Acyl-homoserine Lactone Production Research Articles

Microfluidic static droplet array for analyzing microbial communication on a population gradient.

Quorum sensing (QS) is a type of cell-cell communication using signal molecules that are released and detected by cells, which respond to changes in their population density. A few studies explain that QS may operate in a density-dependent manner; however, due to experimental challenges, this fundamental hypothesis has never been investigated. Here, we present a microfluidic static droplet array (SDA) that combines a droplet generator with hydrodynamic traps to independently generate a bacterial population gradient into a parallel series of droplets under complete chemical and physical isolation. The SDA independently manipulates both a chemical concentration gradient and a bacterial population density. In addition, the bacterial population gradient in the SDA can be tuned by a simple change in the number of sample plug loading. Finally, the method allows the direct analysis of complicated biological events in an addressable droplet to enable the characterization of bacterial communication in response to the ratio of two microbial populations, including two genetically engineered QS circuits, such as the signal sender for acyl-homoserine lactone (AHL) production and the signal receiver bacteria for green fluorescent protein (GFP) expression induced by AHL. For the first time, we found that the population ratio of the signal sender and receiver indicates a significant and potentially interesting partnership between microbial communities. Therefore, we envision that this simple SDA could be a useful platform in various research fields, including analytical chemistry, combinatorial chemistry, synthetic biology, microbiology, and molecular biology.

Applications of thin-layer chromatography in extraction and characterisation of ajoene from garlic bulbs

Novel and inexpensive methods of thin-layer chromatography (TLC) were employed for the extraction, characterisation and mechanism of quorum sensing inhibition by ajoene, a component from toluene garlic bulb (Allium sativum L.) extract (TGE). TLC profiling of TGE was carried out using ethyl acetate as solvent. Out of total spots extracted from TLC, four spots exhibited quorum sensing inhibitory (QSI) potential. Among those, spot 5 was identified as Z-ajoene by TLC and confirmed by NMR and MS. HPLC analysis indicated 97.7% purity for purified ajoene. TLC densitometric analysis quantified 221.08 μmol/g of ajoene in TGE and indicated that ajoene is stable at 4°C and at acidic pH. HPTLC profiling showed that ajoene exhibits QSI effect by inhibiting the production of both long-chain acyl homoserine lactones and Pseudomonas quinolone signal (PQS) by P. aeruginosa and also by inactivating PQS molecules.

AHL-type quorum sensing and its regulation on symplasmata formation in Pantoea agglomerans YS19.

Pantoea agglomerans YS19, an endophytic diazotrophic bacterium isolated from rice, is characterized by the formation of multicellular aggregate structure called symplasmata, which not only bestow the strong stress-resistance of the bacterium, but also contribute to the specific adaptation in the endophyte-host association. Acyl-homoserine lactones (AHLs), as the important signal molecule in the quorum sensing (QS) system of gram-negative bacteria, were demonstrated to regulate motility, cell-aggregation, and other bacterial behaviors. Here, the production of AHL by P. agglomerans YS19 and its regulation on the symplasmata formation were studied. It was revealed that the production of AHL by YS19 was initiated at the exponential growth stage and from then on, reached the peak values at the stationary growth stage in LB medium. The AHL was identified as N-3-oxooctanoyl-L-homoserine lactone (OOHL) by MALDI-TOF-MS analysis. The AHL synthesis gene pagI and receptor gene pagR in YS19 were cloned and phylogenetic analysis showed that they were high conservative among strains in species of P. agglomerans. It was revealed that AHL promoted the bacterial growth and symplasmata formation of YS19. Meanwhile, the colonization ability and growth-promoting effect of YS19 on the host plant were also enhanced by AHL. These results strongly suggest the pleiotropic effects of the AHL-type QS system in endophytic life of the strain.

Quorum sensing signal production and microbial interactions in a polymicrobial disease of corals and the coral surface mucopolysaccharide layer.

Black band disease (BBD) of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS) and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community) samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs) and for autoinducer-2 (AI-2) activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community) samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153) of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S)-4,5-dihydroxy-2,3-pentanedione (DPD), the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome.

Quorum Sensing Signalling and Biofilm Formation of Brewery-Derived Bacteria, and Inhibition of Signalling by Natural Compounds

Bacteria use quorum sensing signalling in various functions, e.g. while forming biofilms, and inhibition of this signalling could be one way to control biofilm formation. The aim of this study was to evaluate the production of signalling molecules and its correlation with the biofilm formation capability of bacteria isolated from brewery filling process. A further aim was to study berry extracts and wood-derived terpenes for their possible quorum sensing inhibitory effects. Out of the twenty bacteria studied, five produced short-chain and five long-chain AHL (acyl homoserine lactone) signalling molecules when tested with the Chromobacterium violaceum CV026 reporter bacterium. Production of AI-2 (autoinducer-2) signalling molecules was detected from nine strains with the Vibrio harveyi BB170 bioassay. Over half of the strains produced biofilm in the microtitre plate assay, but the production of AHL and AI-2 signalling molecules and biofilm formation capability did not directly correlate with each other. Out of the 13 berry extracts and wood-derived terpenes screened, four compounds decreased AHL signalling without effect on growth. These were betulin, raspberry extract and two cloudberry extracts. The effect of these compounds on biofilm formation of the selected six bacterial strains varied. The phenolic extract of freeze-dried cloudberry fruit caused a statistically significant reduction of biofilm formation of Obesumbacterium proteus strain. Further experiments should aim at identifying the active compounds and revealing whether quorum sensing inhibition causes structural changes in the biofilms formed.

Characterization of N-Acyl-homoserine Lactones (AHLs)-Deficient Clinical Isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen causing severe respiratory infections. Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing, QS) in a variety of Gram-negative bacteria, and several virulence genes of bacterial pathogens are known to be controlled by QS. Hence, fitness mutant of virulent factors is beneficial for natural selection. In this study, strains of P. aeruginosa isolated from chronic lung infection of cystic fibrosis patients, were screened for AHLs production by using indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens strain At136. Four AHLs defective strains were selected from fifty-three clinical isolates. PCR analysis revealed that only one isolate was negative for lasR gene. These four AHLs defective isolates produced less virulence factors and forming less biofilm than PAO1. Only isolate PA41 produce little more pyocyanin than PAO1. The results indicate that, despite the pivotal role of QS in the pathogenesis of P. aeruginosa infections, AHLs-deficient strains are still capable of causing infections in human.

Antiquorum from Freshwater Zoosporic Isolated Fungi: Maximization of some Ameliorative Factors

Many bacteria detect their critical cell numbers through a mechanism known as quorum sensing (QS). Acyl homoserine lactones (AHLs) mediated QS systems have been shown to operate in important human pathogens. The aim of this study was to evaluate the inhibitory effect generated by zoosporic freshwater fungi on AHLs production. A potent strain of zoosporic fungi, Leptolegniella keratinophilum, was isolated after extended screening study. A new cheap medium was used to support the anti-AHLs activities. Statistical strategy depending on two levels factorial model and three levels quadratic model were applied to guide a production maximization process. The three levels Box-Behnken model possessed the ability to optimize the significant nutritional and cultural conditions and to predict the maximum that can be achieved. In the course of study, we reached 88% anti-AHLs activity, while the initial activity was 63%. There was no previous maximization methodology for either the experimental medium, or the fungi. The results suggest that freshwater zoosporic fungi can be potentially used against bacterial infections.

Pantoea sp. isolated from tropical fresh water exhibiting N-acyl homoserine lactone production.

N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulate various physiological activities including pathogenicity. With the aim of isolating freshwater-borne bacteria that can cause outbreak of disease in plants and portrayed QS properties, environmental water sampling was conducted. Here we report the preliminary screening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors. The 16S rDNA gene sequence of isolate M009 showed the highest sequence similarity to Pantoea stewartii S9-116, which is a plant pathogen. The isolated Pantoea sp. was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolution mass tandem mass spectrometry.

Quorum Sensing-Modulated AND-Gate Promoters Control Gene Expression in Response to a Combination of Endogenous and Exogenous Signals

We have constructed and characterized two synthetic AND-gate promoters that require both a quorum-sensing (QS) signal and an exogenously added inducer to turn on gene expression. The engineered promoters, LEE and TTE, contain binding sites for the QS-dependent repressor, EsaR, and either LacI or TetR, and they are induced by an acyl-homoserine lactone (AHL) signal and IPTG or aTc. Although repression of both LEE and TTE by wild-type EsaR was observed, induction of gene expression at physiologically relevant concentrations of AHL required the use of an EsaR variant with higher signal sensitivity. Gene expression from both LEE and TTE was shown to require both signal molecules, and gene expression above background levels was not observed with either signal alone. We added endogenous production of AHL to evaluate the ability of the promoters to function in a QS-dependent manner and observed that gene expression increased as a function of cell density only in the presence of exogenously added IPTG or aTc. Cell-cell communication-dependent AND-gate behaviors were demonstrated using an agar plate assay, where cells containing the engineered promoters were shown to respond to AHL produced by a second E. coli strain only in the presence of exogenously added IPTG or aTc. The promoters described in this work demonstrate that EsaR and its target DNA sequence can be used to engineer new promoters to respond to cell density or cell-cell communication. Further, the AND-gate promoters described here may serve as a template for new regulatory systems that integrate QS and the presence of key metabolites or other environmental cues to enable dynamic changes in gene expression for metabolic engineering applications.

Application of Mathematical Model for the Quantification of Acylated Homoserine Lactones Produces by Pseudomonas aeruginosa in Chicken Breast Meat and Broth

AbstractAs storage temperature had no influence on the acylated homoserine lactones (AHLs) production, the main AHLs (C8‐HSL and C6‐HSL) were used as reference standards. We proposed to describe the relationship between AHLs and β‐galactosidase of AHL biosensor with an equation. The equation could fit the β‐galactosidase data well with a high determination coefficient (0.994) and low root mean squared error (0.016). In all experiment, there was no significant difference (P > 0.05) between the observed and calculated AHL values in chicken meat, when different type of AHLs was used. Hence, based on the equation the AHLs similar to Pseudomonas aeruginosa production can be calculated. This approach simplified the calculation of the AHL production in chicken meat and broth. Based on this method, we found that during bacterial growth, the AHLs accumulate in the exponential phase, but decreased in the stationary phase. The highest AHLs were determined during late exponential and early stationary phase.Practical ApplicationIn present study, we propose to simplify the calculation of the AHL produced by P. aeruginosa in chicken meat and broth based on the microplate bioassay. We developed a nonlinear model to describe the relationship between the β‐galactosidase expression of Agrobacterium tumefaeiens KYC55 and AHLs addition. The model can be applied to quantify the AHLs‐extraction concentration in the range 0–6 ug/mL.

Characterization of the Quorum Sensing Regulon in <i>Acidithiobacillus ferrooxidans</i>

Bioleaching is the solubilization of metals from ores by microorganisms. This process is more efficient than conventional methods in the metal recovery from low grade ores while decreasing the operating cost and the environmental impact. Among the bioleaching bacteria, the Gram-negative chemolithoautotrophic acidophilic Acidithiobacillus ferrooxidans has the ability to adhere to mineral surface and to form biofilm. The genes involved in the production of this biofilm are controlled by a cellular communication system called Quorum Sensing (QS). The QS occurs mainly through the production of acyl homoserine lactones (AHLs). A functional QS system has been identified in At. ferrooxidans with an acyl synthase (AfeI) and a transcriptional regulator (AfeR). Here, our purpose is to characterize the quorum sensing regulon and particularly the genes involved in biofilm formation in At. ferrooxidans. This has been done by cell adhesion experiments comparing At. ferrooxidans grown with or without Tetrazol, an AHL analogue that activates the cell adhesion to mineral surface. The second step has been to compare the expression of some genes predicted to be involved in the QS regulon in At. ferrooxidans grown in the presence/absence of Tetrazol using real-time PCR. These two different strategies are expected to give insight on the moment at which the At. ferrooxidans QS regulon is switched on. The formation of biofilm could improve the bioleaching operation and therefore the understanding of the QS regulon is of primordial importance.

The Acyl-Homoserine Lactone Synthase YenI from Yersinia enterocolitica Modulates Virulence Gene Expression in Enterohemorrhagic Escherichia coli O157:H7

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes the rectoanal junction (RAJ) in cattle, its natural reservoir. Colonization at the RAJ poses a serious risk for fecal shedding and contamination of the environment. We previously demonstrated that EHEC senses acyl-homoserine lactones (AHLs) produced by the microbiota in the rumen to activate the gad acid resistance genes necessary for survival through the acidic stomachs in cattle and to repress the locus of enterocyte effacement (LEE) genes important for colonization of the RAJ, but unnecessary in the rumen. Devoid of AHLs, the RAJ is the prominent site of colonization of EHEC in cattle. To determine if the presence of AHLs in the RAJ could repress colonization at this site, we engineered EHEC to express the Yersinia enterocolitica AHL synthase gene yenI, which constitutively produces AHLs, to mimic a constant exposure of AHLs in the environment. The yenI(+) EHEC produces oxo-C6-homoserine lactone (oxo-C6-HSL) and had a significant reduction in LEE expression, effector protein secretion, and attaching and effacing (A/E) lesion formation in vitro compared to the wild type (WT). The yenI(+) EHEC also activated expression of the gad genes. To assess whether AHL production, which decreases LEE expression, would decrease RAJ colonization by EHEC, cattle were challenged at the RAJ with WT or yenI(+) EHEC. Although the yenI(+) EHEC colonized the RAJ with efficiency equal to that of the WT, there was a trend for the cattle to shed the WT strain longer than the yenI(+) EHEC.

LuxR- and LuxI-Type Quorum-Sensing Circuits Are Prevalent in Members of the Populus deltoides Microbiome

We are interested in the root microbiome of the fast-growing Eastern cottonwood tree, Populus deltoides. There is a large bank of bacterial isolates from P. deltoides, and there are 44 draft genomes of bacterial endophyte and rhizosphere isolates. As a first step in efforts to understand the roles of bacterial communication and plant-bacterial signaling in P. deltoides, we focused on the prevalence of acyl-homoserine lactone (AHL) quorum-sensing-signal production and reception in members of the P. deltoides microbiome. We screened 129 bacterial isolates for AHL production using a broad-spectrum bioassay that responds to many but not all AHLs, and we queried the available genome sequences of microbiome isolates for homologs of AHL synthase and receptor genes. AHL signal production was detected in 40% of 129 strains tested. Positive isolates included members of the Alpha-, Beta-, and Gammaproteobacteria. Members of the luxI family of AHL synthases were identified in 18 of 39 proteobacterial genomes, including genomes of some isolates that tested negative in the bioassay. Members of the luxR family of transcription factors, which includes AHL-responsive factors, were more abundant than luxI homologs. There were 72 in the 39 proteobacterial genomes. Some of the luxR homologs appear to be members of a subfamily of LuxRs that respond to as-yet-unknown plant signals rather than bacterial AHLs. Apparently, there is a substantial capacity for AHL cell-to-cell communication in proteobacteria of the P. deltoides microbiota, and there are also Proteobacteria with LuxR homologs of the type hypothesized to respond to plant signals or cues.

Presence of acyl‐homoserine lactones in 57 members of theVibrionaceaefamily

Aims The aim of this study was to use a sensitive method to screen and quantify 57 Vibrionaceae strains for the production of acyl-homoserine lactones (AHLs) and map the resulting AHL profiles onto a host phylogeny.Methods and Results We used a high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) protocol to measure AHLs in spent media after bacterial growth. First, the presence/absence of AHLs (qualitative analysis) was measured to choose internal standard for subsequent quantitative AHL measurements. We screened 57 strains from three genera (Aliivibrio,Photobacterium and Vibrio) of the same family (i.e. Vibrionaceae). Our results show that about half of the isolates produced multiple AHLs, typically at 25–5000 nmol l−1.Conclusions This work shows that production of AHL quorum sensing signals is found widespread among Vibrionaceae bacteria and that closely related strains typically produce similar AHL profiles.Significance and Impact of the Study The AHL detection protocol presented in this study can be applied to a broad range of bacterial samples and may contribute to a wider mapping of AHL production in bacteria, for example, in clinically relevant strains.

The GacS-regulated sigma factor RpoS governs production of several factors involved in biocontrol activity of the rhizobacterium Pseudomonas chlororaphis O6

Pseudomonas chlororaphis O6 possesses many beneficial traits involved in biocontrol of plant diseases. In this paper, we examined the effect of a mutation in rpoS encoding a stress-related alternative sigma factor to better understand the regulation of these traits. Biochemical studies indicated that production of acyl homoserine lactones was altered and phenazine was increased in the P. chlororaphis O6 rpoS mutant. The rpoS mutation reduced hydrogen cyanide levels, but the rpoS mutant still displayed a level of in vitro antifungal activity against Fusarium graminearum and Alternaria alternata. Tomato root colonization by the rpoS mutant was lower than that by the wild type at 5, 7, and 13 days after inoculation. The rpoS mutant was less effective than the wild type in induction of systemic resistance to two foliar pathogens after root inoculation of the tomato plants. Our findings demonstrate that the stationary-phase sigma factor RpoS regulates production of several key factors involved in the biocontrol potential of P. chlororaphis O6, some independently of the global regulator GacS.

Contribution of the production of quormones to some phenotypic characteristics of Pseudomonas aeruginosa clinical strains

The contribution of quorum sensing in some phenotypic and pathogenic characteristics of Pseudomonas aeruginosa was studied. The production of acylhomoserine lactones (AHL) by planktonic cultures of eight clinical and reference strains of P. aeruginosa was evaluated using two biosensors. The adhesion of the bacteria on a surface (Biofilm Ring Test ®, BFRT), their capacity to develop a biofilm (crystal violet staining method, CVSM), their sensitivity to tobramycin and their secretion of proteases or of rhamnolipids were also measured. The production and the release of AHL widely varied among the eight strains. An analysis of the extracts by TLC showed that 3-oxo-C8-HSL, 3-oxo-C10-HSL and 3-oxo-C12-HSL were released by the five strains producing the highest amount of Cn≥6-HSL. The genes lasI and lasR involved in the synthesis and response to 3-oxo-C12-HSL were detected in the genomes of all strains. Two clinical strains had deletions in the lasR gene leading to truncation of the protein. One subpopulation of the PAO1 strain had a major deletion (98 bp) of the lasR gene. Strains with significant mutations of lasR secreted the lowest amount of AHL, probably due to deficiencies in the self-induction and amplification of the synthesis of the lactone. These strains formed a biofilm with low biomass. C4-HSL production also differed among the strains and was correlated with rhamnolipid production and biofilm formation. Whereas the production of AHL varied among P. aeruginosa strains, few correlations were observed with their phenotypic properties except with their ability to form a biofilm.

The Presence of Quorum-Sensing Genes in Pseudomonas isolates Infecting Cystic Fibrosis and Non-cystic Fibrosis Patients

Ninety-one Pseudomonas aeruginosa isolates recovered from cystic fibrosis and non-cystic fibrosis patients were evaluated regarding the ability to form biofilm and acyl-homoserine lactones production and for the presence of five quorum-sensing (QS) regulatory genes (lasI, lasR, rhlI, rhlR, and vfr). Most isolates (90.1 %) presented all five QS genes. Five isolates shown to be lasI/lasR-deficient were not able to produce biofilm in vitro. Moreover, one isolate harboring all five QS genes was also not able to form a biofilm. The function of rhlR gene may be compensated by the las QS system. However, in our study, all isolates which were deficient for the rhlR gene were also deficient for the lasI/lasR system. This may point to some hierarchy in QS regulation which may pose a potential for controlling biofilm infections due to P. aeruginosa.

Virulence factor regulator (Vfr) controls virulence‐associated phenotypes in Pseudomonas syringae pv. tabaci 6605 by a quorum sensing‐independent mechanism

Virulence factor regulator (Vfr) is a member of the cyclic 3',5'-adenosine monophosphate (cAMP) receptor proteins that regulate the expression of many important virulence genes in Pseudomonas aeruginosa. The role of Vfr in pathogenicity has not been elucidated fully in phytopathogenic bacteria. To investigate the function of Vfr in Pseudomonas syringae pv. tabaci 6605, the vfr gene was disrupted. The virulence of the vfr mutant towards host tobacco plants was attenuated significantly, and the intracellular cAMP level was decreased. The vfr mutant reduced the expression of flagella-, pili- and type III secretion system-related genes and the defence response in nonhost Arabidopsis leaves. Furthermore, the expression levels of achromobactin-related genes and the iron uptake ability were decreased, suggesting that Vfr regulates positively these virulence-related genes. In contrast, the vfr mutant showed higher tolerance to antimicrobial compounds as a result of the enhanced expression of the resistance-nodulation-division family members, the mexA, mexB and oprM genes. We further demonstrated that the mutant strains of vfr and cyaA, an adenylate cyclase gene responsible for cAMP synthesis, showed a similar phenotype, suggesting that Vfr regulates virulence factors in a cAMP-dependent manner. Because there was no significant difference in the production of acylhomoserine lactone (AHL) quorum sensing molecules in the wild-type, vfr and cyaA mutant strains, Vfr might control important virulence factors by an AHL-independent mechanism in an early stage of infection by this bacterium.

Tunability of the ratio of cell states after the synthetic diversification by the diversity generator

The autonomous generation of phenotypic diversity in embryonic cell populations can be explained by Waddington’s landscape. The landscape proposes that intra- and inter-cellular interactions mediate the generation of cellular diversity. Recently, we implemented, in a population of Escherichia coli, a synthetic diversification, which is governed by inter-cellular signaling mediated by acyl-homoserine lactone (AHL). The cells with the diversity generator diversified into two distinct cell states, “high” and “low,” if all of the cells started from the low state. The ratio of the states after the diversification was affected by the velocity of autonomous signal accumulation, which depends on the cell density and the AHL production rate of individual cells. The dependency of the ratio on the initial cell density is reminiscent of the community effect, which is observed in animal development and is important for ES-cell differentiation. Therefore, it is worthwhile reviewing the roles of natural animal gene networks with similar topologies to the diversity generator design. The diversity generator design will also be the basis for a tool to direct cell fates on the population level in tissue engineering. Here, we discuss the tunability of the ratio of cell states by our synthetic circuit design.

Isolation of Bacteria from the Rhizosphere and Rhizoplane of Potato (Solanum tuberosum) Grown in Two Distinct Soils Using Semi Selective Media and Characterization of Their Biological Properties

The objective of this study was to isolate and characterize genera of bacteria that had been identified as being the most common residents in the rhizosphere of potato using cpn60 pyrosequencing analysis. Using various semi-selective media targeted to specific genera of interest, 200 isolates of bacteria were collected from rhizosphere soil and the rhizoplane of potatoes grown in soils obtained from a potato farm in Prince Edward Island and Ontario. The procedures employed were successful in selecting out representative bacteria suggested by pyrosequencing to be common in the potato rhizoplane. Results of 16S rRNA sequencing showed that 44 % of the isolates represented new species. All isolates were tested for biological and biochemical activities including phosphate solubilization, phytohormone metabolism, nitrogen fixation, antibiosis, exoenzyme production, and production of acyl-homoserine lactones. Massilia spp. and Chryseobacterium spp. showed the strongest exoenzyme activities. A greater proportion of Agrobacterium tumefaciens rhizosphere strains produced acyl-homoserine lactones compared to rhizoplane strains. Pseudomonas spp. and Lysobacter capsici had the greatest antagonistic activity on laboratory media towards six potato pathogens, and also significantly decreased disease in plants grown in pathogen-infested soil. Four isolates significantly increased growth of potato nodal explants in tissue culture. By using preliminary results derived from next generation sequencing technology and a targeted cultural technique, we were able to gain a better understanding of the biological activities of the most abundant bacterial species in the rhizosphere and rhizoplane of a cultivated crop.