Shedding light on bacteria-host interactions with the aid of TnSeq approaches.

Bacteria are highly adaptable and grow in diverse niches, where they often interact with eukaryotic organisms. These interactions with different hosts span the entire spectrum from symbiosis to pathogenicity and thus determine the lifestyle of the bacterium. Knowledge of the genetic determinants involved in animal and plant host colonization by pathogenic and mutualistic bacteria is not only crucial to discover new drug targets for disease management but also for developing novel biostimulant strategies. In the last decades, significant progress in genome-wide high-throughput technologies such as transposon insertion sequencing has led to the identification of pathways that enable efficient host colonization. However, the extent to which similar genes play a role in this process in different bacteria is yet unclear. This review highlights the commonalities and specificities of bacterial determinants important for bacteria-host interaction.

RNA-seq analysis in simulated microgravity unveils down-regulation of the beta-rhizobial siderophore phymabactin.

Exploiting the symbiotic interaction between crops and nitrogen-fixing bacteria is a simple and ecological method to promote plant growth in prospective extraterrestrial human outposts. In this study, we performed an RNA-seq analysis to investigate the adaptation of the legume symbiont Paraburkholderia phymatum STM815T to simulated microgravity (s0-g) at the transcriptome level. The results revealed a drastic effect on gene expression, with roughly 23% of P. phymatum genes being differentially regulated in s0-g. Among those, 951 genes were upregulated and 858 downregulated in the cells grown in s0-g compared to terrestrial gravity (1 g). Several genes involved in posttranslational modification, protein turnover or chaperones encoding were upregulated in s0-g, while those involved in translation, ribosomal structure and biosynthesis, motility or inorganic ions transport were downregulated. Specifically, the whole phm gene cluster, previously bioinformatically predicted to be involved in the production of a hypothetical malleobactin-like siderophore, phymabactin, was 20-fold downregulated in microgravity. By constructing a mutant strain (ΔphmJK) we confirmed that the phm gene cluster codes for the only siderophore secreted by P. phymatum as assessed by the complete lack of iron chelating activity of the P. phymatum ΔphmJK mutant on chrome azurol S (CAS) agar plates. These results not only provide a deeper understanding of the physiology of symbiotic organisms exposed to space-like conditions, but also increase our knowledge of iron acquisition mechanisms in rhizobia.

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.

A VirB4 ATPase of the mobile accessory genome orchestrates core genome-encoded features of physiology, metabolism, and virulence of Pseudomonas aeruginosa TBCF10839.

Pseudomonas aeruginosa TBCF10839 is a highly virulent strain that can persist and replicate in human neutrophils. Screening of a signature-tagged mutagenesis (STM) TBCF10839 transposon library in phagocytosis tests identified a mutant that carried the transposon in the VirB4 homolog 5PG21 of an integrative and conjugative element (ICE)-associated type IV secretion system of the pKLC102 subtype. 5P21 TBCF10839 insertion mutants were deficient in metabolic versatility, secretion, quorum sensing, and virulence. The mutants were efficiently killed in phagocytosis tests in vitro and were avirulent in an acute murine airway infection model in vivo. The inactivation of 5PG21 silenced the rhl, las, and pqs operons and the gene expression for the synthesis of hydrogen cyanide, the antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid, and the H2- and H3-type VI secretion systems and their associated effectors. The mutants were impaired in the utilization of carbon sources and stored compounds that are not funneled into intermediary metabolism. This showcase demonstrates that a single gene of the mobile accessory genome can become an essential element to operate the core genome-encoded features of metabolism and virulence.

Paraburkholderia sabiae Uses One Type VI Secretion System (T6SS-1) as a Powerful Weapon against Notorious Plant Pathogens.

Paraburkholderia sabiae LMG24235 is a nitrogen-fixing betaproteobacterium originally isolated from a root nodule of Mimosa caesalpiniifolia in Brazil. We show here that this strain effectively kills strains from several bacterial families (Burkholderiaceae, Pseudomonadaceae, Enterobacteriaceae) which include important plant pathogens in a contact-dependent manner. De novo assembly of the first complete genome of P. sabiae using long sequencing reads and subsequent annotation revealed two gene clusters predicted to encode type VI secretion systems (T6SS), which we named T6SS-1 and T6SS-3 according to previous classification methods (G. Shalom, J. G. Shaw, and M. S. Thomas, Microbiology, 153:2689-2699, 2007, https://doi.org/10.1099/mic.0.2007/006585-0). We created P. sabiae with mutations in each of the two T6SS gene clusters that abrogated their function, and the T6SS-1 mutant was no longer able to outcompete other strains in a contact-dependent manner. Notably, our analysis revealed that T6SS-1 is essential for competition against several important plant pathogens in vitro, including Burkholderia plantarii, Ralstonia solanacearum, Pseudomonas syringae, and Pectobacterium carotovorum. The 9-log reduction in P. syringae cells in the presence of P. sabiae was particularly remarkable. Importantly, in an in vivo assay, P. sabiae was able to protect potato tubers from bacterial soft rot disease caused by P. carotovorum, and this protection was partly dependent on T6SS-1. IMPORTANCE Rhizobia often display additional beneficial traits such as the production of plant hormones and the acquisition of limited essential nutrients that improve plant growth and enhance plant yields. Here, we show that the rhizobial strain P. sabiae antagonizes important phytopathogens such as P. carotovorum, P. syringae, and R. solanacearum and that this effect is due to contact-dependent killing mediated by one of two T6SS systems identified in the complete, de novo assembled genome sequence of P. sabiae. Importantly, co-inoculation of Solanum tuberosum tubers with P. sabiae also resulted in a drastic reduction of soft rot caused by P. carotovorum in an in vivo model system. This result highlights the protective potential of P. sabiae against important bacterial plant diseases, which makes it a valuable candidate for application as a biocontrol agent. It also emphasizes the particular potential of rhizobial inoculants that combine several beneficial effects such as plant growth promotion and biocontrol for sustainable agriculture.

Allylic Carbocyclic Inhibitors Covalently Bind Glycoside Hydrolases.

Allylic cyclitols were investigated as covalent inhibitors of glycoside hydrolases by chemical, enzymatic, proteomic, and computational methods. This approach was inspired by the C7 cyclitol natural product streptol glucoside, which features a potential carbohydrate leaving group in the 4-position (carbohydrate numbering). To test this hypothesis, carbocyclic inhibitors with leaving groups in the 4- and 6- positions were prepared. The results of enzyme kinetics analyses demonstrated that dinitrophenyl ethers covalently inhibit α-glucosidases of the GH13 family without reactivation. The labeled enzyme was studied by proteomics, and the active site residue Asp214 was identified as modified. Additionally, computational studies, including enzyme homology modeling and density functional theory (DFT) calculations, further delineate the electronic and structural requirements for activity. This study demonstrates that previously unexplored 4- and 6-positions can be exploited for successful inhibitor design.

Composition and functions of bacterial membrane vesicles.

Extracellular vesicles are produced by species across all domains of life, suggesting that vesiculation represents a fundamental principle of living matter. In Gram-negative bacteria, membrane vesicles (MVs) can originate either from blebs of the outer membrane or from endolysin-triggered explosive cell lysis, which is often induced by genotoxic stress. Although less is known about the mechanisms of vesiculation in Gram-positive and Gram-neutral bacteria, recent research has shown that both lysis and blebbing mechanisms also exist in these organisms. Evidence has accumulated over the past years that different biogenesis routes lead to distinct types of MV with varied structure and composition. In this Review, we discuss the different types of MV and their potential cargo packaging mechanisms. We summarize current knowledge regarding how MV composition determines their various functions including support of bacterial growth via the disposal of waste material, nutrient scavenging, export of bioactive molecules, DNA transfer, neutralization of phages, antibiotics and bactericidal functions, delivery of virulence factors and toxins to host cells and inflammatory and immunomodulatory effects. We also discuss the advantages of MV-mediated secretion compared with classic bacterial secretion systems and we introduce the concept of quantal secretion.

In Situ Investigation of Pseudomonas aeruginosa Biofilm Development: Interplay between Flow, Growth Medium, and Mechanical Properties of Substrate.

To better understand the impact of biomaterial mechanical properties and growth medium on bacterial adhesion and biofilm formation under flow, we investigated the biofilm formation ability of Pseudomonas aeruginosa in different media on polydimethylsiloxane (PDMS) of different stiffness in real time using a microfluidic platform. P. aeruginosa colonization was recorded with optical microscopy and automated image analysis. The bacterial intracellular level of cyclic diguanylate (c-di-GMP), which regulates biofilm formation, was monitored using the transcription of the putative adhesin gene (cdrA) as a proxy. Contrary to the previous supposition, we revealed that PDMS material stiffness within the tested range has negligible impact on biofilm development and biofilm structures, whereas culture media not only influence the kinetics of biofilm development but also affect the biofilm morphology and structure dramatically. Interestingly, magnesium rather than previously reported calcium was identified here to play a decisive role in the formation of dense P. aeruginosa aggregates and high levels of c-di-GMP. These results demonstrate that although short-term adhesion assays bring valuable insight into bacterial and material interactions, long-term evaluations are essential to better predict overall biofilm outcome. The microfluidic system developed here presents a valuable application potential for studying biofilm development in situ. .

Investigating the Biocontrol Potential of the Natural Microbiota of the Apple Blossom.

Erwinia amylovora, the causative agent of fire blight, leads to important economic losses of apple and pear crops worldwide. This study aimed to investigate the potential of the resident microbiota of the apple blossom in combatting plant disease-causing organisms, with a focus on controlling fire blight. We obtained 538 isolates from sites around Canton Zurich, which we tested for activity against Pectobacterium carotovorum and E. amylovora. We also evaluated the isolates' activity against oomycete and fungal pathogens. Nine isolates showed activity against P. carotovorum, and eight of these against E. amylovora. Furthermore, 117 showed antifungal, and 161 anti-oomycete, activity. We assigned genera and in some cases species to 238 of the isolates by sequencing their 16S RNA-encoding gene. Five strains showed activity against all pathogens and were tested in a detached apple model for anti-E. amylovora activity. Of these five strains, two were able to antagonize E. amylovora, namely Bacillus velezensis #124 and Pantoea agglomerans #378. We sequenced the P. agglomerans #378 genome and analyzed it for secondary metabolite clusters using antiSMASH, revealing the presence of a putative bacteriocin cluster. We also showed that B. velezensis #124 exhibits strong activity against three different fungi and two oomycetes in vitro, suggesting a broader capacity for biocontrol. Our results showcase the protective potential of the natural apple blossom microbiota. We isolated two candidate biocontrol strains from apple blossoms, suggesting that they might persist at the most common entry point for the causative agent of fire blight. Furthermore, they are probably already part of the human diet, suggesting they might be safe for consumption, and thus are promising candidates for biocontrol applications.

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.

A novel function of the key nitrogen-fixation activator NifA in beta-rhizobia: Repression of bacterial auxin synthesis during symbiosis.

Rhizobia fix nitrogen within root nodules of host plants where nitrogenase expression is strictly controlled by its key regulator NifA. We recently discovered that in nodules infected by the beta-rhizobial strain Paraburkholderia phymatum STM815, NifA controls expression of two bacterial auxin synthesis genes. Both the iaaM and iaaH transcripts, as well as the metabolites indole-acetamide (IAM) and indole-3-acetic acid (IAA) showed increased abundance in nodules occupied by a nifA mutant compared to wild-type nodules. Here, we document the structural changes that a P. phymatum nifA mutant induces in common bean (Phaseolus vulgaris) nodules, eventually leading to hypernodulation. To investigate the role of the P. phymatum iaaMH genes during symbiosis, we monitored their expression in presence and absence of NifA over different stages of the symbiosis. The iaaMH genes were found to be under negative control of NifA in all symbiotic stages. While a P. phymatum iaaMH mutant produced the same number of nodules and nitrogenase activity as the wild-type strain, the nifA mutant produced more nodules than the wild-type that clustered into regularly-patterned root zones. Mutation of the iaaMH genes in a nifA mutant background reduced the presence of these nodule clusters on the root. We further show that the P. phymatum iaaMH genes are located in a region of the symbiotic plasmid with a significantly lower GC content and exhibit high similarity to two genes of the IAM pathway often used by bacterial phytopathogens to deploy IAA as a virulence factor. Overall, our data suggest that the increased abundance of rhizobial auxin in the non-fixing nifA mutant strain enables greater root infection rates and a role for bacterial auxin production in the control of early stage symbiotic interactions.

Pseudomonas putida mediates bacterial killing, biofilm invasion and biocontrol with a type IVB secretion system.

Many bacteria utilize contact-dependent killing machineries to eliminate rivals in their environmental niches. Here we show that the plant root colonizer Pseudomonas putida strain IsoF is able to kill a wide range of soil and plant-associated Gram-negative bacteria with the aid of a type IVB secretion system (T4BSS) that delivers a toxic effector into bacterial competitors in a contact-dependent manner. This extends the range of targets of T4BSSs-so far thought to transfer effectors only into eukaryotic cells-to prokaryotes. Bioinformatic and genetic analyses showed that this killing machine is entirely encoded by the kib gene cluster located within a rare genomic island, which was recently acquired by horizontal gene transfer. P. putida IsoF utilizes this secretion system not only as a defensive weapon to kill bacterial competitors but also as an offensive weapon to invade existing biofilms, allowing the strain to persist in its natural environment. Furthermore, we show that strain IsoF can protect tomato plants against the phytopathogen Ralstonia solanacearum in a T4BSS-dependent manner, suggesting that IsoF can be exploited for pest control and sustainable agriculture.

Identification of Key Factors for Anoxic Survival of B. cenocepacia H111.

Burkholderia cenocepacia is an opportunistic pathogen that can lead to severe infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Being an obligate aerobe, B. cenocepacia is unable to grow in the absence of oxygen. In this study, we show that the CF isolate B. cenocepacia H111 can survive in the absence of oxygen. Using a transposon sequencing (Tn-seq) approach, we identified 71 fitness determinants involved in anoxic survival, including a Crp-Fnr family transcriptional regulatory gene (anr2), genes coding for the sensor kinase RoxS and its response regulator RoxR, the sigma factor for flagella biosynthesis (FliA) and subunits of a cytochrome bd oxidase (CydA, CydB and the potentially novel subunit CydP). Individual knockouts of these fitness determinants significantly reduced anoxic survival, and inactivation of both anr copies is shown to be lethal under anoxic conditions. We also show that the two-component system RoxS/RoxR and FliA are important for virulence and swarming/swimming, respectively.

The structural role of bacterial eDNA in the formation of biofilm streamers.

Across diverse habitats, bacteria are mainly found as biofilms, surface-attached communities embedded in a self-secreted matrix of extracellular polymeric substances (EPS), which enhance bacterial recalcitrance to antimicrobial treatment and mechanical stresses. In the presence of flow and geometric constraints such as corners or constrictions, biofilms can take the form of long, suspended filaments (streamers), which bear important consequences in industrial and clinical settings by causing clogging and fouling. The formation of streamers is thought to be driven by the viscoelastic nature of the biofilm matrix. Yet, little is known about the structural composition of streamers and how it affects their mechanical properties. Here, using a microfluidic platform that allows growing and precisely examining biofilm streamers, we show that extracellular DNA (eDNA) constitutes the backbone and is essential for the mechanical stability of Pseudomonas aeruginosa streamers. This finding is supported by the observations that DNA-degrading enzymes prevent the formation of streamers and clear already formed ones and that the antibiotic ciprofloxacin promotes their formation by increasing the release of eDNA. Furthermore, using mutants for the production of the exopolysaccharide Pel, an important component of P. aeruginosa EPS, we reveal an concurring role of Pel in tuning the mechanical properties of the streamers. Taken together, these results highlight the importance of eDNA and of its interplay with Pel in determining the mechanical properties of P. aeruginosa streamers and suggest that targeting the composition of streamers can be an effective approach to control the formation of these biofilm structures.

The role of peptidoglycan hydrolases in the formation and toxicity of Pseudomonas aeruginosa membrane vesicles.

Bacterial membrane vesicles (MVs) have been reported to kill other bacteria. In the case of Pseudomonas aeruginosa the bactericidal activity has been attributed to an unidentified 26 kDa peptidoglycan (PG) hydrolase that is associated with MVs and gives rise to a lytic band on zymograms using murein sacculi as substrate. In this study, we employed a proteomics approach to show that this PG hydrolase is the AmphD3 amidase. The analysis of an amphD3 mutant as well as of an AmphD3 overexpression derivative revealed that this enzyme is not required for the bactericidal activity of P. aeruginosa MVs but is involved in cell wall recycling and thus protects the cell against PG damage. Another 23 kDa PG hydrolase, which we observed on zymograms of SOS-induced MVs, was identified as the endolysin Lys, which triggers explosive cell lysis but is shown to be dispensable for MV-mediated killing. We conclude that the lytic activities observed on zymograms do not correlate with the bactericidal potential of MVs. We demonstrate that P. aeruginosa MVs are enriched for several autolysins, suggesting that the predatory activity of MVs depends on the combined action of different murein hydrolases.

Identification of genes required for gold and silver tolerance in Burkholderia cenocepacia H111 by transposon sequencing.

Members of the genus Burkholderia show remarkable abilities to adapt to a wide range of environmental conditions and is frequently isolated from soils contaminated with heavy metals. In this study, we used a transposon sequencing approach to identify 138 and 164 genes that provide a benefit for growth of the opportunistic pathogen Burkholderia cenocepacia H111 in the presence of silver and gold ions respectively. The data suggest that arginine metabolism and citrate biosynthesis are important for silver tolerance, while components of an ABC transporter (BCAL0307-BCAL0308) and de novo cysteine biosynthesis are required for tolerance to gold ions. We show that determinants that affect tolerance to both metal ions include the two-component systems BCAL0497/99 and BCAL2830/31 and genes that are involved in maintaining the integrity of the cell envelope, suggesting that membrane proteins represent important targets of silver and gold ions. Furthermore, we show that that the P-type ATPase CadA (BCAL0055), which confers tolerance to cadmium contributes to silver but not gold tolerance. Our results may be useful for improving the antibacterial effect of silver and gold ions to combat drug-resistant pathogens.

Leaf nodule endosymbiotic Burkholderia confer targeted allelopathy to their Psychotria hosts.

After a century of investigations, the function of the obligate betaproteobacterial endosymbionts accommodated in leaf nodules of tropical Rubiaceae remained enigmatic. We report that the α-D-glucose analogue (+)-streptol, systemically supplied by mature Ca. Burkholderia kirkii nodules to their Psychotria hosts, exhibits potent and selective root growth inhibiting activity. We provide compelling evidence that (+)-streptol specifically affects meristematic root cells transitioning to anisotropic elongation by disrupting cell wall organization in a mechanism of action that is distinct from canonical cellulose biosynthesis inhibitors. We observed no inhibitory or cytotoxic effects on organisms other than seed plants, further suggesting (+)-streptol as a bona fide allelochemical. We propose that the suppression of growth of plant competitors is a major driver of the formation and maintenance of the Psychotria-Burkholderia association. In addition to potential agricultural applications as a herbicidal agent, (+)-streptol might also prove useful to dissect plant cell and organ growth processes.

Mitigation of Pseudomonas syringae virulence by signal inactivation.

Pseudomonas syringae is an important plant pathogen of many valuable crops worldwide, with more than 60 identified pathovars. The phytotoxins produced by these organisms were related to the severity of the damage caused to the plant. An emerging strategy to treat bacterial infections relies on interference with their signaling systems. In this study, we investigated P. syringae pv. syringae, which produces the virulence factor mangotoxin that causes bacterial apical necrosis on mango leaves. A previously unknown signaling molecule named leudiazen was identified, determined to be unstable and volatile, and responsible for mangotoxin production. A strategy using potassium permanganate, compatible with organic farming, was developed to degrade leudiazen and thus to attenuate the pathogenicity of P. syringae pv. syringae.

Bacterial surface properties influence the activity of the TAT-RasGAP317-326 antimicrobial peptide.

Antibiotic resistance is an increasing threat for public health, underscoring the need for new antibacterial agents. Antimicrobial peptides (AMPs) represent an alternative to classical antibiotics. TAT-RasGAP317-326 is a recently described AMP effective against a broad range of bacteria, but little is known about the conditions that may influence its activity. Using RNA-sequencing and screening of mutant libraries, we show that Escherichia coli and Pseudomonas aeruginosa respond to TAT-RasGAP317-326 by regulating metabolic and stress response pathways, possibly implicating two-component systems. Our results also indicate that bacterial surface properties, in particular integrity of the lipopolysaccharide layer, influence peptide binding and entry. Finally, we found differences between bacterial species with respect to their rate of resistance emergence against this peptide. Our findings provide the basis for future investigation on the mode of action of TAT-RasGAP317-326, which may help developing antimicrobial treatments based on this peptide.