Cell envelope structural and functional contributions to antibiotic resistance in Burkholderia cenocepacia.

Antibiotic activity is limited by the physical construction of the Gram-negative cell envelope. Species of the Burkholderia cepacia complex (Bcc) are known as intrinsically multidrug-resistant opportunistic pathogens with low permeability cell envelopes. Here, we re-examined a previously performed chemical-genetic screen of barcoded transposon mutants in B. cenocepacia K56-2, focusing on cell envelope structural and functional processes. We identified structures mechanistically important for resistance to singular and multiple antibiotic classes. For example, susceptibility to novobiocin, avibactam, and the LpxC inhibitor, PF-04753299, was linked to the BpeAB-OprB efflux pump, suggesting these drugs are substrates for this pump in B. cenocepacia. Defects in peptidoglycan precursor synthesis specifically increased susceptibility to cycloserine and revealed a new putative amino acid racemase, while defects in divisome accessory proteins increased susceptibility to multiple ß-lactams. Additionally, disruption of the periplasmic disulfide bond formation system caused pleiotropic defects on outer membrane integrity and ß-lactamase activity. Our findings highlight the layering of resistance mechanisms in the structure and function of the cell envelope. Consequently, we point out processes that can be targeted for developing antibiotic potentiators.IMPORTANCEThe Gram-negative cell envelope is a double-layered physical barrier that protects cells from extracellular stressors, such as antibiotics. The Burkholderia cell envelope is known to contain additional modifications that reduce permeability. We investigated Burkholderia cell envelope factors contributing to antibiotic resistance from a genome-wide view by re-examining data from a transposon mutant library exposed to an antibiotic panel. We identified susceptible phenotypes for defects in structures and functions in the outer membrane, periplasm, and cytoplasm. Overall, we show that resistance linked to the cell envelope is multifaceted and provides new targets for the development of antibiotic potentiators.

Protozoan predation enhances stress resistance and antibiotic tolerance in Burkholderia cenocepacia by triggering the SOS response.

Bacterivorous protists are thought to serve as training grounds for bacterial pathogens by subjecting them to the same hostile conditions that they will encounter in the human host. Bacteria that survive intracellular digestion exhibit enhanced virulence and stress resistance after successful passage through protozoa but the underlying mechanisms are unknown. Here we show that the opportunistic pathogen Burkholderia cenocepacia survives phagocytosis by ciliates found in domestic and hospital sink drains, and viable bacteria are expelled packaged in respirable membrane vesicles with enhanced resistance to oxidative stress, desiccation, and antibiotics, thereby contributing to pathogen dissemination in the environment. Reactive oxygen species generated within the protozoan phagosome promote the formation of persisters tolerant to ciprofloxacin by activating the bacterial SOS response. In addition, we show that genes encoding antioxidant enzymes are upregulated during passage through ciliates increasing bacterial resistance to oxidative radicals. We prove that suppression of the SOS response impairs bacterial intracellular survival and persister formation within protists. This study highlights the significance of protozoan food vacuoles as niches that foster bacterial adaptation in natural and built environments and suggests that persister switch within phagosomes may be a widespread phenomenon in bacteria surviving intracellular digestion.

Identification of a system for hydroxamate xenosiderophore-mediated iron transport in Burkholderia cenocepacia.

One of the mechanisms employed by the opportunistic pathogen Burkholderia cenocepacia to acquire the essential element iron is the production and release of two ferric iron chelating compounds (siderophores), ornibactin and pyochelin. Here we show that B. cenocepacia is also able to take advantage of a range of siderophores produced by other bacteria and fungi ('xenosiderophores') that chelate iron exclusively by means of hydroxamate groups. These include the tris-hydroxamate siderophores ferrioxamine B, ferrichrome, ferricrocin and triacetylfusarinine C, the bis-hydroxamates alcaligin and rhodotorulic acid, and the monohydroxamate siderophore cepabactin. We also show that of the 24 TonB-dependent transporters encoded by the B. cenocepacia genome, two (FhuA and FeuA) are involved in the uptake of hydroxamate xenosiderophores, with FhuA serving as the exclusive transporter of iron-loaded ferrioxamine B, triacetylfusarinine C, alcaligin and rhodotorulic acid, while both FhuA and FeuA are able to translocate ferrichrome-type siderophores across the outer membrane. Finally, we identified FhuB, a putative cytoplasmic membrane-anchored ferric-siderophore reductase, as being obligatory for utilization of all of the tested bis- and tris-hydroxamate xenosiderophores apart from alcaligin.

Genotoxic stress stimulates eDNA release via explosive cell lysis and thereby promotes streamer formation of Burkholderia cenocepacia H111 cultured in a microfluidic device.

DNA is a component of biofilms, but the triggers of DNA release during biofilm formation and how DNA contributes to biofilm development are poorly investigated. One key mechanism involved in DNA release is explosive cell lysis, which is a consequence of prophage induction. In this article, the role of explosive cell lysis in biofilm formation was investigated in the opportunistic human pathogen Burkholderia cenocepacia H111 (H111). Biofilm streamers, flow-suspended biofilm filaments, were used as a biofilm model in this study, as DNA is an essential component of their matrix. H111 contains three prophages on chromosome 1 of its genome, and the involvement of each prophage in causing explosive cell lysis of the host and subsequent DNA and membrane vesicle (MV) release, as well as their contribution to streamer formation, were studied in the presence and absence of genotoxic stress. The results show that two of the three prophages of H111 encode functional lytic prophages that can be induced by genotoxic stress and their activation causes DNA and MVs release by explosive cell lysis. Furthermore, it is shown that the released DNA enables the strain to develop biofilm streamers, and streamer formation can be enhanced by genotoxic stress. Overall, this study demonstrates the involvement of prophages in streamer formation and uncovers an often-overlooked problem with the use of antibiotics that trigger the bacterial SOS response for the treatment of bacterial infections.

Tyramine, one quorum sensing inhibitor, reduces pathogenicity and restores tetracycline susceptibility in Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic respiratory pathogen of particular relevance to patients with cystic fibrosis (CF), primarily regulating its biological functions and virulence factors through two quorum sensing (QS) systems (CepI/R and CciI/R). The highly persistent incidence of multidrug resistant Burkholderia cenocepacia poses a global threat to public health. In this study, we investigated the effects of tyramine, one biogenic amine, on the QS systems of Burkholderia cenocepacia. Genetic and biochemical analyses revealed that tyramine inhibited the production of N-hexanoyl-homoserine (AHL) signaling molecules (C8-HSL and C6-HSL) by blocking the CepI/R and CciI/R systems. As a result, the inhibition of QS systems leads to reduced production of various virulence factors, such as biofilm formation, extracellular polysaccharides, lipase, and swarming motility. Notably, as a potential quorum sensing inhibitor, tyramine exhibits low toxicity in vivo in Galleria mellonella larvae and is well characterized by Lipinski's five rules. It also shows high gastrointestinal absorption and the ability to cross the blood-brain barrier according to SwissADME database and ProTox-II server. Additionally, tyramine was found to enhance the efficacy of tetracycline in reducing the infectivity of Burkholderia cenocepacia in Galleria mellonella larvae infection model. Therefore, tyramine could be a promising candidate for combination therapy with traditional antimicrobials to improve their effectiveness against Burkholderia cenocepacia.

Membrane-enclosed Pseudomonas quinolone signal attenuates bacterial virulence by interfering with quorum sensing.

Outer membrane vesicle (OMV)-delivered Pseudomonas quinolone signal (PQS) plays a critical role in cell-cell communication in Pseudomonas aeruginosa. However, the functions and mechanisms of membrane-enclosed PQS in interspecies communication in microbial communities are not clear. Here, we demonstrate that PQS delivered by both OMVs from P. aeruginosa and liposome reduces the competitiveness of Burkholderia cenocepacia, which usually shares the same niche in the lungs of cystic fibrosis patients, by interfering with quorum sensing (QS) in B. cenocepacia through the LysR-type regulator ShvR. Intriguingly, we found that ShvR regulates the production of the QS signals cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) by directly binding to the promoters of signal synthase-encoding genes. Perception of PQS influences the regulatory activity of ShvR and thus ultimately reduces QS signal production and virulence in B. cenocepacia. Our findings provide insights into the interspecies communication mediated by the membrane-enclosed QS signal among bacterial species residing in the same microbial community.IMPORTANCEQuorum sensing (QS) is a ubiquitous cell-to-cell communication mechanism. Previous studies showed that Burkholderia cenocepacia mainly employs cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) QS systems to regulate biological functions and virulence. Here, we demonstrate that Pseudomonas quinolone signal (PQS) delivered by outer membrane vesicles from Pseudomonas aeruginosa or liposome attenuates B. cenocepacia virulence by targeting the LysR-type regulator ShvR, which regulates the production of the QS signals BDSF and AHL in B. cenocepacia. Our results not only suggest the important roles of membrane-enclosed PQS in interspecies and interkingdom communications but also provide a new perspective on the use of functional nanocarriers loaded with QS inhibitors for treating pathogen infections.

Transcriptional Response of Burkholderia cenocepacia H111 to Severe Zinc Starvation.

Burkholderia cenocepacia is an opportunistic pathogen that is primarily associated with severe respiratory infections in people with cystic fibrosis. These bacteria have significant intrinsic resistance to antimicrobial therapy, and there is a need for more effective treatments. Bacterial zinc uptake and homeostasis systems are attractive targets for new drugs, yet our understanding of how bacteria acquire and utilise zinc remains incomplete. Here we have used RNA-sequencing and differential gene expression analysis to investigate how B. cenocepacia H111 is able to survive in zinc poor environments, such as those expected to be encountered within the host. The data shows that 201 genes are significantly differentially expressed when zinc supply is severely limited. Included in the 85 upregulated genes, are genes encoding a putative ZnuABC high affinity zinc importer, two TonB-dependent outer membrane receptors that may facilitate zinc uptake across the outer cell membrane, and a COG0523 family zinc metallochaperone. Amongst the 116 downregulated genes, are several zinc-dependent enzymes suggesting a mechanism of zinc sparring to reduce the cells demand for zinc when bioavailability is low.

Genomic features, antimicrobial susceptibility, and epidemiological insights into Burkholderia cenocepacia clonal complex 31 isolates from bloodstream infections in India.

Introduction: Burkholderia cepacia complex (Bcc) clonal complex (CC) 31, the predominant lineage causing devastating outbreaks globally, has been a growing concern of infections in non-cystic fibrosis (NCF) patients in India. B. cenocepacia is very challenging to treat owing to its virulence determinants and antibiotic resistance. Improving the management of these infections requires a better knowledge of their resistance patterns and mechanisms. Methods: Whole-genome sequences of 35 CC31 isolates obtained from patient samples, were analyzed against available 210 CC31 genomes in the NCBI database to glean details of resistance, virulence, mobile elements, and phylogenetic markers to study genomic diversity and evolution of CC31 lineage in India. Results: Genomic analysis revealed that 35 isolates belonging to CC31 were categorized into 11 sequence types (ST), of which five STs were reported exclusively from India. Phylogenetic analysis classified 245 CC31 isolates into eight distinct clades (I-VIII) and unveiled that NCF isolates are evolving independently from the global cystic fibrosis (CF) isolates forming a distinct clade. The detection rate of seven classes of antibiotic-related genes in 35 isolates was 35 (100%) for tetracyclines, aminoglycosides, and fluoroquinolones; 26 (74.2%) for sulphonamides and phenicols; 7 (20%) for beta-lactamases; and 1 (2.8%) for trimethoprim resistance genes. Additionally, 3 (8.5%) NCF isolates were resistant to disinfecting agents and antiseptics. Antimicrobial susceptibility testing revealed that majority of NCF isolates were resistant to chloramphenicol (77%) and levofloxacin (34%). NCF isolates have a comparable number of virulence genes to CF isolates. A well-studied pathogenicity island of B. cenocepacia, GI11 is present in ST628 and ST709 isolates from the Indian Bcc population. In contrast, genomic island GI15 (highly similar to the island found in B. pseudomallei strain EY1) is exclusively reported in ST839 and ST824 isolates from two different locations in India. Horizontal acquisition of lytic phage ST79 of pathogenic B. pseudomallei is demonstrated in ST628 isolates Bcc1463, Bcc29163, and BccR4654 amongst CC31 lineage. Discussion: The study reveals a high diversity of CC31 lineages among B. cenocepacia isolates from India. The extensive information from this study will facilitate the development of rapid diagnostic and novel therapeutic approaches to manage B. cenocepacia infections.

Identification of Burkholderia cenocepacia non-coding RNAs expressed during Caenorhabditis elegans infection.

Small non-coding RNAs (sRNAs) are key regulators of post-transcriptional gene expression in bacteria. Despite the identification of hundreds of bacterial sRNAs, their roles on bacterial physiology and virulence remain largely unknown, as is the case of bacteria of the Burkholderia cepacia complex (Bcc). Bcc is a group of opportunistic pathogens with relatively large genomes that can cause lethal lung infections amongst cystic fibrosis (CF) patients. To characterise sRNAs expressed by Bcc bacteria when infecting a host, the nematode Caenorhabditis elegans was used as an infection model by the epidemic CF strain B. cenocepacia J2315. A total of 108 new and 31 previously described sRNAs with a predicted Rho independent terminator were identified, most of them located on chromosome 1. RIT11b, a sRNA downregulated under C. elegans infection conditions, was shown to directly affect B. cenocepacia virulence, biofilm formation, and swimming motility. RIT11b overexpression reduced the expression of the direct targets dusA and pyrC, involved in biofilm formation, epithelial cell adherence, and chronic infections in other organisms. The in vitro direct interaction of RIT11b with the dusA and pyrC messengers was demonstrated by electrophoretic mobility shift assays. To the best of our knowledge this is the first report on the functional characterization of a sRNA directly involved in B. cenocepacia virulence. KEY POINTS: • 139 sRNAs expressed by B. cenocepacia during C. elegans infection were identified • The sRNA RIT11b affects B. cenocepacia virulence, biofilm formation, and motility • RIT11b directly binds to and regulates dusA and pyrC mRNAs.

The Small RNA NcS25 Regulates Biological Amine-Transporting Outer Membrane Porin BCAL3473 in Burkholderia cenocepacia.

Regulation of porin expression in bacteria is complex and often involves small-RNA regulators. Several small-RNA regulators have been described for Burkholderia cenocepacia, and this study aimed to characterize the biological role of the conserved small RNA NcS25 and its cognate target, outer membrane protein BCAL3473. The B. cenocepacia genome carries a large number of genes encoding porins with yet-uncharacterized functions. Expression of the porin BCAL3473 is strongly repressed by NcS25 and activated by other factors, such as a LysR-type regulator and nitrogen-depleted growth conditions. The porin is involved in transport of arginine, tyrosine, tyramine, and putrescine across the outer membrane. Porin BCAL3473, with NcS25 as a major regulator, plays an important role in the nitrogen metabolism of B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is a Gram-negative bacterium which causes infections in immunocompromised individuals and in people with cystic fibrosis. A low outer membrane permeability is one of the factors giving it a high level of innate resistance to antibiotics. Porins provide selective permeability for nutrients, and antibiotics can also traverse the outer membrane by this means. Knowing the properties and specificities of porin channels is therefore important for understanding resistance mechanisms and for developing new antibiotics and could help in overcoming permeability issues in antibiotic treatment.

Phage-antibiotic synergy reduces Burkholderia cenocepacia population.

BACKGROUND: Burkholderia cenocepacia is an opportunistic pathogen that can cause acute and chronic infections in patients with weakened immune systems and in patients with cystic fibrosis. B. cenocepacia is resistant to many antibiotics making treatment challenging. Consequently, there is a critical need for alternative strategies to treat B. cenocepacia infections such as using bacteriophages and/or bacteriophages with subinhibitory doses of antibiotic called phage-antibiotic synergy. RESULTS: We isolated a bacteriophage, KP1, from raw sewage that infects B. cenocepacia. Its morphological characteristics indicate it belongs in the family Siphoviridae, it has a 52 Kb ds DNA genome, and it has a narrow host range. We determined it rescued infections in Lemna minor (duckweed) and moderately reduced bacterial populations in our artificial sputum medium model. CONCLUSION: These results suggest that KP1 phage alone in the duckweed model or in combination with antibiotics in the ASMDM model improves the efficacy of reducing B. cenocepacia populations.

The Bep gene cluster in Burkholderia cenocepacia H111 codes for a water-insoluble exopolysaccharide essential for biofilm formation.

Burkholderia cenocepacia is an opportunistic pathogen isolated from cystic fibrosis patients where it causes infections that are extremely difficult to treat with antibiotics, and sometimes have a fatal outcome. Biofilm is a virulence trait of B. cenocepacia, and is associated with infection persistence and increased tolerance to antibiotics. In biofilms exopolysaccharides have an important role, conferring mechanical stability and antibiotic tolerance. Two different exopolysaccharides were isolated from B. cenocepacia H111 biofilms: a water-soluble polysaccharide rich in rhamnose and containing an L-Man residue, and a water-insoluble polymer made of glucose, galactose and mannose. In the present work, the product encoded by B. cenocepacia H111 bepA-L gene cluster was identified as the water-insoluble exopolysaccharide, using mutant strains and NMR spectroscopy of the purified polysaccharides. It was also demonstrated that the B. cenocepacia H111 wild type strain produces the water-insoluble exopolysaccharide in pellicles, thus underlining its potential importance in in vivo infections.

The BDSF quorum sensing receptor RpfR regulates Bep exopolysaccharide synthesis in Burkholderia cenocepacia via interaction with the transcriptional regulator BerB.

The polysaccharide Bep is essential for in vitro biofilm formation of the opportunistic pathogen Burkholderia cenocepacia. We found that the Burkholderia diffusible signaling factor (BDSF) quorum sensing receptor RpfR is a negative regulator of the bep gene cluster in B. cenocepacia. An rpfR mutant formed wrinkled colonies, whereas additional mutations in the bep genes or known bep regulators like berA and berB restored the wild-type smooth colony morphology. We found that there is a good correlation between intracellular c-di-GMP levels and bep expression when the c-di-GMP level is increased or decreased through ectopic expression of a diguanylate cyclase or a c-di-GMP phosphodiesterase, respectively. However, when the intracellular c-di-GMP level is changed by site directed mutagenesis of the EAL or GGDEF domain of RpfR there is no correlation between intracellular c-di-GMP levels and bep expression. Except for rpfR, deletion mutants of all 25 c-di-GMP phosphodiesterase and diguanylate cyclase genes encoded by B. cenocepacia showed no change to berA and bep gene expression. Moreover, bacterial two-hybrid assays provided evidence that RpfR and BerB physically interact and give specificity to the regulation of the bep genes. We suggest a model where RpfR binds BerB at low c-di-GMP levels to sequester this RpoN-dependent activator to an RpfR/RpfF complex. If the c-di-GMP levels rise, possibly by the enzymatic action of RpfR, BerB binds c-di-GMP and is released from the RpfR/RpfF complex and associates with RpoN to activate transcription of berA, and the BerA protein subsequently activates transcription of the bep genes.

Surface Motility Favors Codependent Interaction between Pseudomonas aeruginosa and Burkholderia cenocepacia.

Interactions between different bacterial species shape bacterial communities and their environments. The opportunistic pathogens Pseudomonas aeruginosa and Burkholderia cenocepacia both can colonize the lungs of individuals affected by cystic fibrosis. Using the social surface behavior called swarming motility as a study model, we noticed intricate interactions between B. cenocepacia K56-2 and P. aeruginosa PA14. While strain K56-2 does not swarm under P. aeruginosa favorable swarming conditions, co-inoculation with a nonmotile PA14 flagellum-less ΔfliC mutant restored spreading for both strains. We show that P. aeruginosa provides the wetting agent rhamnolipids allowing K56-2 to perform swarming motility, while aflagellated PA14 appears to "hitchhike" along with K56-2 cells in the swarming colony. IMPORTANCE Pseudomonas aeruginosa and Burkholderia cenocepacia are important opportunistic pathogens often found together in the airways of persons with cystic fibrosis. Laboratory cocultures of both species often ends with one taking over the other. We used a surface motility assay to study the social interactions between populations of these bacterial species. Under our conditions, B. cenocepacia cannot swarm without supplementation of the wetting agent produced by P. aeruginosa. In a mixed colony of both species, an aflagellated mutant of P. aeruginosa provides the necessary wetting agent to B. cenocepacia, allowing both bacteria to swarm and colonize a surface. We highlight this peculiar interaction where both bacteria set aside their antagonistic tendencies to travel together.

Oridonin Attenuates Burkholderia cenocepacia Virulence by Suppressing Quorum-Sensing Signaling.

Burkholderia cenocepacia is a human opportunistic pathogen that mostly employs two types of quorum-sensing (QS) systems to regulate its various biological functions and pathogenicity: the cis-2-dodecenoic acid (BDSF) system and the N-acyl homoserine lactone (AHL) system. In this study, we reported that oridonin, which was screened from a collection of natural products, disrupted important B. cenocepacia phenotypes, including motility, biofilm formation, protease production, and virulence. Genetic and biochemical analyses showed that oridonin inhibited the production of BDSF and AHL signals by decreasing the expression of their synthase-encoding genes. Furthermore, we revealed that oridonin directly binds to the regulator RqpR of the two-component system RqpSR that dominates the above-mentioned QS systems to inhibit the expression of the BDSF and AHL signal synthase-encoding genes. Oridonin also binds to the transcriptional regulator CepR of the cep AHL system to inhibit its binding to the promoter of bclACB. These findings suggest that oridonin could potentially be developed as a new QS inhibitor against pathogenic B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is an important human opportunistic pathogen that can cause life-threatening infections in susceptible individuals. It employs quorum-sensing (QS) systems to regulate biological functions and virulence. In this study, we have identified a lead compound, oridonin, that is capable of interfering with B. cenocepacia QS signaling and physiology. We demonstrate that oridonin suppressed cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) signal production and attenuated virulence in B. cenocepacia. Oridonin also impaired QS-regulated phenotypes in various Burkholderia species. These results suggest that oridonin could interfere with QS signaling in many Burkholderia species and might be developed as a new antibacterial agent.

The anti-virulence activity of the non-mevalonate pathway inhibitor FR900098 towards Burkholderia cenocepacia is maintained during experimental evolution.

Burkholderia cenocepacia infections are difficult to treat and there is an urgent need for alternative (combination) treatments. The use of anti-virulence therapies in combination with antibiotics is a possible strategy to increase the antimicrobial susceptibility of the pathogen and to slow down the development of resistance. In the present study we evaluated the ß-lactam and colistin-potentiating activity, and anti-virulence effect of the non-mevalonate pathway inhibitor FR900098 against B. cenocepacia in various in vitro and in vivo models. In addition, we evaluated whether repeated exposure to FR900098 alone or when combined with ceftazidime leads to increased resistance. FR900098 potentiated the activity of colistin and several ß-lactam antibiotics (aztreonam, cefepime, cefotaxime, ceftazidime, mecillinam and piperacillin) but not of imipenem and meropenem. When used alone or in combination with ceftazidime, FR900098 increased the survival of infected Galleria mellonella and Caenorhabditis elegans. Furthermore, combining ceftazidime with FR900098 resulted in a significant inhibition of the biofilm formation of B. cenocepacia. Repeated exposure to FR900098 in the C. elegans infection model did not lead to decreased activity, and the susceptibility of the evolved B. cenocepacia HI2424 lineages to ceftazidime, FR900098 and the combination of both remained unchanged. In conclusion, FR900098 reduces B. cenocepacia virulence and potentiates ceftazidime in an in vivo C. elegans model, and this activity is not lost during the experimental evolution experiment carried out in the present study.

The Burkholderia cenocepacia iron starvation σ factor, OrbS, possesses an on-board iron sensor.

Burkholderia cenocepacia is an opportunistic pathogen that causes severe infections of the cystic fibrosis (CF) lung. To acquire iron, B. cenocepacia secretes the Fe(III)-binding compound, ornibactin. Genes for synthesis and utilisation of ornibactin are served by the iron starvation (IS) extracytoplasmic function (ECF) σ factor, OrbS. Transcription of orbS is regulated in response to the prevailing iron concentration by the ferric uptake regulator (Fur), such that orbS expression is repressed under iron-sufficient conditions. Here we show that, in addition to Fur-mediated regulation of orbS, the OrbS protein itself responds to intracellular iron availability. Substitution of cysteine residues in the C-terminal region of OrbS diminished the ability to respond to Fe(II) in vivo. Accordingly, whilst Fe(II) impaired transcription from and recognition of OrbS-dependent promoters in vitro by inhibiting the binding of OrbS to core RNA polymerase (RNAP), the cysteine-substituted OrbS variant was less responsive to Fe(II). Thus, the cysteine residues within the C-terminal region of OrbS contribute to an iron-sensing motif that serves as an on-board 'anti-σ factor' in the presence of Fe(II). A model to account for the presence two regulators (Fur and OrbS) that respond to the same intracellular Fe(II) signal to control ornibactin synthesis and utilisation is discussed.

Protein with negative surface charge distribution, Bnr1, shows characteristics of a DNA-mimic protein and may be involved in the adaptation of Burkholderia cenocepacia.

Adaptation of opportunistic pathogens to their host environment requires reprogramming of a vast array of genes to facilitate survival in the host. Burkholderia cenocepacia, a Gram-negative bacterium with a large genome of ∼8 Mb that colonizes environmental niches, is exquisitely adaptable to the hypoxic environment of the cystic fibrosis lung and survives in macrophages. We previously identified an immunoreactive acidic protein encoded on replicon 3, BCAS0292. Deletion of the BCAS0292 gene significantly altered the abundance of 979 proteins by 1.5-fold or more; 19 proteins became undetectable while 545 proteins showed ≥1.5-fold reduced abundance, suggesting the BCAS0292 protein is a global regulator. Moreover, the ∆BCAS0292 mutant showed a range of pleiotropic effects: virulence and host-cell attachment were reduced, antibiotic susceptibility was altered, and biofilm formation enhanced. Its growth and survival were impaired in 6% oxygen. In silico prediction of its three-dimensional structure revealed BCAS0292 presents a dimeric ß-structure with a negative surface charge. The ΔBCAS0292 mutant displayed altered DNA supercoiling, implicated in global regulation of gene expression. Three proteins were identified in pull-downs with FLAG-tagged BCAS0292, including the Histone H1-like protein, HctB, which is recognized as a global transcriptional regulator. We propose that BCAS0292 protein, which we have named Burkholderia negatively surface-charged regulatory protein 1 (Bnr1), acts as a DNA-mimic and binds to DNA-binding proteins, altering DNA topology and regulating the expression of multiple genes, thereby enabling the adaptation of B. cenocepacia to highly diverse environments.

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.

Characterization of glyphosate-resistant Burkholderia anthina and Burkholderia cenocepacia isolates from a commercial Roundup® solution.

Roundup® is the brand name for herbicide solutions containing glyphosate, which specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase of the shikimate pathway. The inhibition of the EPSP synthase causes plant death because EPSP is required for biosynthesis of aromatic amino acids. Glyphosate also inhibits the growth of archaea, bacteria, Apicomplexa, algae and fungi possessing an EPSP synthase. Here, we have characterized two glyphosate-resistant bacteria from a Roundup solution. Taxonomic classification revealed that the isolates 1CH1 and 2CH1 are Burkholderia anthina and Burkholderia cenocepacia strains respectively. Both isolates cannot utilize glyphosate as a source of phosphorus and synthesize glyphosate-sensitive EPSP synthase variants. Burkholderia. anthina 1CH1 and B. cenocepacia 2CH1 tolerate high levels of glyphosate because the herbicide is not taken up by the bacteria. Previously, it has been observed that the exposure of soil bacteria to herbicides like glyphosate promotes the development of antibiotic resistances. Antibiotic sensitivity testing revealed that the only the B. cenocepacia 2CH1 isolate showed increased resistance to a variety of antibiotics. Thus, the adaptation of B. anthina 1CH1 and B. cenocepacia 2CH1 to glyphosate did not generally increase the antibiotic resistance of both bacteria. However, our study confirms the genomic adaptability of bacteria belonging to the genus Burkholderia.