Heterologous overexpression of quorum-sensing regulators to study cell-density-dependent phenotypes in a symbiotic plant bacterium <Emphasis Type="Italic">Mesorhizobium huakuii</Emphasis>

Quorum-sensing is widespread among many prokaryotic lineages. In order to investigate quorum regulation in the plant bacterium Mesorhizobium huakuii which produces an N-acyl homoserine lactone (AHL) quorum signal, the Agrobacterium quorum-sensing regulator TraR was heterologously expressed in this bacterium. The resulting strains showed reduced AHL production in the supernatant compared to wild-type, but similar intracellular levels of AHLs were detected, suggesting that M. huakuii AHLs can be bound to intracellular TraR proteins and thus become unavailable for its own quorum systems. M. huakuii overexpressing TraR formed thinner biofilms than the wild-type, suggesting a role played by quorum-sensing in biofilm formation.Hui Wang and Zengtao Zhong contributed equally to this work.     

Complex quorum-sensing regulatory systems regulate bacterial growth and symbiotic nodulation in <Emphasis Type="Italic">Mesorhizobium tianshanense</Emphasis>

LuxR/LuxI-type quorum-sensing systems have been shown to be important for symbiotic interactions between a number of rhizobium species and host legumes. In this study, we found that different isolates of Mesorhizobium tianshanense, a moderately-growing Rhizobium that forms nodules on a number of types of licorice plants, produces several different N-acyl homoserine lactone-like molecules. In M. tianshanense CCBAU060A, we performed a genetic screen and identified a network of regulatory components including a set of LuxI/LuxR-family regulators as well as a MarR-family regulator that is required for quorum-sensing regulation. Furthermore, compared with the wild-type strains, quorum-sensing deficient mutants showed a reduced growth rate and were defective in nodule formation on their host plant Glycyrrhiza uralensis. These data suggest that different M. tianshanense strains may use diverse quorum-sensing systems to regulate symbiotic process. H. Cao, M. Yang, and H. Zheng contributed equally to this work.     

The quorum-sensing systems of Vibrio campbellii DS40M4 and BB120 are genetically and functionally distinct

Vibrio campbellii BB120 (previously classified as Vibrio harveyi) is a fundamental model strain for studying quorum sensing in vibrios. A phylogenetic evaluation of sequenced Vibrio strains in Genbank revealed that BB120 is closely related to the environmental isolate V. campbellii DS40M4. We exploited DS40M4's competence for exogenous DNA uptake to rapidly generate greater than 30 isogenic strains with deletions of genes encoding BB120 quorum-sensing system homologues. Our results show that the quorum-sensing circuit of DS40M4 is distinct from BB120 in three ways: (i) DS40M4 does not produce an acyl homoserine lactone autoinducer but encodes an active orphan LuxN receptor, (ii) the quorum regulatory small RNAs (Qrrs) are not solely regulated by autoinducer signalling through the response regulator LuxO and (iii) the DS40M4 quorum-sensing regulon is much smaller than BB120 (~100 genes vs. ~400 genes, respectively). Using comparative genomics to expand our understanding of quorum-sensing circuit diversity, we observe that conservation of LuxM/LuxN proteins differs widely both between and within Vibrio species. These strains are also phenotypically distinct: DS40M4 exhibits stronger interbacterial cell killing, whereas BB120 forms more robust biofilms and is bioluminescent. These results underscore the need to examine wild isolates for a broader view of bacterial diversity in the marine ecosystem.     

Vibrio fischeri uses two quorum-sensing systems for the regulation of early and late colonization factors

Vibrio fischeri possesses two quorum-sensing systems, ain and lux, using acyl homoserine lactones as signaling molecules. We have demonstrated previously that the ain system activates luminescence gene expression at lower cell densities than those required for lux system activation and that both systems are essential for persistent colonization of the squid host, Euprymna scolopes. Here, we asked whether the relative contributions of the two systems are also important at different colonization stages. Inactivation of ain, but not lux, quorum-sensing genes delayed initiation of the symbiotic relationship. In addition, our data suggest that lux quorum sensing is not fully active in the early stages of colonization, implying that this system is not required until later in the symbiosis. The V. fischeri luxI mutant does not express detectable light levels in symbiosis yet initiates colonization as well as the wild type, suggesting that ain quorum sensing regulates colonization factors other than luminescence. We used a recently developed V. fischeri microarray to identify genes that are controlled by ain quorum sensing and could be responsible for the initiation defect. We found 30 differentially regulated genes, including the repression of a number of motility genes. Consistent with these data, ain quorum-sensing mutants displayed an altered motility behavior in vitro. Taken together, these data suggest that the sequential activation of these two quorum-sensing systems with increasing cell density allows the specific regulation of early colonization factors (e.g., motility) by ain quorum sensing, whereas late colonization factors (e.g., luminescence) are preferentially regulated by lux quorum sensing.     

Bacterial quorum sensing and cell surface electrokinetic properties

The hypothesis tested in this paper is that quorum sensing influences the microbial surface electrokinetic properties. Escherichia coli MG1655 and MG1655 LuxS- mutant (lacking quorum-sensing gene for Autoinducer synthase AI-2) were used for this study. AI-2 production (or lack of) in both strains was analyzed using the Vibrio harveyi bioassay. The levels of extracellular AI-2 with and without glucose in the growth medium were consistent with previously published work. The surface electrokinetic properties were determined for each strain of E. coli MG1655 by measuring the electrophoretic mobility using a phase amplitude light-scattering (PALS) Zeta potential analyser. The findings show that the surface charge of the cells is dependent upon the stage in the growth phase as well as the ability to participate in quorum sensing. In addition, significant differences in the electrophoretic mobility were observed between both strains of E. coli. These findings suggest that quorum sensing plays a significant role in the surface chemistry of bacteria during their growth.     

BacA is indispensable for successful <Emphasis Type="Italic">Mesorhizobium</Emphasis>–<Emphasis Type="Italic">Astragalus</Emphasis> symbiosis

A homologue of Sinorhizobium meliloti bacA was isolated from Mesorhizobium huakuii 7653R, which is capable of fixing atmospheric nitrogen in symbiotic association with leguminous Astragalus sinicus (Chinese milk vetch). Inactivation of the bacA gene abolished the ability of M. huakuii 7653R to establish a successful symbiosis with its host plant. Simultaneously, compared with wild-type M. huakuii 7653R, the bacA mutant was more sensitive to cell envelope-disrupting agents (acidic solution, ethanol, SDS, and crystal violet). Mass spectrometry analysis revealed that the very-long-chain fatty acid (27-OHC-28:0 and 29-OHC-30:0) contents of lipid A was reduced in the M. huakuii 7653R bacA mutant. Taken together, our data suggest that the cell envelope was altered in the M. huakuii 7653R bacA mutant, which might deteriorate bacterial adaption to acute environmental changes encountered in host cells and ultimately result in the failure of Mesorhizobium–legume symbiosis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Producing mechanism of an algicidal compound against red tide phytoplankton in a marine bacterium γ-proteobacterium

Strain MS-02-063, γ-proteobacterium, isolated from a coast area of Nagasaki, Japan, produced a red pigment which belongs to prodigiosin members. This pigment, PG-L-1, showed potent algicidal activity against various red tide phytoplanktons in a concentration-dependent manner. An understanding of a mechanism of PG-L-1 production by this marine bacterium may yield important new insights and strategies for preventing blooms of harmful flagellate algae in natural marine environments. Therefore, we analyzed the mechanisms of PG-L-1 production. In our previous study, the pigment production by this marine bacterium was completely inhibited at 1.56 μg/ml of erythromycin or 3.13 μg/ml of chloramphenicol, while minimal inhibitory concentrations for cell growth of erythromycin and chloramphenicol against this bacterium were >100 and 25 μg/ml, respectively. It is interesting to note that the ability of the pigment production in erythromycin-treated bacterium recovered by an addition of homoserine lactone. In fact, the pigment production was inhibited by β-cyclodextrin that inhibits autoinducer activities by a complex with N-acyl homoserine lactones. N-acyl homoserine lactones with autoinducer activities are ubiquitous bacterial signaling molecules that regulate gene expression in a cell density dependent process known as quorum sensing. Therefore, it was suggested that PG-L-1 produced by strain MS-02-063 is controlled by the homoserine lactone quorum sensing. It is speculated that this quorum sensing is involved in the production of algicidal agents of other marine bacteria. This bacterium and other algicidal bacteria might be concerned in regulating the blooms of harmful flagellate algae through the quorum sensing system.     

The quorum-sensing system AvsR-AvsI regulates both long-chain and short-chain acyl-homoserine lactones in <Emphasis Type="Italic">Agrobacterium vitis</Emphasis> E26

Agrobacterium vitis strain E26 is a nonpathogenic bacterium isolated from grape crown gall. In this study, the identification of a luxR-luxI type quorum-sensing system in strain E26 is reported. This system is involved in the induction of hypersensitive response on tobacco, but not its biocontrol activity. The deduced components AvsI_E26 and AvsR_E26 show the greatest similarity to AvsI_F2/5 and AvsR_F2/5, respectively from A. vitis strain F2/5. The mutant in AvsI_E26 abolished the production of both long-chain and short-chain acyl-homoserine lactones signals as well as the ability to cause hypersensitive response on tobacco. Complementaion of avsI _E26 and avsR _E26 genes restored the lost phenotypes to the level of wild type E26. In pot trial, no significant difference on biocontrol efficiency against grapevine crown gall was found between the wide type E26 and its quorum sensing negative mutants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24

Pseudomonas fluorescens 2P24 is a biocontrol agent isolated from a wheat take-all decline soil in China. This strain produces several antifungal compounds, such as 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide and siderophore(s). Our recent work revealed that strain 2P24 employs a quorum-sensing system to regulate its biocontrol activity. In this study, we identified a quorum-sensing system consisting of PcoR and PcoI of the LuxR–LuxI family from strain 2P24. Deletion of pcoI from 2P24 abolishes the production of the quorum-sensing signals, but does not detectably affect the production of antifungal metabolites. However, the mutant is significantly defective in biofilm formation, colonization on wheat rhizosphere and biocontrol ability against wheat take-all, whilst complementation of pcoI restores the biocontrol activity to the wild-type level. Our data indicate that quorum sensing is involved in regulation of biocontrol activity in P. fluorescens 2P24.     

Biological characteristics of plasmids of <Emphasis Type="Italic">Mesorhizobium huakuii</Emphasis> HN3015 from <Emphasis Type="Italic">Astragalus sinicus</Emphasis>

A Mesorhizobium huakuii strain HN3015 was isolated from Astragalus sinicus in a rice-growing field of Southern China. Strain HN3015 contained three large plasmids. The three indigenous plasmids, named as pMhHN3015a, pMhHN3015b and pMhHN3015c of M. huakuii HN3015, were, respectively, cured by Tn5-sacB insertion. The mutant strain HN3015-1 cured with its largest plasmid pMhHN3015c formed only white null nodules. Mutant HN3015-3 cured with its smallest plasmid pMhHN3015a could form pink effective nodules. However, mutant HN3015-2 cured of the second largest plasmid pMhHN3015b lost nodulation ability. Furthermore, curing of pMhHN3015a had enhanced competitive nodulation ability and symbiotic efficiency of HN3015-3. The results from acidity tolerance assays indicated that the three plasmids in M. huakuii HN3015 had a positive control effect on acidity tolerance of HN3015, and all indigenous plasmids of M. huakuii HN3015 had a negative control effect on the alkali tolerance capacity of HN3015. Surprisingly, all plasmids in M. huakuii HN3015 had also a negative control effect on its growth rate. The results showed an interactive and functional complexity of plasmids in strain HN3015.     

Regulation of motility by the ExpR/Sin quorum-sensing system in Sinorhizobium meliloti

A successful symbiotic relationship between Sinorhizobium meliloti and its host Medicago sativa (alfalfa) depends on several signaling mechanisms, such as the biosynthesis of exopolysaccharides (EPS) by S. meliloti. Previous work in our laboratory has shown that a quorum-sensing mechanism controls the production of the symbiotically active EPS II. Recent microarray analysis of the whole-genome expression profile of S. meliloti reveals that the ExpR/Sin quorum-sensing system regulates additional physiological processes that include low-molecular-weight succinoglycan production, nitrogen utilization, metal transport, motility, and chemotaxis. Nearly half of the flagellar genes and their dependence on quorum sensing are prominently displayed in our microarray analyses. We extend those observations in this work and confirm the findings by real-time PCR expression analysis of selected genes, including the flaF, flbT, flaC, cheY1, and flgB genes, involved in motility and chemotaxis. These genes code for regulators of flagellum synthesis, the chemotactic response, or parts of the flagellar apparatus. Gene expression analyses and visualization of flagella by electron microscopy performed at different points in the growth phase support our proposed model in which quorum sensing downregulates motility in S. meliloti. We demonstrate that the ExpR/Sin quorum-sensing system controls motility gene expression through the VisN/VisR/Rem relay. We also show that the ExoS-dependent two-component system suppresses motility gene expression through VisN and Rem in parallel to quorum sensing. This study contributes to our understanding of the mechanisms that govern motility in S. meliloti.     

The role of luxS in the fire blight pathogen Erwinia amylovora is limited to metabolism and does not involve quorum sensing

Erwinia amylovora is a gram-negative phytopathogen that causes fire blight of pome fruit and related members of the family Rosaceae. We sequenced the putative autoinducer-2 (AI-2) synthase gene luxS from E. amylovora. Diversity analysis indicated that this gene is extremely conserved among E. amylovora strains. Quorum sensing mediated by LuxS has been implicated in coordinated gene expression, growth, and virulence in other enterobacteria; however, our evidence suggests this is not the function in E. amylovora. Mutational analysis pointed to a role in colonization of apple blossoms, the primary infection court for fire blight, although little if any role in virulence on apple shoots and pear fruit was observed. Expression of key virulence genes hrpL and dspA/E was reduced in mutants of two E. amylovora strains. Stronger effects on gene expression were observed for metabolic genes involved in the activated methyl cycle with mutants having greater levels of expression. No quorum-sensing effect was observed in coculture experiments with wild-type and mutant strains either in vitro or in apple blossoms. Known receptors essential for AI-2 quorum sensing, the LuxPQ sensor kinase or the Lsr ABC-transporter, are absent in E. amylovora, further suggesting a primarily metabolic role for luxS in this bacterium.     

Genetic Determinants of Swarming in Rhizobium etli

Swarming motility is considered to be a social phenomenon that enables groups of bacteria to move coordinately atop solid surfaces. The differentiated swarmer cell population is embedded in an extracellular slime layer, and the phenomenon has previously been linked with biofilm formation and virulence. The gram-negative nitrogen-fixing soil bacterium Rhizobium etli CNPAF512 was previously shown to display swarming behavior on soft agar plates. In a search for novel genetic determinants of swarming, a detailed analysis of the swarming behavior of 700 miniTn5 mutants of R. etli was performed. Twenty-four mutants defective in swarming or displaying abnormal swarming patterns were identified and could be divided into three groups based on their swarming pattern. Fourteen mutants were completely swarming deficient, five mutants showed an atypical swarming pattern with no completely smooth edge and local extrusions, and five mutants displayed an intermediate swarming phenotype. Sequence analysis of the targeted genes indicated that the mutants were likely affected in quorum-sensing, polysaccharide composition or export, motility, and amino acid and polyamines metabolism. Several of the identified mutants displayed a reduced symbiotic nitrogen fixation activity.     

Quorum-sensing in Rhizobium

Quorum-sensing signals are found in many species of legume-nodulating rhizobia. In a well-characterized strain of R. leguminosarum biovar viciae, a variety of autoinducers are synthesised, and all have been identified as N-acyl-homoserine lactones. One of these N-acyl-homoserine lactones, is N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone, previously known as small bacteriocin, which inhibits the growth of several R. leguminosarum strains. The cinRI locus is responsible for the production of small bacteriocin. CinR induces cinI in response to the AHL made by CinI, thus forming a positive autoregulatory induction loop. A complex cascade of quorum-sensing loops was characterized, in which the cinIR locus appears to be the master control for three other AHL-dependent quorum-sensing control systems. These systems include the raiI/raiR, traI/triR and rhiI/rhiR. Other rhizobial strains appear to share some of these quorum sensing loci, but not all loci are found in all strains. Small bacteriocin along with the other N-acyl-homoserine lactones produced by these three AHL-based control systems regulate (i) growth inhibition of sensitive strains, (ii) transfer of the symbiotic plasmid pRL1JI, and (iii) expression of the rhizosphere-expressed (rhi) genes that influence nodulation. Some of the genes regulated by these systems have been identified. While the functions of some, such as the trb operon regulated by triR are clear, several of the regulated genes have no homologues of known function. It is anticipated that several other genes regulated by these systems have yet to be identified. Therefore, despite the regulation of one of the most complex quorum-sensing cascade being understood, several of the functions regulated by the quorum-sensing genes remain to be elucidated. This revised version was published online in August 2006 with corrections to the Cover Date.     

Exopolysaccharide biosynthesis is important for <Emphasis Type="Italic">Mesorhizobium tianshanense</Emphasis>: plant host interaction

Mesorhizobium tianshanense is a nitrogen-fixing bacterium that can establish symbiotic associations with Glycyrrhiza uralensis in the form of root nodules. Nodule formation in rhizobia often requires various secreted carbohydrates. To investigate exopolysaccharide (EPS) production and function in M. tianshanense, we performed a genome-wide screen using transposon mutagenesis to identify genes involved in EPS production. We identified seven mutants that produced significantly lower amounts of EPS as well as a two-component sensor kinase/response regulator system that is involved in the activation of EPS synthesis. EPS mutants formed significantly less biofilm and displayed severely reduced nodulation capacity than wild type bacteria, suggesting that EPS synthesis can play important roles in the symbiosis process. Peng Wang, Zengtao Zhong and Jing Zhou have contributed equally to this work.     

Incompatibility behavior of a megaplasmid pMhHN3015c in Mesorhizobium huakuii HN3015

The Tn5-sacB-labeled symbiotic megaplasmid pMhHN3015c of Mesorhizobium huakuii HN3015 was, respectively, transferred into M. huakuii HN308SR containing three large plasmids of pMhHN308a, pMhHN308b and pMhHN308c, and 7653R-1SR, a symbiotic plasmid pMh7653Rb deleted mutant from M. huakuii 7653R by tri-parent mating. The stable indigenous plasmid pMhHN308c of HN308SR was cured by the introduction of pMhHN3015c and the transconjugant was named as HN308SRN18. The results implied that pMhHN3015c and pMhHN308c were incompatible and might be ascribed to the same incompatibility group. Furthermore, the results from plasmid curing tests of HN308SRN18 containing pMhHN3015c, pMhHN308b, and pMhHN308a showed that not only was pMhHN3015c deleted, but that pMhHN308a was also cured simultaneously. The plasmid profiles of transconjugant 7653R-1SRN18 showed pMhHN3015c could coexist with pMh7563Ra. The plasmid replication repC-like gene sequences were detected by polymerase chain reaction from 7653R-1SRN18, HN308SRN18 and its plasmid-curing derivatives, but failed to detect from plasmid-curing derivatives of 7653R-1SRN18. The repC gene sequence similarities of strains tested were up to 99%. Results from plant nodulation tests showed that introduction of pMhHN3015c failed to restore the nitrogen fixation ability of HN308SRN18 and 7653R-1SRN18.