To streamline the elucidation of antibacterial compounds’ mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing.

Burkholderia cenocepacia K56-2 belongs to the Burkholderia cepacia complex, a group of Gram-negative opportunistic pathogens that have large and dynamic genomes. In this work, we identified the essential genome of B. cenocepacia K56-2 using high-density transposon mutagenesis and insertion site sequencing (Tn-seq circle). We constructed a library of one million transposon mutants and identified the transposon insertions at an average of one insertion per 27 bp. The probability of gene essentiality was determined by comparing of the insertion density per gene with the variance of neutral datasets generated by Monte Carlo simulations. Five hundred and eight genes were not significantly disrupted, suggesting that these genes are essential for survival in rich, undefined medium. Comparison of the B. cenocepacia K56-2 essential genome with that of the closely related B. cenocepacia J2315 revealed partial overlapping, suggesting that some essential genes are strain-specific. Furthermore, 158 essential genes were conserved in B. cenocepacia and two species belonging to the Burkholderia pseudomallei complex, B. pseudomallei K96243 and Burkholderia thailandensis E264. Porins, including OpcC, a lysophospholipid transporter, LplT, and a protein involved in the modification of lipid A with aminoarabinose were found to be essential in Burkholderia genomes but not in other bacterial essential genomes identified so far. Our results highlight the existence of cell envelope processes that are uniquely essential in species of the genus Burkholderia for which the essential genomes have been identified by Tn-seq.

Identification of essential genes by construction of conditional knockouts with inducible promoters allows the identification of essential genes and creation of conditional growth (CG) mutants that are then available as genetic tools for further studies. We used large-scale transposon delivery of the rhamnose-inducible promoter, PrhaB followed by robotic screening of rhamnose-dependent growth to construct a genomic library of 106 Burkholderia cenocepacia CG mutants. Transposon insertions were found where PrhaB was in the same orientation of widely conserved, well-characterized essential genes as well as genes with no previous records of essentiality in other microorganisms. Using previously reported global gene-expression analyses, we demonstrate that PrhaB can achieve the wide dynamic range of expression levels required for essential genes when the promoter is delivered randomly and mutants with rhamnose-dependent growth are selected. We also show specific detection of the target of an antibiotic, novobiocin, by enhanced sensitivity of the corresponding CG mutant (PrhaB controlling gyrB expression) within the library. Modulation of gene expression to achieve 30–60% of wild-type growth created conditions for specific hypersensitivity demonstrating the value of the CG mutant library for chemogenomic experiments. In summary, CG mutants can be obtained on a large scale by random delivery of a tightly regulated inducible promoter into the bacterial chromosome followed by a simple screening for the CG phenotype, without previous information on gene essentiality.

Chemogenetic approaches to profile an antibiotic mode of action are based on detecting differential sensitivities of engineered bacterial strains in which the antibacterial target (usually encoded by an essential gene) or an associated process is regulated. We previously developed an essential-gene knockdown mutant library in the multidrug-resistant Burkholderia cenocepacia by transposon delivery of a rhamnose-inducible promoter. In this work, we used Illumina sequencing of multiplex-PCR-amplified transposon junctions to track individual mutants during pooled growth in the presence of antibiotics. We found that competition from nontarget mutants magnified the hypersensitivity of a clone underexpressing gyrB to novobiocin by 8-fold compared with hypersensitivity measured during clonal growth. Additional profiling of various antibiotics against a pilot library representing most categories of essential genes revealed a two-component system with unknown function, which, upon depletion of the response regulator, sensitized B. cenocepacia to novobiocin, ciprofloxacin, tetracycline, chloramphenicol, kanamycin, meropenem, and carbonyl cyanide 3-chlorophenylhydrazone, but not to colistin, hydrogen peroxide, and dimethyl sulfoxide. We named the gene cluster esaSR for enhanced sensitivity to antibiotics sensor and response regulator. Mutational analysis and efflux activity assays revealed that while esaS is not essential and is involved in antibiotic-induced efflux, esaR is an essential gene and regulates efflux independently of antibiotic-mediated induction. Furthermore, microscopic analysis of cells stained with propidium iodide provided evidence that depletion of EsaR has a profound effect on the integrity of cell membranes. In summary, we unraveled a previously uncharacterized two-component system that can be targeted to reduce antibiotic resistance in B. cenocepacia.

Pseudomonas brassicacearum is one of over fifty species of bacteria classified into the P. fluorescens group. Generally considered a harmless commensal, these bacteria are studied for their plant-growth promotion (PGP) and biocontrol characteristics. Intriguingly, P. brassicacearum is closely related to P. corrugata , which is classified as an opportunistic phytopathogen. Twenty-one P. brassicacearum genomes have been sequenced to date. In the current review, genomes of P. brassicacearum and strains from the P. corrugata clade were mined for regions associated with PGP, biocontrol and pathogenicity. We discovered that ‘beneficial’ bacteria and those classified as plant pathogens have many genes in common; thus, only a fine line separates beneficial/harmless commensals from those capable of causing disease in plants. The genotype and physiological state of the plant, the presence of biotic/abiotic stressors, and the ability of bacteria to manipulate the plant immune system collectively contribute to how the bacterial-plant interaction plays out. Because production of extracellular metabolites is energetically costly, these compounds are expected to impart a fitness advantage to the producer. P. brassicacearum is able to reduce the threat of nematode predation through release of metabolites involved in biocontrol. Moreover this bacterium has the unique ability to form biofilms on the head of Caenorhabditis elegans, as a second mechanism of predator avoidance. Rhizobacteria, plants, fungi, and microfaunal predators have occupied a shared niche for millions of years and, in many ways, they function as a single organism. Accordingly, it is essential that we appreciate the dynamic interplay among these members of the community.

The increasing emergence of antimicrobial multiresistant bacteria is of great concern to public health. While these bacteria are becoming an ever more prominent cause of nosocomial and community-acquired infections worldwide, the antibiotic discovery pipeline has been stalled in the last few years with very few efforts in the research and development of novel antibacterial therapies. Some of the root causes that have hampered current antibiotic drug development are the lack of understanding of the mode of action (MOA) of novel antibiotic molecules and the poor characterization of the bacterial physiological response to antibiotics that ultimately causes resistance. Here, we review how bacterial genetic tools can be applied at the genomic level with the goal of profiling resistance to antibiotics and elucidating antibiotic MOAs. Specifically, we highlight how chemical genomic detection of the MOA of novel antibiotic molecules and antibiotic profiling by next-generation sequencing are leveraging basic antibiotic research to unprecedented levels with great opportunities for knowledge translation.

Pseudomonas chlororaphis PA23 is a biocontrol agent that, in addition to producing antifungal compounds, synthesizes polyhydroxyalkanoate (PHA) polymers as a carbon and energy sink. Quorum sensing (QS) and the anaerobic regulator (ANR) are required for PA23-mediated fungal suppression; however, the role of these regulators in PHA production is unknown. Strains lacking either QS or ANR accumulated less PHA polymers when propagated on Ramsay's Minimal Medium (RMM) with glucose or octanoate as the carbon source. In the AHL-deficient background, all six of the genes in the pha locus (phaC1, phaC2, phaZ, phaD, phaF, phaI) showed reduced expression in RMM-glucose, and all except phaC2 were repressed in RMM-octanoate. While changes in gene activity were observed in the anr mutant, they were less pronounced. Analysis of the promoter regions for QS- and ANR-binding consensus sequences revealed putative phz-boxes upstream of phaZ and phaI, but no anr-boxes were identified. Our findings indicate that altered pha gene expression likely contributes to the lower PHA accumulation in the QS- and ANR-deficient strains, which may be in part indirectly mediated. This study is the first to show that mcl-PHA production is under QS and ANR control.

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum. PA23 produces several inhibitory compounds that are under control of a complex regulatory network. Included in this cascade is the PhzRI quorum sensing (QS) system, which plays an essential role in PA23 biocontrol, as well as CsaRI and AurRI, which have not yet been characterized in PA23. The focus of the current study was to employ RNA sequencing to explore the spectrum of PA23 genes under QS control. In this work, we investigated genes under the control of the main QS transcriptional regulator, PhzR, as well as those differentially expressed in an AHL-deficient strain, PA23-6863, which constitutively expresses an AiiA lactonase, rendering the strain QS defective. Transcriptomic profiling revealed 545 differentially expressed genes (365 downregulated; 180 upregulated) in the phzR mutant and 534 genes (382 downregulated; 152 upregulated) in the AHL-deficient PA23-6863. In both strains, decreased expression of phenazine, pyrrolnitrin, and exoprotease biosynthetic genes was observed. We have previously reported that QS activates expression of these genes and their encoded products. In addition, elevated siderophore and decreased chitinase gene expression was observed in the QS-deficient stains, which was confirmed by phenotypic analysis. Inspection of the promoter regions revealed the presence of "phz-box" sequences in only 58 of the 807 differentially expressed genes, suggesting that much of the QS regulon is indirectly regulated. Consistent with this notion, 41 transcriptional regulators displayed altered expression in one or both of the QS-deficient strains. Collectively, our findings indicate that QS governs expression of approximately 13% of the PA23 genome affecting diverse functions ranging from secondary metabolite production to general metabolism.