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• W2113122980 abstract "Rhizobium etli CNPAF512 produces an autoinducer that inhibits growth of Rhizobium leguminosarum bv. viciae 248 and activates theAgrobacterium tumefaciens tra reporter system. Production of this compound in R. etli is dependent on two genes, named cinR and cinI, postulated to code for a transcriptional regulator and an autoinducer synthase, respectively. NMR analysis of the purified molecule indicates that the R. etli autoinducer produced by CinI is a saturated long chain 3-hydroxy-acyl-homoserine lactone, abbreviated as 3OH-(slc)-HSL. Usingcin-gusA fusions, expression ofcinI and cinR was shown to be growth phase-dependent. Deletion analysis of the cinIpromoter region indicates that a regulatory element negatively controlscinI expression. Mutational analysis revealed that expression of the cinI gene is positively regulated by the CinR/3OH-(slc)-HSL complex. Besides 3OH-(slc)-HSL, R. etliproduces at least six other autoinducer molecules, for which the structures have not yet been revealed, and of which the synthesis requires the previously identified raiI andraiR genes. At least three different autoinducers, including a compound co-migrating with 3OH-(slc)-HSL, are produced inR. etli bacteroids isolated from bean nodules. This is further substantiated by the observation that cinI andcinR are both expressed under symbiotic conditions. Acetylene reduction activity of nodules induced by the cinmutants was reduced with 60–70% compared with wild-type nodules, indicating that the R. etli 3OH-(slc)-HSL is involved in the symbiotic process. This was further confirmed by transmission electron microscopy of nodules induced by the wild type and thecinI mutant. Symbiosomes carrying cinI mutant bacteroids did not fully differentiate compared with wild-type symbiosomes. Finally, it was observed that the cinRgene and raiR control growth of R. etli. Rhizobium etli CNPAF512 produces an autoinducer that inhibits growth of Rhizobium leguminosarum bv. viciae 248 and activates theAgrobacterium tumefaciens tra reporter system. Production of this compound in R. etli is dependent on two genes, named cinR and cinI, postulated to code for a transcriptional regulator and an autoinducer synthase, respectively. NMR analysis of the purified molecule indicates that the R. etli autoinducer produced by CinI is a saturated long chain 3-hydroxy-acyl-homoserine lactone, abbreviated as 3OH-(slc)-HSL. Usingcin-gusA fusions, expression ofcinI and cinR was shown to be growth phase-dependent. Deletion analysis of the cinIpromoter region indicates that a regulatory element negatively controlscinI expression. Mutational analysis revealed that expression of the cinI gene is positively regulated by the CinR/3OH-(slc)-HSL complex. Besides 3OH-(slc)-HSL, R. etliproduces at least six other autoinducer molecules, for which the structures have not yet been revealed, and of which the synthesis requires the previously identified raiI andraiR genes. At least three different autoinducers, including a compound co-migrating with 3OH-(slc)-HSL, are produced inR. etli bacteroids isolated from bean nodules. This is further substantiated by the observation that cinI andcinR are both expressed under symbiotic conditions. Acetylene reduction activity of nodules induced by the cinmutants was reduced with 60–70% compared with wild-type nodules, indicating that the R. etli 3OH-(slc)-HSL is involved in the symbiotic process. This was further confirmed by transmission electron microscopy of nodules induced by the wild type and thecinI mutant. Symbiosomes carrying cinI mutant bacteroids did not fully differentiate compared with wild-type symbiosomes. Finally, it was observed that the cinRgene and raiR control growth of R. etli. N-acyl-homoserine lactone transmission electron microscopic analysis open-reading frame high-performance liquid chromatography 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside acetylene reduction activity thin layer chromatography diketopiperasines Although bacteria are unicellular organisms, they often show group behavior. For this, bacteria have to monitor their own population size. This can be achieved by means of autoinduction. Cell-cell communication using N-acyl-homoserine lactone (AHL)1 signals is one of the few known mechanisms through which bacteria can communicate with each other and is a widespread phenomenon in Gram-negative bacteria (1Fuqua C. Winans S.C. Greenberg E.P. Annu. Rev. Microbiol. 1996; 50: 727-751Crossref PubMed Scopus (924) Google Scholar, 2Swift S. Williams P. Stewart G.S.A.B. Cell Cell Signaling in Bacteria.. ASM Press, Washington, D.C.1999: 291-313Google Scholar), including plant-associated bacteria (3Shaw P.D. Ping G. Daly S.L. Cha C. Cronan J.E. Rinehart K.L. Farrand S.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6036-6041Crossref PubMed Scopus (679) Google Scholar, 4Pierson L.S. Wood D.W. Pierson E.A. Annu. Rev. Phytopathol. 1998; 36: 207-225Crossref PubMed Scopus (116) Google Scholar). AHLs mainly vary with respect to the length (4–14 carbons) and the substituent (H, O, or OH) at the third carbon of the acyl side chain. The AHL signal is released into the environment, either by passive diffusion, as observed for 3O-C6-HSL in Vibrio fischeri andEscherichia coli cells (5Kaplan H.B. Greenberg E.P. J. Bacteriol. 1985; 163: 1210-1214Crossref PubMed Google Scholar) or by a combination of diffusion and active efflux in Pseudomonas aeruginosa (6Pearson J.P. Van Delden C. Iglewski B.H. J. Bacteriol. 1999; 181: 1203-1210Crossref PubMed Google Scholar) and accumulates with growth of the bacterial population. At least inV. fischeri, the signal freely diffuses back into the cells such that its intracellular concentration also rises as a function of the increase in bacterial population. Transduction of this information to response regulators of gene expression leads to the elaboration of an appropriate phenotype at high cell densities. Using the Agrobacterium tumefaciens tra reporter system to detect autoinducer molecules, members of the genus Rhizobiumshowed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals (7Cha C. Gao P. Chen Y.C. Shaw P.D. Farrand S.K. Mol. Plant Microbe Interact. 1998; 11: 1119-1129Crossref PubMed Scopus (512) Google Scholar). InRhizobium leguminosarum bv. viciae, thecin locus encodes a master regulatory system. Mutation ofcinIR abolishes the production ofN-(3R)-hydroxy-7-cis-tetradecenoyl-l-homoserine lactone (3OH-C14:1-HSL), also termed “small”, and reduces the synthesis of AHLs produced by the enzymes encoded byraiI, traI-like, or rhiI (8Lithgow J.K. Wilkinson A. Hardman A. Rodelas B. Wisniewski-Dye F. Williams P. Downie J.A. Mol. Microbiol. 2000; 37: 81-97Crossref PubMed Scopus (171) Google Scholar). The reduced levels of C6-HSL and C8-HSL and decreased rhiR expression cause a repression of the rhizosphere-expressed genes in cinI or cinRmutants (8Lithgow J.K. Wilkinson A. Hardman A. Rodelas B. Wisniewski-Dye F. Williams P. Downie J.A. Mol. Microbiol. 2000; 37: 81-97Crossref PubMed Scopus (171) Google Scholar, 9Gray K.M. Pearson J.P. Downie J.A. Boboye B.E.A. Greenberg E.P. J. Bacteriol. 1996; 178: 372-376Crossref PubMed Google Scholar, 10Rodelas B. Lithgow J.K. Wisniewski-Dye F. Hardman A. Wilkinson A. Economou A. Williams P. Downie J.A. J. Bacteriol. 1999; 181: 3816-3823Crossref PubMed Google Scholar). Furthermore, 3OH-C14:1-HSL induces the stationary phase (9Gray K.M. Pearson J.P. Downie J.A. Boboye B.E.A. Greenberg E.P. J. Bacteriol. 1996; 178: 372-376Crossref PubMed Google Scholar) whereas mutation of cinI has little effect on growth or nodulation of the host plant (8Lithgow J.K. Wilkinson A. Hardman A. Rodelas B. Wisniewski-Dye F. Williams P. Downie J.A. Mol. Microbiol. 2000; 37: 81-97Crossref PubMed Scopus (171) Google Scholar, 11van Brussel A.A. Zaat S.A. Wijffelman C.A. Pees E. Lugtenberg B.J. J. Bacteriol. 1985; 162: 1079-1082Crossref PubMed Google Scholar). Rhizobium etli CNPAF512, a nitrogen-fixing symbiont ofPhaseolus vulgaris, produces at least seven different autoinducer molecules (12Rosemeyer V. Michiels J. Verreth C. Vanderleyden J. J. Bacteriol. 1998; 180: 815-821Crossref PubMed Google Scholar). Rosemeyer et al. (12Rosemeyer V. Michiels J. Verreth C. Vanderleyden J. J. Bacteriol. 1998; 180: 815-821Crossref PubMed Google Scholar) identified the raiIR quorum-sensing system in R. etli. Examination of different rai mutants for nodulation of beans showed that raiI is involved in the restriction of nodule number, whereas nitrogen-fixing activity per nodule is not affected. The culture supernatant of a raiI mutant revealed only three different autoinducer molecules. One of them induces a growth-inhibitory effect on R. leguminosarum bv.viciae 248, similar to the low molecular weight bacteriocin “small”, which is common in fast-growing rhizobia. The properties, growth inhibition, and autoinducer activity, are features reported for 3OH-C14:1-HSL, produced by R. leguminosarum bv.viciae (13Schripsema J. de Rudder K.E. van Vliet T.B. Lankhorst P.P. de Vroom E. Kijne J.W. van Brussel A.A. J. Bacteriol. 1996; 178: 366-371Crossref PubMed Google Scholar). Here we report on the cin locus, the second quorum-sensing system in R. etli CNPAF512 that is expressed under both free-living and symbiotic conditions and is involved in the production of a 3OH-(slc)-HSL (slc, saturated long chain). Despite high sequence conservation, the cin locus of R. etli andR. leguminosarum bv. viciae appear to control different functions. Mutational analysis of R. etli revealed that the cin system regulates growth and fulfills a key role in bacteroid differentiation and nitrogen fixation. E. coli was grown in Luria-Bertani (LB) medium at 37 °C (14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1989Google Scholar). Rhizobium was grown at 30 °C in TY or AMS medium (15Michiels J. Van Soom T. D'hooghe I. Dombrecht B. Benhassine T. de Wilde P. Vanderleyden J. J. Bacteriol. 1998; 180: 1729-1740Crossref PubMed Google Scholar). A. tumefaciens NT1 was grown in AB medium at 28 °C (16Chilton M.D. Currier T.C. Farrand S.K. Bendich A.J. Gordon M.P. Nester E.W. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3672-3676Crossref PubMed Scopus (545) Google Scholar). Antibiotics were added as appropriate. To study bacterial growth over a long period of time, overnight cultures of the strains were diluted in 10 mm MgSO4 to an absorbance at 595 nm (A595) of 0.3 (dilution ∼10-fold). Subsequently, these cultures were diluted 10-fold after which 295 μl of growth medium was inoculated with 5 μl of bacterial suspension (total dilution ∼6000-fold). Bacteria were grown, and the absorbance was measured automatically each 30 min during at least 6 days in a BioscreenC (Labsystems Oy). For each time point, the average optical density was calculated from five independent measurements. Phaseolus vulgaris cv. Limburgse vroege seedlings were planted in Snoeck medium, which is optimized for in vitro growth of common bean. 2C. Snoeck, unpublished data. Plants were inoculated and grown essentially as described by Michiels et al. (17Michiels J. Moris M. Dombrecht B. Verreth C. Vanderleyden J. J. Bacteriol. 1998; 180: 3620-3628Crossref PubMed Google Scholar). Acetylene reduction activity was determined 3 weeks after inoculation. For expression analysis during symbiosis, bacteroids were purified from plant material by differential centrifugation (17Michiels J. Moris M. Dombrecht B. Verreth C. Vanderleyden J. J. Bacteriol. 1998; 180: 3620-3628Crossref PubMed Google Scholar). This protocol was slightly adapted for the extraction of bacteroid autoinducers. Nodules from 1–2 plants (± 1.5 g) were collected in a Falcon tube (15 ml) containing 0.2 g of polyvinyl polypyrolidone, and magnesium phosphate buffer was added to a final volume of 6 ml and subsequently crushed. Next, the plant material was removed by differential centrifugation. To lyse the bacteria, the suspension was supplemented with SDS (final concentration, 1%) and proteinase K (final concentration, 100 μg/ml) and incubated for 1 h at 37 °C. After incubation, the cell debris was removed by centrifugation at 6000 rpm after which the cell-free supernatant was immediately extracted to isolate the autoinducers. Qualitative and quantitative analysis of β-glucuronidase (GusA) activity was performed as described elsewhere (17Michiels J. Moris M. Dombrecht B. Verreth C. Vanderleyden J. J. Bacteriol. 1998; 180: 3620-3628Crossref PubMed Google Scholar, 18Vande Broek A. Vanderleyden J. Mol. Plant Microbe Interact. 1995; 8: 800-810Crossref Google Scholar). For transmission electron microscopic analysis (TEM), thin sections of 3-week-old nodules were prepared as described by Xi et al. (19Xi C. Schoeters E. Vanderleyden J. Michiels J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11114-11119Crossref PubMed Scopus (50) Google Scholar), and analyzed in a Zeiss EM 900 electron microscope. Rhizobium strains and A. tumefaciens transformants were tested for activation of theA. tumefaciens tra reporter system and for bacteriostatic activity toward the sensitive strain R. leguminosarum bv.viciae 248 as described by Schripsema et al. (13Schripsema J. de Rudder K.E. van Vliet T.B. Lankhorst P.P. de Vroom E. Kijne J.W. van Brussel A.A. J. Bacteriol. 1996; 178: 366-371Crossref PubMed Google Scholar). To extract autoinducers, strains were first grown to the stationary phase. Cell-free supernatant from either a free-living liquid culture or from symbiotic bacteroids (see above) was extracted, and the autoinducers were detected on TLC by the trareporter system as described by Rosemeyer et al. (12Rosemeyer V. Michiels J. Verreth C. Vanderleyden J. J. Bacteriol. 1998; 180: 815-821Crossref PubMed Google Scholar). Ethyl acetate extracts from 10 liters of stationary phase cultures of FAJ4010 were reconstituted in 50% acetonitrile in water and subjected to solid-phase extraction using Waters OASIS HLB cartridges. Fractions were eluted with an increasing concentration of methanol in water (50–100%, v/v). Positive fractions on the A. tumefaciens tra reporter and R. leguminosarum bv. viciae248 growth inhibition assays were collected, dried, and redissolved in 50% acetonitrile in water and applied to a C18 Phenomenex Bondclone HPLC column. Fractions were eluted with a linear gradient of acetonitrile in water (40–100%, v/v) over a 30-min period at a flow rate of 1 ml/min and monitored at 200 nm. Positive fractions were re-chromatographed using an isocratic mobile phase (50% acetonitrile in water; 1 ml/min), and the active subfraction was analyzed by NMR spectroscopy. Standard techniques were used for DNA manipulations (14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1989Google Scholar). Restriction enzymes were used according to the manufacturer's instructions. DNA probes for Southern hybridization were labeled with digoxigenin. An ordered series of sequencing clones was obtained via restriction enzyme mapping of pFAJ4003 and ExoIII deletion procedures (Erase-A-Base® Promega). Nucleotide sequencing ofcinR, cinI, and the flanking regions was accomplished by using the A.L.F. sequencer (Amersham Biosciences). The 5.3-kb EcoRI fragment from pFAJ4000 containing cinR, cinI, orf123, andorf140 was first cloned into pBluescriptIIKs+yielding pFAJ4003. A PstI-EcoRI fragment of pFAJ4003, lacking the 5′-end of orf140, wasEcoRI-PstI subcloned in the broad host range vector pLAFR3 (pFAJ4012; Fig. 1). Furthermore, pFAJ4013 was made by insertion of the 5.3-kb EcoRI fragment from pFAJ4003 in pPZP200, in which part of the multiple cloning site betweenXhoI and PacI was deleted. A fragment containing cinR and cinI was amplified via PCR using primers Rhi15 (5′-ATGGGAATTCATCCAGTGCCGAGGAGATAC-3′) and Rhi16 (5′-TAGAGGATCCTCGGCATCATCATCACCTCG-3′) and pFAJ4003 as template DNA. The resulting 3-kb fragment was digested with EcoRI andBamHI and cloned in pUCNotI-ΔS (pUCNotI derivative lacking the SphI recognition site via restriction digest and blunt ligation) yielding plasmid pFAJ4004 (Fig. 1). The 2.2-kbBamHI fragment from pHP45Ω-Km containing a Kmrcassette was ligated into the unique SphI site ofcinI in pFAJ4004 after blunting of the fragments. ThecinRcinI::Km locus was further cloned into thesacB suicide vector pJQ200uc1 as a 5.2-kb NotI fragment to obtain pFAJ4006. The non-polar cinR point mutation and an additional frameshift were introduced via the QuickChange™ site-directed mutagenesis (Stratagene) using primers Rhi32 (5′-CTATGCCATGCTGGTACCATGCCCAGAAGCACG-3′) and Rhi33 (5′-CGTGCTTCTGGGCATGGTACCAGCATGGCATAG-3′) on pFAJ4004. As a result of the mutation, a new and unique KpnI site (GGTACC; mutations are shown in bold: insertion of A; substitution G with C) was created in cinR. Subsequently, the 3-kb NotI fragment containing the mutatedcinR gene was inserted into pJQ200uc1 creating pFAJ4007. ThecinR::Sp mutation was made by introducing the 2-kb spectinomycin resistance cartridge from pHP45Ω into the uniqueKpnI site within cinR of pFAJ4007 after blunting of the fragments, resulting in pFAJ4009. The plasmids pFAJ4006, pFAJ4007, and pFAJ4009 were introduced into R. etliCNPAF512, and double recombinants were selected as described previously (12Rosemeyer V. Michiels J. Verreth C. Vanderleyden J. J. Bacteriol. 1998; 180: 815-821Crossref PubMed Google Scholar) creating a cinI mutant (FAJ4006) and twocinR mutants (FAJ4007, FAJ4009; Fig. 1). A cartridge containing the promoterless gusA gene and a spectinomycin resistance gene from pWM5 was removed with SmaI and ligated into the XhoI site of pFAJ1327 (containing raiI,raiR, and orf1) after blunting of the vector, yielding plasmid pFAJ4010. For the construction of a R. etli raiI mutant strain, pFAJ4010 was introduced into the R. etli CNPAF512 wild-type strain, yielding FAJ4010. Tri-parental conjugation of pFAJ4006 (cinI::Km) into FAJ4010 resulted in the raiI::SpcinI::Km double mutant FAJ4013. Plasmid-bornePcinR-gusA andPcinI-gusA fusions were constructed in the promoter probe vector pFAJ1703 (20Dombrecht B. Vanderleyden J. Michiels J. Mol. Plant Microbe Interact. 2001; 14: 426-430Crossref PubMed Scopus (103) Google Scholar). The cinRpromoter region was amplified by PCR with FAJ1337 as a template using primers Rhi15 and Tn5-B (5′-GGTTCCGTTCAGGACGCTAC-3′). The resulting 1.2-kb fragment was cloned into the HpaI site of pFAJ1703 after blunting of the fragments, yielding plasmid pFAJ4011. To construct pFAJ4014 (cinI-gusA fusion carrying a mutant cinR gene region), the 1.8-kbEcoRI-SphI fragment of pFAJ4007 was blunt-end ligated into the HpaI site of pFAJ1703. pFAJ4015 is aKpnI deletion derivative of thecinI-gusA fusion pFAJ4014 (Fig. 1). A Tn5-induced mutant library of R. etli CNPAF512 was screened using the growth inhibition assay (13Schripsema J. de Rudder K.E. van Vliet T.B. Lankhorst P.P. de Vroom E. Kijne J.W. van Brussel A.A. J. Bacteriol. 1996; 178: 366-371Crossref PubMed Google Scholar). The mutant FAJ1337 no longer inhibited growth of R. leguminosarum bv.viciae 248. By means of inverse PCR onEcoRI-digested genomic DNA of FAJ1337 with transposon-specific primers, ∼10 kb of the transposon flanking DNA of FAJ1337 was amplified and cloned. Subsequently, PstI fragments of the amplified DNA were subcloned into pUC18 and tested for autoinducer production in E. coli using the trasystem of A. tumefaciens as a reporter (7Cha C. Gao P. Chen Y.C. Shaw P.D. Farrand S.K. Mol. Plant Microbe Interact. 1998; 11: 1119-1129Crossref PubMed Scopus (512) Google Scholar). One positive clone was obtained, and the corresponding 7.5-kb PstI fragment, carrying part of the cloning vector, was subsequently used as a probe in a Southern hybridization of a R. etli CNPAF512 genomic library, constructed in pLAFR1 (21D'hooghe I. Michiels J. Vlassak K. Verreth C. Waelkens F. Vanderleyden J. Mol. Gen. Genet. 1995; 249: 117-126Crossref PubMed Scopus (39) Google Scholar). A 5.3-kb EcoRI fragment from clone pFAJ4000 was found to give a positive hybridization signal. FAJ1337 was complemented for growth inhibition of the sensitive strain R. leguminosarum bv. viciae 248 by both pFAJ4000 and pFAJ4012 (Fig. 1). DNA sequence analysis of the cloned 5.3-kb EcoRI fragment revealed five complete open reading frames (ORFs) as illustrated in Fig. 1. The ORFs were identified and the start codon was assigned on the basis of the GC content (22Bibb M.J. Findlay P.R. Johnson M.W. Gene (Amst.). 1984; 30: 157-166Crossref PubMed Scopus (648) Google Scholar), the preferential codon usage (searchcutg, GCG-package Wisconsin), and similarity with known genes. One ORF (726 bp) codes for a putative protein of 241 amino acids with a calculated molecular mass of 27.3 kDa. The putative protein is similar to several LuxR-type transcriptional activators such as CinR (96% amino acid identity) of R. leguminosarum bv.viciae (AAF89989), CerR (30% identity) of Rhodobacter sphaeroides (AAC46021) and RaiR (31% identity) of R. etli (AAC38173). Because of the high amino acid identity of theR. etli putative protein with CinR, it was given the same name. Alignment of R. etli CinR with E. coli NarL (23Baikalov I. Schroder I. Kaczor-Grzeskowiak M. Grzeskowiak K. Gunsalus R.P. Dickerson R.E. Biochemistry. 1996; 35: 11053-11061Crossref PubMed Scopus (280) Google Scholar), designates a helix-turn-helix motif between residues 196 and 220.In silico analysis of the cinR non-coding region revealed a putative terminator (nucleotides 2615–2650, ΔG = −26.3). PCR analysis with a Tn5-specific primer combined with primers within the coding sequence of cinR or cinI (see below) indicated that the transposon in FAJ1337 is inserted between nucleotides 423 and 424 of cinR. A second ORF of 666 bp, which is unidirectional with cinR, is found 224-bp downstream of cinR. While the deduced amino acid sequence is most related to CinI (95% identity) of R. leguminosarum bv. viciae (AAF89990), it is also similar to CerI (33% identity) of R. sphaeroides (AAC46022) and RaiI of R. etli (39% identity) (AAC38172). The putative protein with a calculated molecular mass of 25.0 kDa was named CinI. CinI contains 10 invariant amino acids typical for the LuxI family of autoinducer synthases (24Parsek M.R. Schaefer A.L. Greenberg E.P. Mol. Microbiol. 1997; 26: 301-310Crossref PubMed Scopus (73) Google Scholar) of which seven (R24, E43, D45, D48, R70, E101, R104; numbered with respect to R. etli CinI) may take part in the S-adenosyl-methionine binding site (25Fuqua C. Greenberg E.P. Curr. Opin. Microbiol. 1998; 1: 183-189Crossref PubMed Scopus (248) Google Scholar). In the intergenic region between cinI and ORF123 (see below), two putative terminators (nucleotides 3520–3576, ΔG = −23.8; nucleotide 3694–3735, ΔG = −32.4) were found. Immediately downstream of cinI, a short ORF123 (368 bp), located on the opposite strand, encodes a putative response regulator of the CheY family with 94% identity to the CheY like protein ofR. leguminosarum bv. viciae (AAF89991), 40% identity to a probable response regulator of Mesorhizobium loti (BAB49462) and 35% identity to the FixL receiver domain ofR. etli (AAG00949). The R. etli response regulator encoded by ORF123 contains the conserved residues D14, D58, T86 en K106 (numbered with respect to ORF123), which are part of the essential active site of CheY (26Volz K. Biochemistry. 1993; 32: 11741-11753Crossref PubMed Scopus (255) Google Scholar) in which D58 can be phosphorylated. ORF140 (420 bp) is located upstream of cinR and codes for a protein similar to a hypothetical protein (AAG2039) ofHalobacterium sp. NRC-1 (51% identity), and an unknown protein of Bacillus subtilis (CAB11811) (39% identity). Upstream of ORF123, a Met-tRNA gene (74 bp) (tRNAscan-S.E. v. 1.11) is found with the anticodon (CAT) located between nucleotides 4364 and 4366 of the 5.3-kb EcoRI fragment. This gene shows perfect (100%) DNA sequence identity to the Met-tRNA gene of R. leguminosarum bv. viciae (AF210630) and a M. loti sequence (AP002999) and is similar to a Rhizobiumsp. NGR234 sequence (AE000079) (91% identity). Analysis of the intergenic region between ORF123 and the Met-tRNA gene indicates the presence of a putative terminator sequence downstream of the tRNA gene (nucleotides 5002–5068, ΔG = −22.9). An NMR analysis of the compound produced by R. etli CinI was conducted. As a control, 3OH-C14:1-HSL was synthesized (data not shown) and analyzed. The NMR data of the synthetic compound are in agreement with previously recorded data (13Schripsema J. de Rudder K.E. van Vliet T.B. Lankhorst P.P. de Vroom E. Kijne J.W. van Brussel A.A. J. Bacteriol. 1996; 178: 366-371Crossref PubMed Google Scholar). The 1H NMR spectrum of the R. etli compound contains all characteristic signals of a 3-hydroxyacyl-homoserine lactone. Evidence for the homoserine lactone moiety is constituted by the signal at 6.33–6.24 ppm (amide NH) and the characteristic butyrolactone signals at 4.52, 4.47, 4.27, 2.77, and 2.10 ppm. The line shapes and splitting patterns are in good agreement with those of synthetic acyl-homoserine lactones. Moreover, the line at 3.98 ppm is similar to the CH(OH) resonance in 3OH-C14:1-HSL. However, the characteristic double bond signals between 5 and 6 ppm observed for 3OH-C14:1-HSL, as well as the signals around 2.00 ppm of the protons on adjacent carbon atoms are absent. On the basis of its chromatographic properties (TLC, HPLC), the R. etli HSL is likely to possess a long chain fatty acid group. This allows to tentatively assign the spectrum of the R. etli autoinducer produced by CinI to a saturated long chain 3-hydroxy-acyl-homoserine lactone, which is clearly different from the structure of R. leguminosarum 3OH-C14:1-HSL. In the subsequent part we will refer to the R. etli autoinducer as 3OH-(slc)-HSL. To study the cell density-dependent expression of the cin locus,cinR-gusA (pFAJ4011) andcinI-gusA (pFAJ4014) fusions were constructed. The cinR gene in pFAJ4014 was inactivated by site-directed mutagenesis. To determine whether a promoter is present in thecinR-cinI intergenic region, a secondcinI-gusA (pFAJ4015) fusion, containing a 632-bp upstream region of cinI (Fig. 1), was also constructed. As shown in Fig. 2A,cinI expression from pFAJ4014 under free-living conditions in a wild-type background increased with the cell density and reached a plateau (1500–2400 units) as the culture entered into the stationary phase. cinI expression from pFAJ4015 displayed a similar cell density-dependent pattern of expression (Fig.2B). However, two differences between the two fusions can be noticed. Firstly, induction of cinI expression from pFAJ4015 starts at a lower absorbance compared with pFAJ4014. Secondly, the maximum expression level of cinI from pFAJ4015 is approximately 4-fold higher, compared with that of pFAJ4014. None of the cinI-gusA fusions are expressed in cinR orcinI mutant backgrounds (Fig. 2, A andB), demonstrating that transcription of cinIrequires both CinI and CinR. A threshold cell density (approximately A595 = 0.6) seems to be required to observe a very low cinRexpression in wild-type and cinR or cinI mutant backgrounds (Fig. 2C). Although expression ofcinR remains low, it reaches a maximum (∼20 units) as soon as cells enter the stationary phase. The observation that bothcinR and cinI expression is maximal during the same stage of growth is in agreement with a role of CinR in the regulation of cinI expression. The observation that cinI is expressed in either the presence or absence of the cinR promoter region, demonstrates that cinR and cinI are likely organized into different transcriptional units. Furthermore, the overall high expression level of cinI (minimal 100-fold higher than cinR), suggests that transcription ofcinI in both pFAJ4014 and pFAJ4015 is controlled by a promoter in the cinR-cinI intergenic region. This is in agreement with the presence of a putative terminator downstream of cinR. The difference in cinI expression levels between pFAJ4014 and pFAJ4015, is likely caused by a negative regulation at the level of the putative cinI promoter. Expression of thecinI-gusA and cinR-gusAfusions was monitored in isolated bacteroids, obtained from 21-day-old bean nodules, induced by wild-type CNPAF512, the cinR(FAJ4007), and cinI mutant (FAJ4006). The presence of the plasmid-borne gusA-fusions pFAJ4011, pFAJ4014, and pFAJ4015 in the different strains did not affect their symbiotic performance (data not shown). As illustrated in Fig.3, expression of the cinRfusion (pFAJ4011) is low (∼40 units) and does not significantly differ in the three genetic backgrounds. Expression of the shortcinI fusion (pFAJ4015) is significantly higher (∼8-fold) compared with the long cinI fusion (pFAJ4014) in wild-type background, similar to what was observed under free-living conditions. However, in contrast to free-living conditions, expression of the shortcinI fusion is less dependent on the presence of CinI because cinI expression from pFAJ4015 is still observed incinI mutant (FAJ4006) bacteroids. Similarly to free-living conditions, cinI gene expression requires the presence of CinR. Finally, it should be noted that the expression level ofcinI in bacteroids is at least 10-fold lower compared with free-living conditions. Shortly after inoculation (24 h), cinI-gusAexpression (pFAJ4015) was observed on the surface of root hairs during colonization of wild-type and the cinI mutant (FAJ4006) (data not shown). Light microscopic analysis, 48 h after inoculation of the roots, localized pFAJ4015 expression in the infection tread, formed by wild-type and the FAJ4006 mutant (Fig.4, A and B). However, no cinI-gusA expression from pFAJ4015 was observed in the cinR mutant (FAJ4007) during colonization (data" @default.
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• W2113122980 title "The cin Quorum Sensing Locus of Rhizobium etli CNPAF512 Affects Growth and Symbiotic Nitrogen Fixation" @default.
• W2113122980 cites W1500430545 @default.
• W2113122980 cites W1567514429 @default.
• W2113122980 cites W1593334283 @default.
• W2113122980 cites W1632889752 @default.
• W2113122980 cites W1641345914 @default.
• W2113122980 cites W1681269431 @default.
• W2113122980 cites W1766151574 @default.
• W2113122980 cites W1821097090 @default.
• W2113122980 cites W1971085025 @default.
• W2113122980 cites W1992050565 @default.
• W2113122980 cites W1994441123 @default.
• W2113122980 cites W1995133494 @default.
• W2113122980 cites W2011240457 @default.
• W2113122980 cites W2021582328 @default.
• W2113122980 cites W2030074619 @default.
• W2113122980 cites W2035184066 @default.
• W2113122980 cites W2038309376 @default.
• W2113122980 cites W2047630852 @default.
• W2113122980 cites W2057427118 @default.
• W2113122980 cites W2064612941 @default.
• W2113122980 cites W2074687201 @default.
• W2113122980 cites W2077818909 @default.
• W2113122980 cites W2097754260 @default.
• W2113122980 cites W2098626852 @default.
• W2113122980 cites W2099952268 @default.
• W2113122980 cites W2107368851 @default.
• W2113122980 cites W2113177287 @default.
• W2113122980 cites W2120473134 @default.
• W2113122980 cites W2145048594 @default.
• W2113122980 cites W2148816582 @default.
• W2113122980 cites W2156626134 @default.
• W2113122980 cites W2165611106 @default.
• W2113122980 cites W2166662833 @default.
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