Fimbrolide Natural Products Disrupt Bioluminescence of Vibrio By Targeting Autoinducer Biosynthesis and Luciferase Activity.

Vibrio is a model organism for the study of quorum sensing (QS) signaling and is used to identify QS-interfering drugs. Naturally occurring fimbrolides are important tool compounds known to affect QS in various organisms; however, their cellular targets have so far remained elusive. Here we identify the irreversible fimbrolide targets in the proteome of living V. harveyi and V. campbellii via quantitative mass spectrometry utilizing customized probes. Among the major hits are two protein targets with essential roles in Vibrio QS and bioluminescence. LuxS, responsible for autoinducer 2 biosynthesis, and LuxE, a subunit of the luciferase complex, were both covalently modified at their active-site cysteines leading to inhibition of activity. The identification of LuxE unifies previous reports suggesting inhibition of bioluminescence downstream of the signaling cascade and thus contributes to a better mechanistic understanding of these QS tool compounds.

The phosphorylation flow of the Vibrio harveyi quorum-sensing cascade determines levels of phenotypic heterogeneity in the population.

UNLABELLED: Quorum sensing (QS) is a communication process that enables a bacterial population to coordinate and synchronize specific behaviors. The bioluminescent marine bacterium Vibrio harveyi integrates three autoinducer (AI) signals into one quorum-sensing cascade comprising a phosphorelay involving three hybrid sensor kinases: LuxU; LuxO, an Hfq/small RNA (sRNA) switch; and the transcriptional regulator LuxR. Using a new set of V. harveyi mutants lacking genes for the AI synthases and/or sensors, we assayed the activity of the quorum-sensing cascade at the population and single-cell levels, with a specific focus on signal integration and noise levels. We found that the ratios of kinase activities to phosphatase activities of the three sensors and, hence, the extent of phosphorylation of LuxU/LuxO are important not only for the signaling output but also for the degree of noise in the system. The pools of phosphorylated LuxU/LuxO per cell directly determine the amounts of sRNAs produced and, consequently, the copy number of LuxR, generating heterogeneous quorum-sensing activation at the single-cell level. We conclude that the ability to drive the heterogeneous expression of QS-regulated genes in V. harveyi is an inherent feature of the architecture of the QS cascade. IMPORTANCE: V. harveyi possesses one of the most complex quorum-sensing (QS) cascades known, using three different autoinducers (AIs) to control the induction of, e.g., bioluminescence, virulence factors, and biofilm and exoprotease production. We constructed various V. harveyi mutants to study the impact of each component and subsystem of the QS signaling cascade on QS activation at the population and single-cell levels. We found that the output was homogeneous only in the presence of all AIs. In the absence of any one AI, QS activation varied from cell to cell, resulting in phenotypic heterogeneity. This study elucidates a molecular design principle which enables a tightly integrated signaling cascade to control the expression of diverse phenotypes within a genetically homogeneous population.

Variation in archaeal and bacterial community profiles and their functional metabolic predictions under the influence of pure and mixed fertilizers in paddy soil.

Impact of environmental perturbations i.e., nitrogen (N), phosphorus (P), potassium (K), and rice straw (Rs) on the dynamics of soil bacterial and archaeal communities are multifactor dependent and seeks a contemporary approach to study underlying mechanisms. The current study investigates the effect of pure and mixed fertilizers on soil physicochemical properties, the microbial community structure, and their functional metabolic predictions. It involved amendments with distinct combinations of N as C(H2N)2O, P and K as KH2PO4, K as KCl, and Rs in paddy soil microcosms with concentrations common in rice fields agriculture. Soil pH, electrical conductivity (EC), total carbon (TC), total nitrogen (TN), organic matter (OM), available K (AK), and total extractable P (TEP) were evaluated. To comprehend community variation and functional predictions, 16S rRNA-based high throughput sequencing (HTS) and phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) were employed, respectively. Our findings showed enhanced community richness and diversity in all amendments compared to control. Proteobacteria, Actinobacteria, and Firmicutes were dominant bacterial phyla. Regarding relative abundance, Chloroflexi, Bacteroidetes, and Verrucomicrobia showed positive while Actinobacteria, Acidobacteria, and Gemmatimonadetes showed negative trends compared to controls. Thaumarchaeota and Euryarchaeota were dominant archaeal phyla and exhibited increasing and decreasing trends, respectively. The PICRUSt analysis indicated functional prediction more towards amino acid, carbohydrate, energy, and lipid metabolism while less towards others. Concerning energy metabolism, most and least responsive treatments were KP and controls, respectively. These outcomes enhanced our understanding regarding soil quality, fertilizer composition and application, and functional metabolomics of archaea and bacteria.

Activity, Abundance, and Localization of Quorum Sensing Receptors in Vibrio harveyi.

Quorum sensing (QS) is a process enabling a bacterial population to communicate via small molecules called autoinducers (AIs). This intercellular communication process allows single cells to synchronize their behavior within a population. The marine bacterium Vibrio harveyi ATCC BAA-1116 channels the information of three AI signals into one QS cascade. Three receptors perceive these AIs, the hybrid histidine kinases LuxN, Lux(P)Q and CqsS, to transduce the information to the histidine phosphotransfer (HPt) protein LuxU via phosphorelay, and finally to the response regulator LuxO. Hence, the level of phosphorylated LuxO depends on the AI concentrations. The phosphorylated LuxO (P-LuxO) controls the expression of small regulatory RNAs (sRNAs), which together with the RNA chaperon Hfq, destabilize the transcript of the master regulator luxR. LuxR is responsible for the induction and repression of several genes (e.g., for bioluminescence, exoprotease and siderophore production). In vivo studies with various mutants have demonstrated that the ratio between kinase and phosphatase activities of the individual QS receptors and therefore the P-LuxO/LuxO ratio is crucial not only for the output strength but also for the degree of noise. This study was undertaken to better understand the inherent design principles of this complex signaling cascade, which allows sensing and integration of different signals, but also the differentiated output in individual cells. Therefore, we quantitatively analyzed not only the enzymatic activities, but also the abundance and localization of the three QS receptors. We found that LuxN presents the highest capacity to phosphorylate LuxU, while the phosphatase activity was comparable to LuxQ and CqsS in vitro. In whole cells the copy number of LuxN was higher than that of LuxQ and CqsS, and further increased in the late exponential growth phase. Microscopy experiments indicate that LuxN and LuxQ form independent clusters. Altogether, these results suggest, that the three QS receptors act in parallel, and V. harveyi has developed with LuxN the most dynamic sensing range for HAI-1, the species-specific AI.

Mechanistic analysis of aliphatic ß-lactones in Vibrio harveyi reveals a quorum sensing independent mode of action.

N-Acylhomoserine lactones are autoinducers of quorum sensing (QS) in Gram-negative bacteria. We exploit here the role of structurally related ß-lactones in the inhibition of Vibrio harveyi bioluminescence and identify a derivative with nanomolar potency. Surprisingly, QS was not affected and combined proteomic/biochemical studies revealed insights into the cellular mode of action.

Identification and Initial Characterization of Prophages in Vibrio campbellii.

Phages are bacteria targeting viruses and represent the most abundant biological entities on earth. Marine environments are exceptionally rich in bacteriophages, harboring a total of 4x10(30) viruses. Nevertheless, marine phages remain poorly characterized. Here we describe the identification of intact prophage sequences in the genome of the marine γ-proteobacterium Vibrio campbellii ATCC BAA-1116 (formerly known as V. harveyi ATCC BAA-1116), which presumably belong to the family of Myoviridae. One prophage was found on chromosome I and shows significant similarities to the previously identified phage ΦHAP-1. The second prophage region is located on chromosome II and is related to Vibrio phage kappa. Exposure of V. campbellii to mitomycin C induced the lytic cycle of two morphologically distinct phages and, as expected, extracellular DNA from induced cultures was found to be specifically enriched for the sequences previously identified as prophage regions. Heat stress (50°C, 30 min) was also found to induce phage release in V. campbellii. Notably, promoter activity of two representative phage genes indicated heterogeneous phage induction within the population.

Novel volatiles of skin-borne bacteria inhibit the growth of Gram-positive bacteria and affect quorum-sensing controlled phenotypes of Gram-negative bacteria.

The skin microbiota is import for body protection. Here we present the first comprehensive analysis of the volatile organic compound (VOC) profiles of typical skin-resident corynebacterial and staphylococcal species. The VOC profile of Staphylococcus schleiferi DSMZ 4807 was of particular interest as it is dominated by two compounds, 3-(phenylamino)butan-2-one and 3-(phenylimino)butan-2-one (schleiferon A and B, respectively). Neither of these has previously been reported from natural sources. Schleiferon A and B inhibited the growth of various Gram-positive species and affected two quorum-sensing-dependent phenotypes - prodigiosin accumulation and bioluminescence - of Gram-negative bacteria. Both compounds were found to inhibit the expression of prodigiosin biosynthetic genes and stimulate the expression of prodigiosin regulatory genes pigP and pigS. This study demonstrates that the volatile schleiferons A and B emitted by the skin bacterium S. schleiferi modulate differentially and specifically its interactions with members of diverse bacterial communities. A network of VOC-mediated interspecies interactions and communications must be considered in the establishment of the (skin) microbiome and both compounds are interesting candidates for further investigations to better understand how VOCs emitted by skin bacteria influence and modulate the local microbiota and determine whether they are relevant to antibiotic and anti-virulence therapies.

Changes in rhizosphere bacterial gene expression following glyphosate treatment.

In commercial agriculture, populations and interactions of rhizosphere microflora are potentially affected by the use of specific agrichemicals, possibly by affecting gene expression in these organisms. To investigate this, we examined changes in bacterial gene expression within the rhizosphere of glyphosate-tolerant corn (Zea mays) and soybean (Glycine max) in response to long-term glyphosate (PowerMAX™, Monsanto Company, MO, USA) treatment. A long-term glyphosate application study was carried out using rhizoboxes under greenhouse conditions with soil previously having no history of glyphosate exposure. Rhizosphere soil was collected from the rhizoboxes after four growing periods. Soil microbial community composition was analyzed using microbial phospholipid fatty acid (PLFA) analysis. Total RNA was extracted from rhizosphere soil, and samples were analyzed using RNA-Seq analysis. A total of 20-28 million bacterial sequences were obtained for each sample. Transcript abundance was compared between control and glyphosate-treated samples using edgeR. Overall rhizosphere bacterial metatranscriptomes were dominated by transcripts related to RNA and carbohydrate metabolism. We identified 67 differentially expressed bacterial transcripts from the rhizosphere. Transcripts downregulated following glyphosate treatment involved carbohydrate and amino acid metabolism, and upregulated transcripts involved protein metabolism and respiration. Additionally, bacterial transcripts involving nutrients, including iron, nitrogen, phosphorus, and potassium, were also affected by long-term glyphosate application. Overall, most bacterial and all fungal PLFA biomarkers decreased after glyphosate treatment compared to the control. These results demonstrate that long-term glyphosate use can affect rhizosphere bacterial activities and potentially shift bacterial community composition favoring more glyphosate-tolerant bacteria.

Glyphosate effects on soil rhizosphere-associated bacterial communities.

Glyphosate is one of the most widely used herbicides in agriculture with predictions that 1.35 million metric tons will be used annually by 2017. With the advent of glyphosate tolerant (GT) cropping more than 10 years ago, there is now concern for non-target effects on soil microbial communities that has potential to negatively affect soil functions, plant health, and crop productivity. Although extensive research has been done on short-term response to glyphosate, relatively little information is available on long-term effects. Therefore, the overall objective was to investigate shifts in the rhizosphere bacterial community following long-term glyphosate application on GT corn and soybean in the greenhouse. In this study, rhizosphere soil was sampled from rhizoboxes following 4 growth periods, and bacterial community composition was compared between glyphosate treated and untreated rhizospheres using next-generation barcoded sequencing. In the presence or absence of glyphosate, corn and soybean rhizospheres were dominated by members of the phyla Proteobacteria, Acidobacteria, and Actinobacteria. Proteobacteria (particularly gammaproteobacteria) increased in relative abundance for both crops following glyphosate exposure, and the relative abundance of Acidobacteria decreased in response to glyphosate exposure. Given that some members of the Acidobacteria are involved in biogeochemical processes, a decrease in their abundance could lead to significant changes in nutrient status of the rhizosphere. Our results also highlight the need for applying culture-independent approaches in studying the effects of pesticides on the soil and rhizosphere microbial community.