Glycoproteomic and proteomic analysis of Burkholderia cenocepacia reveals glycosylation events within FliF and MotB are dispensable for motility.

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 ΔfliF (ΔBCAL0525), ΔmotB (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 ΔpglL. 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.

Identification of putative essential protein domains from high-density transposon insertion sequencing.

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.

One gene, multiple ecological strategies: A biofilm regulator is a capacitor for sustainable diversity.

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.

Characterization of Volatile Organic Compounds Emitted from Endophytic Burkholderia cenocepacia ETR-B22 by SPME-GC-MS and Their Inhibitory Activity against Various Plant Fungal Pathogens.

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.

PEGylated mucus-penetrating nanocrystals for lung delivery of a new FtsZ inhibitor against Burkholderia cenocepacia infection.

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-ß-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.

Negative frequency-dependent selection maintains coexisting genotypes during fluctuating selection.

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.

Loss of O-Linked Protein Glycosylation in Burkholderia cenocepacia Impairs Biofilm Formation and Siderophore Activity and Alters Transcriptional Regulators.

O-linked protein glycosylation is a conserved feature of the Burkholderia genus. The addition of the trisaccharide ß-Gal-(1,3)-α-GalNAc-(1,3)-ß-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.

Small RNA NcS27 co-regulates utilization of carbon sources in Burkholderia cenocepacia J2315.

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 Burkholderiadouble-hairpin sRNA regulator bdhR1 as designation for ncS27.

The Eno Gene of Burkholderia cenocepacia Strain 71-2 is Involved in Phosphate Solubilization.

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.

Various Evolutionary Trajectories Lead to Loss of the Tobramycin-Potentiating Activity of the Quorum-Sensing Inhibitor Baicalin Hydrate in Burkholderia cenocepacia Biofilms.

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.

Elucidation of the mechanism behind the potentiating activity of baicalin against Burkholderia cenocepacia biofilms.

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.

Identification of small RNAs abundant in Burkholderia cenocepacia biofilms reveal putative regulators with a potential role in carbon and iron metabolism.

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.

Burkholderia cenocepacia integrates cis-2-dodecenoic acid and cyclic dimeric guanosine monophosphate signals to control virulence.

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.

Novel co-culture plate enables growth dynamic-based assessment of contact-independent microbial interactions.

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.

The Essential Genome of Burkholderia cenocepacia H111.

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.

Regulation of Burkholderia cenocepacia biofilm formation by RpoN and the c-di-GMP effector BerB.

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.

Identification and analysis of genomic islands in Burkholderia cenocepacia AU 1054 with emphasis on pathogenicity islands.

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.

Cheating fosters species co-existence in well-mixed bacterial communities.

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.

The LpxL acyltransferase is required for normal growth and penta-acylation of lipid A in Burkholderia cenocepacia.

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 ß-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.