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Introduction

Rhizobium leguminosarumbiovar viciae isa rod-shaped, motile, gram-negative, nitrogen-fixing bacterium which fixes nitrogen on legumes such as lentils, peas and vetches.^1,2These legumes like others are important in the provision of body-building minerals, calcium, vitamins A and B, thiamine, niacin and oil.^3-7

Various research studies including works onrhi(rhizosphere-expressed) genes are being carried out on legume-rhizobia interaction to improve yields. These genes form an operon comprising ofrhiABC. The operon is located adjacent torhiR on symbiotic plasmid ofR.leguminosarumbiovar viciae.⁸Rhizosphere-expressed genes are under the influence ofrhiR. TherhiR is important in root nodulation. According to Gaoet al.,⁹the quorum-sensing system in a plant bacterium,Mesorhizobium huakuiiaffects growth rate and symbiotic nodulation. TherhiR encodes RhiR, a regulatory protein which functions as a transcriptional activator. RhiR has a homology with LuxR which belongs to LuxR-LuxI family. This class of transcriptional activators is common to a diverse group of gram-negative bacteria^10,11including the quorum-sensing signal molecules and the LuxRI homologs that were identified in fish pathogenEdwardsiella tarda.¹²Comprehensive profiling of N-acylhomoserine lactones formed byYersiniapseudotuberculosisusing liquid chromatographycoupled to hybrid quadrupole–linear ion trap mass spectrometry showed that the microbe belong to this group of transcriptional activators producers.¹³Pseudomonas aeruginosapossesses LasR whichfunctions to activate virulence.Agrobacteriumtumefacienssynthesizes TraR which is responsible for conjugation control.^14-17Also,Agrobacterium vitisbears aluxRhomolog,aviR, that is associated with induction of necrosis on Grape and a hypersensitive response on Tobacco.¹⁸LuxR family protein SpnR which functions as a negative regulator of N-acylhomoserine lactone-dependent quorum sensing is present inSerratia marcescens¹⁹. LuxR inVibrio fischeriregulates the expression of an operonluxICDABEG in the presence of VAI (Vibrioautoinducer, N-3-(oxohexanonyl) homoserine lactone) thus influences luminescence in the microbe. LuxR-LuxI family is a population density-dependent gene activators.

The regulatory mechanism exhibited by LuxR-LuxI family is known as quorum sensing. It involves the interaction of self-produced extracellular signal compounds (autoinducers) with transcriptional activator proteins. In quorum sensing, after an adequate cell density is obtained, autoinducer normally accumulates which enables the bacterium to take a decision to regulate the expression of specific sets of genes^10,20. Based on the homology which exists between RhiR and LuxR family, it was decided that investigation should be carried out on the principle of quorum sensing inR.leguminosarumbv。viciae。为了做到这一点,我们研究了英孚fects of cell density and mutation on the expression ofrhigenes in the bacterium. This could give an indication to the regulatory circuits involved in the symbiotic properties of theR.leguminosarumbv。viciae。

Material and Methods

Organism and growth media

Rhizobium leguminosarumbiovar viciaewas obtained from American Type Culture Collection (Rockville, Md.).Rhizobium melilotiwas kindly provided by Allan Downie with whom this work was carried out at John Innes Institute, Norwich, Great Britain.R. leguminosarumbiovar viciae andR. melilotilacking symbiotic (Sym) plasmids arethese strains that have been cured of their Sym plasmids. They were grown in minimal medium²¹and total yeast (TY) medium when required.²²

Table 1: Strain specificity of R. leguminosarum bv. viciae for the production of rhi genes inducer. Click here to view table

Test for strain specificity for the production ofrhigene inducer.

Late exponential growth phase cultures ofR. leguminosarumbiovar viciae andR. melilotiwith and without regulatory gene (rhiR) were tested for synthesis of therhigene inducer by alkaline phosphatase assay²²using arhiC::phoA fused gene.

Test for the effect of cell density onrhigene expression.

Strain ofR.leguminosarumbiovar viciae carryingrhiA::lacZ fused gene with and without symbiotic plasmid were grown at 28⁰Cin liquid TY medium,²²shaken at 180 rev min^-1for 72 hours. Cell growth by viable plate count method and β-galactosidase activity²²were determined during the incubation period at an interval of 6 hours.

Mutation experiment.

Transposon (Tn5) mutagenesis was carried out on mid-logarithmic phase cells ofR. leguminosarumbiovar viciae. One thousand one hundred and fifty mutants were screened on plate for the production ofrhigene inducer by alkaline phosphatase assay as described by²²Miller. Nineteen mutants found defective in the synthesis of the inducer were tested for alkaline phosphatase activity by liquid assay.²²Two controls (positive and negative) were prepared along with the mutants. The mutants were grouped into classes using respective 0-500, 501-1000, 1001-1500 and 1501- above Miller units of alkaline phosphatase.

Figure 1. Effect of cell density and symbiotic plasmid on rhi genes expression. Click here to view figure

Assay for β-galactosidase and alkaline phosphatase activities.

Methods described by²²米勒被雇佣。b-galactosidase的是slightly modified and carried out as follows. Reaction mixture contained 0.9 ml culture and 0.2 ml isopropyl-thio-b-D-galactoside. The reaction was stopped after five minutes at room temperature (27^oC) with 0.5 ml 1 M Na₂Co₃. Optical density of the mixture was read at 600nm and 420nm. The latter density was corrected to obtain the actual optical density. Units of b-galactosidase activity (Miller units) was defined as the absorbance at 420nm and 600nm per minute per millilitre of the reaction product under this assay condition.

Results

Phosphatase activity for synyhesis ofrhiR inducer byR. melilotiwas low relative to that ofR.leguminosarumwith and without symbiotic plasmidTable 1. Growth phase influencedrhigenes action Fig . 1. The colony forming units increased with incubation time inR. leguminosarumin the presence and absence of symbiotic plasmid. The two bacterial strains showed similar growth pattern without any significant difference. However, there was an increase in the activity of b-galactosidase with increased cell population in the presence of symbiotic plasmid with highest value of 237.5 Miller units in comparison with 12.5-17.5 Miller units for strain lacking symbiotic plasmid Fig. 1. This activity was highest in the exponential phase with pronounced increase in the mid-logarithmic phase than in any other growth phases.

Table 2 shows that the mutants were defective in the production ofrhigenes inducer at various levels. Class I mutants (low-producers) having the inducer synthetic ability of 145-250 Miller units of phosphatase activity respectively comprised of mutant numbers 9, 15 and 16. Class II mutants (moderate-producers) were mutant numbers 1, 3, 4, 5, 6, 8, 11 and 14 with 625-896 Miller units. Class III mutants (high-producers, 1031-1375 Miller units) were made up of mutant numbers 2, 7, 10, 12, 13, 17 and 18 while class IV (super-producer) has a single member, number 19 showing formation ability of inducer at and 1563 Miller units. Relative to the negative and positive controls Table 2.

Table 2: Effect of mutation on rhi genes inducer. Click here to view table

Discussion

Synthesis of large amount ofrhigenes inducer by theR. leguminosarumthanR. melilotiindicates that production of this metabolite is specific for the former bacterium and thus suggests that only certain rhizobial species form the compound. The inability ofR. melilotito express therhigenes at high level was possibly due to presence of other genes located on the symbiotic plasmid which could block the production of appropriate metabolite to induce therhigenes expression.

β-galactosidase activity highest in the exponential phase with pronounced increase in the mid-logarithmic phase than in any other growth phases indicates thatrhigenes function optimally when the cells are in the active stage of their growth. Thus, the regulation ofrhigenes by RhiR is growth phase and cell density dependent. Stationary phase cells appeared less active than logarithmic phase cells. Although, diversity in production of and response to N-acyl homoserine lactones is possible²³; the result obtained here is similar to that reported by Fuquaet al.,¹⁰and Gaoet al.,¹¹Fuquaet al.,¹⁰said that LuxR-LuxI family of transcriptional regulators are cell density dependent inVibrio, Erwinia, Pseudomonas andAgrobacterium. Gaoet al.,¹¹showed that rate ofgrowth and symbiotic nodulation were directly related to the quorum-sensing system inMesorhizobium huakuii. Many plant-associatedbacteria regulate gene expression in a cell density-dependent manner by using quorum sensing viaN-acyl homoserine lactones (AHLs). These AHLspass out of and into bacterial cells. As the population of bacteria increases, so does the concentration of AHLs.²⁴

The data on mutation implies that Class I mutants were strongly defective inrhigenes induction, class II were averagely defective and class IV mutantrhigenes were enhanced by the mutation to express the genes better than the wild type. Class III appeared unaffected being related to the wild-type strains (positive controls 1 and 2). This could be becauserhigenes in this class were not affected by the Tn5 mutation to lack or produce the metabolite at lower or higher level in contrast to the last two Classes I and II.

Conclusion

Synthesis ofrhigenes inducer was specific forR. leguminosarumbiovar viciae. The bacterium expressedrhigenes in a cell density dependent pattern. Symbiotic plasmid is vital for the action ofrhigenes;rhigenes induction is under the influence ofrhiR which is located on the symbiotic plasmid. Tn-5 mutation affected therhigenes induction.

Acknowledgements

The authors thank British Council, John Innes Institute, Great Britain and Federal University of Technology Akure, Nigeria for the financial support and instrumentation needed during this research work.

References

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