Coordinated surface activities in Variovorax paradoxus EPS

<p>Abstract</p> <p>Background</p> <p><it>Variovorax paradoxus </it>is an aerobic soil bacterium frequently associated with important biodegradative processes in nature. Our group has cultivated a mucoid strain of <it>Variovorax paradoxus </it>for study as a model of bacterial development and response to environmental conditions. Colonies of this organism vary widely in appearance depending on agar plate type.</p> <p>Results</p> <p>Surface motility was observed on minimal defined agar plates with 0.5% agarose, similar in nature to swarming motility identified in <it>Pseudomonas aeruginosa </it>PAO1. We examined this motility under several culture conditions, including inhibition of flagellar motility using Congo Red. We demonstrated that the presence of a wetting agent, mineral, and nutrient content of the media altered the swarming phenotype. We also demonstrated that the wetting agent reduces the surface tension of the agar. We were able to directly observe the presence of the wetting agent in the presence and absence of Congo Red, and found that incubation in a humidified chamber inhibited the production of wetting agent, and also slowed the progression of the swarming colony. We observed that swarming was related to both carbon and nitrogen sources, as well as mineral salts base. The phosphate concentration of the mineral base was critical for growth and swarming on glucose, but not succinate. Swarming on other carbon sources was generally only observed using M9 salts mineral base. Rapid swarming was observed on malic acid, d-sorbitol, casamino acids, and succinate. Swarming at a lower but still detectable rate was observed on glucose and sucrose, with weak swarming on maltose. Nitrogen source tests using succinate as carbon source demonstrated two distinct forms of swarming, with very different macroscopic swarm characteristics. Rapid swarming was observed when ammonium ion was provided as nitrogen source, as well as when histidine, tryptophan, or glycine was provided. Slower swarming was observed with methionine, arginine, or tyrosine. Large effects of mineral content on swarming were seen with tyrosine and methionine as nitrogen sources. Biofilms form readily under various culture circumstances, and show wide variance in structure under different conditions. The amount of biofilm as measured by crystal violet retention was dependent on carbon source, but not nitrogen source. Filamentous growth in the biofilm depends on shear stress, and is enhanced by continuous input of nutrients in chemostat culture.</p> <p>Conclusion</p> <p>Our studies have established that the beta-proteobacterium <it>Variovorax paradoxus </it>displays a number of distinct physiologies when grown on surfaces, indicative of a complex response to several growth parameters. We have identified a number of factors that drive sessile and motile surface phenotypes. This work forms a basis for future studies using this genetically tractable soil bacterium to study the regulation of microbial development on surfaces.</p

Genes That Influence Swarming Motility and Biofilm Formation in Variovorax paradoxus EPS

Variovorax paradoxus is an aerobic soil bacterium associated with important biodegradative processes in nature. We use V. paradoxus EPS to study multicellular behaviors on surfaces.We recovered flanking sequence from 123 clones in a Tn5 mutant library, with insertions in 29 different genes, selected based on observed surface behavior phenotypes. We identified three genes, Varpa_4665, Varpa_4680, and Varpa_5900, for further examination. These genes were cloned into pBBR1MCS2 and used to complement the insertion mutants. We also analyzed expression of Varpa_4680 and Varpa_5900 under different growth conditions by qPCR.The 29 genes we identified had diverse predicted functions, many in exopolysaccharide synthesis. Varpa_4680, the most commonly recovered insertion site, encodes a putative N-acetyl-L-fucosamine transferase similar to WbuB. Expression of this gene in trans complemented the mutant fully. Several unique insertions were identified in Varpa_5900, which is one of three predicted pilY1 homologs in the EPS genome. No insertions in the two other putative pilY1 homologs present in the genome were identified. Expression of Varpa_5900 altered the structure of the wild type swarm, as did disruption of the chromosomal gene. The swarming phenotype was complemented by expression of Varpa_5900 from a plasmid, but biofilm formation was not restored. Both Varpa_4680 and Varpa_5900 transcripts were downregulated in biofilms and upregulated during swarming when compared to log phase culture. We identified a putative two component system (Varpa_4664-4665) encoding a response regulator (shkR) and a sensor histidine kinase (shkS), respectively. Biofilm formation increased and swarming was strongly delayed in the Varpa_4665 (shkS) mutant. Complementation of shkS restored the biofilm phenotype but swarming was still delayed. Expression of shkR in trans suppressed biofilm formation in either genetic background, and partially restored swarming in the mutant.The data presented here point to complex regulation of these surface behaviors

Genome-wide analysis of 53,400 people with irritable bowel syndrome highlights shared genetic pathways with mood and anxiety disorders

Funder: Kennedy Trust Rheumatology Research Prize StudentshipFunder: DFG Cluster of Excellence “Precision Medicine in Chronic In-flammation” (PMI; ID: EXC2167)Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: “Ideas” Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199; Grant(s): 715772Funder: NWO-VIDI grant 016.178.056, the Netherlands Heart Foundation CVON grant 2018-27, and NWO Gravitation grant ExposomeNLFunder: Li Ka Shing Foundation (Li Ka Shing Foundation Limited); doi: https://doi.org/10.13039/100007421Abstract: Irritable bowel syndrome (IBS) results from disordered brain–gut interactions. Identifying susceptibility genes could highlight the underlying pathophysiological mechanisms. We designed a digestive health questionnaire for UK Biobank and combined identified cases with IBS with independent cohorts. We conducted a genome-wide association study with 53,400 cases and 433,201 controls and replicated significant associations in a 23andMe panel (205,252 cases and 1,384,055 controls). Our study identified and confirmed six genetic susceptibility loci for IBS. Implicated genes included NCAM1, CADM2, PHF2/FAM120A, DOCK9, CKAP2/TPTE2P3 and BAG6. The first four are associated with mood and anxiety disorders, expressed in the nervous system, or both. Mirroring this, we also found strong genome-wide correlation between the risk of IBS and anxiety, neuroticism and depression (rg > 0.5). Additional analyses suggested this arises due to shared pathogenic pathways rather than, for example, anxiety causing abdominal symptoms. Implicated mechanisms require further exploration to help understand the altered brain–gut interactions underlying IBS

Analysis of surface behaviors to determine efficacy of Varpa_4680 (<i>wbu</i>B) and Varpa_5900 (<i>pil</i>Y1) complementation.

<p>Biofilm levels after 24 h incubation and swarm diameters at 48 h for wt and mutant strains expressing either <i>wbu</i>B (A, B) or <i>pil</i>Y1 (C, D) <i>in trans</i> compared to vector controls. 86 = Varpa_4680::Tn5, 223 = Varpa_5900::Tn5. Error was computed as +/−SEM. All p-values were calculated using the student's unpaired T-test. For all panels a = p<0.01 compared to wt+vec, b = p<0.01 compared to 86+vec (A,B) or 223+vec (C,D).</p

Expression of <i>wbu</i>B (grey bars) and <i>pil</i>Y1(black bars) assessed directly by qPCR.

<p>Planktonic cells, biofilm cells, and plate cultures from 0.5% agarose solidified YE plates or FW swarming plates were harvested at 48 h of growth and compared to aerated liquid culture in log phase (19 h) or stationary phase (26 h). RNA levels were determined in comparison to a luciferase spike added to each sample, and fold expression relative to log phase was assessed using the Pfaffl method.</p

Phylogenetic and nucleotide composition suggests Varpa_4680 entered the <i>V. paradoxus</i> EPS genome by horizontal transfer.

<p>A) Phylogenetic tree based on WbuB amino acid sequences created using ClustalW multiple sequence alignment and subsequent Bayesian inference of phylogeny(Mr. Bayes). B) Phylogenetic tree using the 16s rDNA sequences from the same set of organisms as in (A), using Bayesian inference of phylogeny based on the HKY85 nucleotide substitution model. For both A) and B) Vp EPS = <i>Variovorax paradoxus</i> EPS, Pn = <i>Polaromonas naphthlenivorans</i> CJ2, Bp = <i>Bordatella pertussis</i> Tohama 1, Pao1 = <i>Pseudomonas aeruginosa</i> PAO1, Cv = <i>Chromobacterium violaceum</i> ATCC 12472, Sd = <i>Sulfurimonas denitrificans</i> DSM 1251 C) G/C analysis of 35 kb region of the <i>V. paradoxus</i> EPS genome including <i>wbu</i>B. Total region spans orfs Varpa_4661-4691. Low G/C region from 18.4 kb to 27.1 kb spans orfs Varpa_4679-4684.</p

Swarms of <i>V. paradoxus</i> EPS imaged using a stereomicroscope.

<p>(A) wt+vec, (B) wt+<i>pil</i>Y1, (C) mut223+vec, (D) mut223+<i>pil</i>Y1.</p

Mutants from Colony Morphology Screens.

<p>Mutants from Colony Morphology Screens.</p

Analysis of surface behaviors to examine complementation of Varpa_4664 (<i>shk</i>S).

<p>Biofilm levels after 24 h incubation (A) and swarm diameters (B) at 48 h for wt and mut99 expressing either <i>shk</i>S or <i>shk</i>R <i>in trans</i> compared to vector controls. Error was computed as +/−SEM. P-values were calculated using the student's unpaired T-test. For both panels, a = p<0.01 compared to wt+vec, b = p<0.01 compared to mut99+vec.</p