patients. The putative operon encodes BceS, a sensor kinase, and BceR, a response regulator. Our studies indicated that the bceR mutant showed a statistically significant decrease in protease, swimming motility, and quorum sensing when compared to the wild-type, but there was no significant difference in phospholipase C activity, swarming, and biofilm formation. In addition, the mutant showed a statistically significant reduction in virulence compared to the wild-type using the alfalfa plant model. Examination of the Burkholderia cepacia complex (a group of organisms that are phenotypically similar, but genotypically distinct) revealed that this system is prevalent in B. ambifaria, B. multivorans, B. vietnamiensis and B. dolosa. Interestingly, all these organisms have been associated with CF patients. The collective results indicate that BceSR influences various activities important in Burkholderia physiology and possibly pathogenesis. This information could be important in the design of novel therapeutics for Burkholderia infections.

D-mannitol has been approved in dry powder formulation as an effective antimucolytic agent in patients with cystic fibrosis. What is not known is the effect of adding a metabolisable sugar on the biology of chronic bacterial pathogens in the CF lung. Therefore, a series of simple in vitro experiments were performed to examine the effect of adding D-mannitol on the phenotype of the CF respiratory pathogens Pseudomonas aeruginosa and Burkholderia cenocepacia. Clinical isolates (n = 86) consisting of P. aeruginosa (n = 51), B. cenocepacia (n = 26), P. putida (n = 4), Stenotrophomonas maltophila (n = 3) and Pseudomonas spp. (n = 2) were examined by supplementing basal nutrient agar with varying concentrations of D-mannitol (0-20% [w/v]) and subsequently examining for any change in microbial phenotype. The effect of supplementation with mannitol was four-fold, namely i) To increase the proliferation and increase in cell density of all CF organisms examined, with an optimal concentration of 2-4% (w/v) D-mannitol. No such increase in cell proliferation was observed when mannitol was substituted with sodium chloride. ii) Enhanced pigment production was observed in 2/51 (3.9%) of the P. aeruginosa isolates examined, in one of the P. putida isolates, and in 3/26 (11.5%) of the B. cenocepacia isolates examined. iii). When examined at 4.0% (w/v) supplementation with mannitol, 11/51 (21.6%) P. aeruginosa isolates and 3/26 (11.5%) B. cenocepacia isolates were seen to exhibit the altered adhesion phenotype. iv). With respect to the altered mucoid phenotype, 5/51 (9.8%) P. aeruginosa produced this phenotype when grown at 4% mannitol. Mucoid production was greatest at 4%, was poor at 10% and absent at 20% (w/v) mannitol. The altered mucoid phenotype was not observed in the B. cenocepacia isolates or any of the other clinical taxa examined. Due consideration therefore needs to be given, where there is altered physiology within the small airways, leading to a potentially altered biological state of the colonising microorganisms in novel inhaled pharmaceutical interventions in CF, particularly those, which are not designated as antimicrobial agents.

Burkholderia cenocepacia is notorious for causing respiratory tract infections in people with cystic fibrosis. Infections with this organism are particularly difficult to treat due to its high level of intrinsic resistance to most antibiotics. Multidrug resistance in B. cenocepacia can be ascribed to different mechanisms, including the activity of efflux pumps and biofilm formation. In the present study, the effects of deletion of the 16 operons encoding resistance-nodulation-cell division (RND)-type efflux pumps in B. cenocepacia strain J2315 were investigated by determining the MICs of various antibiotics and by investigating the antibiofilm effect of these antibiotics. Finally, the expression levels of selected RND genes in treated and untreated cultures were investigated using reverse transcriptase quantitative PCR (RT-qPCR). Our data indicate that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonic B. cenocepacia J2315 populations, while the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin. The RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance. Results from the RT-qPCR experiments on the wild-type strain B. cenocepacia J2315 suggest that there is little regulation at the level of mRNA expression for these efflux pumps under the conditions tested., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The phenylacetic acid degradation pathway of Burkholderia cenocepacia is active during cystic fibrosis-like conditions and is necessary for full pathogenicity of B. cenocepacia in nematode and rat infection models; however, the reasons for such requirements are unknown. Here, we show that the attenuated virulence of a phenylacetic acid catabolism mutant is due to quorum sensing inhibition. Unlike wild-type B. cenocepacia, a deletion mutant of the phenylacetyl-CoA monooxygenase complex (ΔpaaABCDE) released phenylacetic acid in the medium that favours infection in Caenorhabditis elegans. Addition of phenylacetic acid further decreased the pathogenicity of the ΔpaaABCDE, which cannot metabolize phenylacetic acid, but did not affect the wild-type, due to phenylacetic acid consumption. In line with reduced detection of acyl-homoserine lactones in spent medium, the ΔpaaABCDE exhibited transcriptional inhibition of the quorum sensing system cepIR. Phenotypes repressed in ΔpaaABCDE, protease activity and pathogenicity against C. elegans, increased with exogenous N-octanoyl-L-homoserine lactone. Thus, we demonstrate that the attenuated phenotype of B. cenocepacia ΔpaaABCDE is due to quorum sensing inhibition by release of phenylacetic acid, affecting N-octanoyl-L-homoserine lactone signalling. Further, we propose that active degradation of phenylacetic acid by B. cenocepacia during growth in cystic fibrosis-like conditions prevents accumulation of a quorum sensing inhibiting compound., (© 2014 John Wiley & Sons Ltd.)

Burkholderia cepacia complex (BCC) presence has been the cause of recalls of both sterile and non-sterile pharmaceutical products since these opportunistic pathogens have been implicated to cause infections to susceptible individuals. BCC are ubiquitous in nature, but in pharmaceutical settings the most common source is contaminated water systems. Some strains of BCC, previously described as Pseudomonas cepacia, were not readily detected by standard culture methods. We have explored different strategies to recover and enrich Burkholderia cenocepacia previously cultured in distilled water for 40 days. Enrichment media of varied nutrient concentrations and composition were used, including modified Tryptic Soy Agar or Broth (TSA or TSB), Reasoner's 2nd Agar or Broth (R2A or R2AB), Brain-Heart Infusion Broth (BHIB), Mueller-Hinton Broth (MHB), and Ashdown's (ASH) medium. Of the various broth media tested, cell growth was significantly greater in TSB and R2AB than in BHIB, MHB, or ASH broth. TSB and R2AB were also compared for their recovery efficiency. Generally, there was no significant difference between the numbers of B. cenocepacia grown on 15 differently modified TSA and five modified R2A solid media. Overall, however, diluted TSA and TSB media, and R2A and R2AB showed better recovery efficiency than TSA and TSB for inocula containing small numbers of cells. All strains persisted in distilled water for 40 days. Broth media were more effective than solid media for recovery of B. cenocepacia from distilled water. These results may assist in improving detection assays with recovery and enrichment strategies to maximize recovery of these fastidious organisms.

Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of relA led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and in vitro directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.

Like many other bacteria, Burkholderia sp. take up iron in its ferric form via siderophore-dependent transporters. We observed that mutant strains of B. cenocepacia H111 unable to synthesize siderophores did not exhibit any growth defect under iron limited conditions. This finding suggested that this opportunistic pathogen can adopt an alternative iron uptake strategy to compensate for the loss of siderophores. We identified a putative iron uptake locus, ftrBcc ABCD, in the genome of B. cenocepacia H111, which is also conserved in other members of the genus Burkholderia. Mutants deficient in both siderophore-dependent and FtrBcc ABCD systems failed to grow under iron-limited conditions and radiolabelled iron transport assays showed that these mutants were impaired in iron uptake. In addition, expression of ftrBcc ABCD can restore growth of an E. coli strain lacking all known high-affinity iron transport systems under iron-limited conditions. We show that all four proteins encoded by ftrBcc ABCD are essential for iron uptake. Furthermore, our results indicate that the expression of ftrBcc ABCD is regulated at the transcriptional level by iron concentration. This study provides evidence of an alternative, siderophore-independent, iron uptake system in Burkholderia species., (© 2013 John Wiley & Sons Ltd.)

How split genomes arise and evolve in bacteria is poorly understood. Since each replicon of such genomes encodes a specific partition (Par) system, the evolution of Par systems could shed light on their evolution. The cystic fibrosis pathogen Burkholderia cenocepacia has three chromosomes (c1, c2, and c3) and one plasmid (pBC), whose compatibility depends on strictly specific interactions of the centromere sequences (parS) with their cognate binding proteins (ParB). However, the Par systems of B. cenocepacia c2, c3, and pBC share many features, suggesting that they arose within an extended family. Database searching revealed seven subfamilies of Par systems like those of B. cenocepacia. All are from plasmids and secondary chromosomes of the Burkholderiales, which reinforces the proposal of an extended family. The subfamily of the Par system of B. cenocepacia c3 includes plasmid variants with parS sequences divergent from that of c3. Using electrophoretic mobility shift assay (EMSA), we found that ParB-c3 binds specifically to centromeres of these variants, despite high DNA sequence divergence. We suggest that the Par system of B. cenocepacia c3 has preserved the features of an ancestral system. In contrast, these features have diverged variably in the plasmid descendants. One such descendant is found both in Ralstonia pickettii 12D, on a free plasmid, and in Ralstonia pickettii 12J, on a plasmid integrated into the main chromosome. These observations suggest that we are witnessing a plasmid-chromosome interaction from which a third chromosome will emerge in a two-chromosome species.

By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice.

Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens that cause multiresistant pulmonary infections in patients with cystic fibrosis (CF). In this study, we evaluated the in vitro antimicrobial efficacy of eight unsaturated fatty acids against Burkholderia cenocepacia K56-2, a CF epidemic strain. Docosahexaenoic acid (DHA) was the most active compound. Its action can be either bacteriostatic or bactericidal, depending upon the concentration used. The effect of DHA was also evaluated on two others B. cenocepacia clinical isolates and compared with one representative member of all the 17 Bcc species. To test whether DHA could have a therapeutic potential, we assessed its efficacy using a Galleria mellonella caterpillar model of B. cenocepacia infection. We observed that the treatment of infected larvae with a single dose of DHA (50 mM) caused an increase in the survival rate as well as a reduced bacterial load. Moreover, DHA administration markedly increases the expression profile of the gene encoding the antimicrobial peptide gallerimycin. Our results demonstrate that DHA has in vitro and in vivo antibacterial activity against Bcc microorganisms. These findings provide evidence that DHA may be a useful nutraceutical for the treatment of CF patients with lung infections caused by antibiotic multiresistant Bcc microorganisms., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Background: Essential genes are absolutely required for the survival of an organism. The identification of essential genes, besides being one of the most fundamental questions in biology, is also of interest for the emerging science of synthetic biology and for the development of novel antimicrobials. New antimicrobial therapies are desperately needed to treat multidrug-resistant pathogens, such as members of the Burkholderia cepacia complex., Methodology/principal Findings: We hypothesize that essential genes may be highly conserved within a group of evolutionary closely related organisms. Using a bioinformatics approach we determined that the core genome of the order Burkholderiales consists of 649 genes. All but two of these identified genes were located on chromosome 1 of Burkholderia cenocepacia. Although many of the 649 core genes of Burkholderiales have been shown to be essential in other bacteria, we were also able to identify a number of novel essential genes present mainly, or exclusively, within this order. The essentiality of some of the core genes, including the known essential genes infB, gyrB, ubiB, and valS, as well as the so far uncharacterized genes BCAL1882, BCAL2769, BCAL3142 and BCAL3369 has been confirmed experimentally in B. cenocepacia., Conclusions/significance: We report on the identification of essential genes using a novel bioinformatics strategy and provide bioinformatics and experimental evidence that the large majority of the identified genes are indeed essential. The essential genes identified here may represent valuable targets for the development of novel antimicrobials and their detailed study may shed new light on the functions required to support life.

Small non-coding regulatory RNAs (sRNAs) post-transcriptionally affect multiple phenotypes in prokaryotes and eukaryotes, yet most of the underlying regulatory mechanisms and the nature of the target mRNAs remain unclear. Here we report the identification and functional analysis of the novel cis-encoded sRNA h2cR, from the human opportunistic pathogen Burkholderia cenocepacia J2315. The sRNA was found to negatively regulate the hfq2 mRNA, through binding to part of the 5'-UTR region of the hfq2 mRNA, resulting in accelerated hfq2 mRNA decay and reduced protein levels in exponentially growing cells. Both the h2cR transcript and the hfq2 mRNA are stabilized by the other B. cenocepacia RNA chaperone, Hfq. Infection experiments using the nematode Caenorhabditis elegans revealed that down-regulation of Hfq2 by h2cR decreases the B. cenocepacia ability to colonize and persist within the nematode, suggesting a role for h2cR on bacterial persistence in the host.

Cystic fibrosis (CF) is the most common inherited lethal disease of Caucasians which results in multi organ dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR ΔF508 mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1β. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type macrophages but not in ΔF508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-ΔF508 (ΔF508) macrophages than in WT macrophages. An autophagosome is a compartment that engulfs non-functional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and ΔF508 macrophages. However, autophagy dysfunction is more pronounced in ΔF508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, Rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.

Burkholderia cepacia complex is a group of bacterial pathogens that cause opportunistic infections in cystic fibrosis (CF). The most virulent of these is Burkholderia cenocepacia. Matrix metalloproteinases (MMPs) are upregulated in CF patients. The aim of this work was to examine the role of MMPs in the pathogenesis of B. cepacia complex, which has not been explored to date. Real-time PCR analysis showed that B. cenocepacia infection upregulated MMP-2 and MMP-9 genes in the CF lung cell line CFBE41o- within 1 h, whereas MMP-2, -7, and -9 genes were upregulated in the non-CF lung cell line 16HBE14o-. Conditioned media from both cell lines showed increased MMP-9 activation following B. cenocepacia infection. Conditioned media from B. cenocepacia-infected cells significantly reduced the rate of wound healing in confluent lung epithelia (P < 0.05), in contrast to conditioned media from Pseudomonas aeruginosa-infected cells, which showed predominant MMP-2 activation. Treatment of control conditioned media from both cell lines with the MMP activator 4-aminophenylmercuric acetate (APMA) also resulted in clear activation of MMP-9 and to a much lesser extent MMP-2. APMA treatment of control media also delayed the repair of wound healing in confluent epithelial cells. Furthermore, specific inhibition of MMP-9 in medium from cells exposed to B. cenocepacia completely reversed the delay in wound repair. These data suggest that MMP-9 plays a role in the reduced epithelial repair observed in response to B. cenocepacia infection and that its activation following B. cenocepacia infection contributes to the pathogenesis of this virulent pathogen.

In cystic fibrosis (CF) high iron concentration in airway secretion plays a pivotal role in bacterial multiplication and biofilm formation as well as in inflammatory response. Burkholderia cenocepacia, an opportunistic facultative pathogen responsible for chronic lung infections and cepacia syndrome, recurrently infects CF patients. Lactoferrin (Lf), an iron binding multifunctional glycoprotein synthesized by exocrine glands and neutrophils, has been found at higher concentration in the airway secretions of infected CF patients than in healthy subjects. Here the influence of milk derivative bovine lactoferrin (bLf), an emerging important regulator of iron and inflammatory homeostasis, on invasiveness of B. cenocepacia iron-modulated biofilm, as well as on inflammatory response by infected CF bronchial (IB3-1) cells, is reported. bLf did not significantly affect invasion efficacy by biofilmforming B. cenocepacia clinical strains. Conversely, the addition of bLf to cell monolayers during infection significantly decreased the pro-inflammatory Interleukin (IL)-1beta and increased the anti-inflammatory IL-11 expression compared to that observed in cells infected in the absence of bLf. The bLf ability to modulate genes expressed following B. cenocepacia infection seems related to its localization to the nucleus of infected IB3-1 cells. These results provide evidence for a role of bLf in the protection of infected CF cells from inflammation-related damage, thus extending the therapeutic potential of this multifunctional natural protein.

Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, phenylpyruvate, and 2-phenylacetamide were usable as sole carbon sources by wild type B. cenocepacia K56-2, but not by a PA catabolism-defective mutant. EMSA analysis showed that the binding of PaaR, the negative regulator protein of B. cenocepacia PA catabolism, to PA regulatory DNA could only be relieved by phenylacetyl-Coenzyme A (PA-CoA), but not by any of the putative phenylalanine degradation intermediates. Taken together, our results show that in B. cenocepacia, phenylalanine is catabolized to PA and induces PA catabolism through PA activation to PA-CoA. Thus, PaaR shares the same inducer with PaaX, the regulator of PA catabolism in Escherichia coli, despite belonging to a different protein family., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis (CF) patients. Previously, we reported that ShvR, a LysR regulator, influences colony morphology, virulence, and biofilm formation and regulates the expression of an adjacent 24-kb genomic region encoding 24 genes. In this study, we report the functional characterization of selected genes in this region. A Tn5 mutant with shiny colony morphology was identified with a polar mutation in BCAS0208, predicted to encode an acyl-coenzyme A dehydrogenase. Mutagenesis of BCAS0208 and complementation analyses revealed that BCAS0208 is required for rough colony morphology, biofilm formation, and virulence on alfalfa seedlings. It was not possible to complement with BCAS0208 containing a mutation in the catalytic site. BCAS0201, encoding a putative flavin adenine dinucleotide (FAD)-dependent oxidoreductase, and BCAS0207, encoding a putative citrate synthase, do not influence colony morphology but are required for optimum levels of biofilm formation and virulence. Both BCAS0208 and BCAS0201 contribute to pellicle formation, although individual mutations in each of these genes had no appreciable effect on pellicle formation. A mutant with a polar insertion in BCAS0208 was significantly less virulent in a rat model of chronic lung infection as well as in the alfalfa model. Genes in this region were shown to influence utilization of branched-chain fatty acids, tricarboxylic acid cycle substrates, l-arabinose, and branched-chain amino acids. Together, our data show that the ShvR-regulated genes BCAS0208 to BCAS0201 are required for the rough colony morphotype, biofilm and pellicle formation, and virulence in B. cenocepacia.

Burkholderia cenocepacia is an important opportunistic pathogen, and one of the most striking features of the Burkholderia genus is the collection of polar lipids present in its membrane, including phosphatidylethanolamine (PE) and ornithine-containing lipids (OLs), as well as the 2-hydroxylated derivatives of PE and OLs (2-OH-PE and 2-OH-OLs, respectively), which differ from the standard versions by virtue of the presence of a hydroxyl group at C2 (2-OH) of an esterified fatty acyl residue. Similarly, a lipid A-esterified myristoyl group from Salmonella typhimurium can have a 2-hydroxy modification that is due to the LpxO enzyme. We thus postulated that 2-hydroxylation of 2-OH-OLs might be catalyzed by a novel dioxygenase homologue of LpxO. In B. cenocepacia, we have now identified two open reading frames (BCAM1214 and BCAM2401) homologous to LpxO from S. typhimurium. The introduction of bcam2401 (designated olsD) into Sinorhizobium meliloti leads to the formation of one new lipid and in B. cenocepacia of two new lipids. Surprisingly, the lipid modifications on OLs due to OlsD occur on the amide-linked fatty acyl chain. This is the first report of a hydroxyl modification of OLs on the amide-linked fatty acyl moiety. Formation of hydroxylated OLs occurs only when the biosynthesis pathway for nonmodified standard OLs is intact. The hydroxyl modification of OLs on the amide-linked fatty acyl moiety occurs only under acid stress conditions. An assay has been developed for the OlsD dioxygenase, and an initial characterization of the enzyme is presented., (© 2011 American Chemical Society)

Aims: To investigate mechanisms of osmotic tolerance in Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc) of closely related strains, which is of clinical as well as environmental importance., Methods and Results: We employed NMR-based metabolic profiling (metabolomics) to elucidate the metabolic consequences of high osmotic stress for five isolates of B. cenocepacia. The strains differed significantly in their levels of osmotic stress tolerance, and we identified three different sets of metabolic responses with the strains least impacted by osmotic stress exhibiting higher levels of the osmo-protective metabolites glycine-betaine and/or trehalose. Strains either increased concentrations or had constitutively high levels of these metabolites., Conclusions: Even within the small set of B. cenocepacia isolates, there was a surprising degree of variability in the metabolic responses to osmotic stress., Significance and Impact of the Study: The metabolic responses, and hence osmotic stress tolerance, vary between different B. cenocepacia isolates. This study provides a first look into the potentially highly diverse physiology of closely related strains of one species of the Bcc and illustrates that physiological or clinically relevant phenotypes are unlikely to be inferable from genetic relatedness within this species group., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)