The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants
© van Dillewijn et al; licensee BioMed Central Ltd. 2009
Received: 16 September 2008
Accepted: 27 January 2009
Published: 27 January 2009
Soil bacteria collectively known as Rhizobium, characterized by their ability to establish beneficial symbiosis with legumes, share several common characteristics with pathogenic bacteria when infecting the host plant. Recently, it was demonstrated that a fadD mutant of Sinorhizobium meliloti is altered in the control of swarming, a type of co-ordinated movement previously associated with pathogenicity, and is also impaired in nodulation efficiency on alfalfa roots. In the phytopathogen Xanthomonas campestris, a fadD homolog (rpfB) forms part of a cluster of genes involved in the r egulation of p athogenicity f actors. In this work, we have investigated the role in swarming and symbiosis of SMc02161, a S. meliloti fadD-linked gene.
The SMc02161 locus in S. meliloti shows similarities with members of the Major Facilitator Superfamily (MFS) of transporters. A S. meliloti null-mutant shows increased sensitivity to chloramphenicol. This indication led us to rename the locus tep1 for t ransmembrane e fflux p rotein. The lack of tep1 does not affect the appearance of swarming motility. Interestingly, nodule formation efficiency on alfalfa plants is improved in the tep1 mutant during the first days of the interaction though nod gene expression is lower than in the wild type strain. Curiously, a nodC mutation or the addition of N-acetyl glucosamine to the wild type strain lead to similar reductions in nod gene expression as in the tep1 mutant. Moreover, aminosugar precursors of Nod factors inhibit nodulation.
tep1 putatively encodes a transmembrane protein which can confer chloramphenicol resistance in S. meliloti by expelling the antibiotic outside the bacteria. The improved nodulation of alfalfa but reduced nod gene expression observed in the tep1 mutant suggests that Tep1 transports compounds which influence nodulation. In contrast to Bradyrhizobium japonicum, we show that in S. meliloti there is no feedback regulation of nodulation genes. Moreover, the Nod factor precursor, N-acetyl glucosamine reduces nod gene expression and nodulation efficiency when present at millimolar concentrations. A role for Tep1 in the efflux of Nod factor precursors could explain the phenotypes associated with tep1 inactivation.
The rhizobia-legume mutualistic symbiosis is characterized by the formation of root nodules in which the bacteria fix atmospheric nitrogen to generate nitrogen sources assimilable by the plant. Although the attack of phytopathogens on plants have a different outcome (i.e. disease), similar efficient strategies have been acquired by pathogenic and mutualistic bacteria to establish compatible associations with their host plants . These include signals involved in cell-cell communication in bacterial populations but also in cross-kingdom communication with host plants .
Recently, swarming has been described in Rhizobiaceae [2, 3]. This type of co-ordinated movement was previously associated with the virulence of pathogens. In Sinorhizobium meliloti, swarming motility was associated with the activity of a long-chain fatty acyl-CoA ligase (FadD) which upon disruption affected nodulation efficiency on alfalfa roots. The authors hypothesized that a fatty acid derivative dependent on FadD activity may act as an intracellular signal controlling motility and symbiotic factors. In fact RpfB, a close homolog of FadD in Xanthomonas campestris , is implicated in the synthesis of cis-11-methyl-2-dodecenoic acid, a low-molecular-mass diffusible signal factor (DSF) involved in the regulation of pathogenicity factors . In X. campestris the homolog of FadD is surrounded by genes which also participate in several ways in the regulation of important virulence determinants . Therefore, a closer look was taken at the genes of S. meliloti in the vicinity of the fadD locus to determine their participation in symbiosis and/or swarming. Of the putative genes in the neighbourhood, the ORF SMc02161 located upstream from fadD and transcribed divergently from this gene, shows significant identity to permeases of the Major Facilitator Superfamily (MFS) . The MFS class of permeases is the second largest family of membrane transporters found, after the ABC transporters. Members of this protein superfamily are typically single-polypeptide secondary carriers, comprising of 10–14 transmembrane α-helices which are able to transport small solutes such as sugars or toxins in response to chemiosmotic ion gradients [7, 8]. In this work, the role of SMc02161 in bacterial resistance to toxics, nod gene expression and nodulation of alfalfa is described.
Results and discussion
S. meliloti ORF Smc02161 potentially codes for a transmembrane transporter with striking homology to MFS permeases
To analyze the region surrounding the fadD gene of S. meliloti, the available sequence of S. meliloti 1021  was used. The analysis using BLAST  revealed an ORF (SMc02163) downstream of fadD with homology to phosphoglucose isomerase (pgi) while upstream a divergently coding ORF (SMc02161) showed high identity to permeases of the Major Facilitator Superfamily (MFS). In this study, we characterize specifically ORF SMc02161. Putatively, this ORF encodes for a 411 amino acid protein with 11 transmembrane motifs typical of inner membrane proteins. This protein has an ATP/GTP binding motif, an alanine rich region (PROSITE ) and has the multi-domain of the MFS that covers most of the protein (from amino acid 73 to 331). The product shows the highest identity (66%) with a putative MFS protein in Beijerinckia indica subsp. indica ATCC9039, and shares most identity to MFS related permeases, transmembrane proteins, sugar transporters and efflux proteins of bacteria belonging to the Rhizobiales and Burkholderiales orders. Unfortunately, the physiological functions of the closest SMc02161 homologs have not been experimentally tested. One of the few SMc02161 homologs with an experimentally assigned function is CmlR (P31141, 29% identity), a chloramphenicol resistance protein of Streptomyces lividans .
The S. meliloti SMc02161 mutant shows higher sensitivity to chloramphenicol
tep1 is not necessary for swarming motility in S. meliloti
A tep1 mutation in S. meliloti improves nodule formation efficiency on alfalfa plants but shows reduced nod gene expression
Expression of transcriptional fusions to lacZ in S. meliloti GR4 and GR4T1.
β-galactosidase activity (Miller U)
465 ± 38
47 ± 12
435 ± 35
45 ± 14
418 ± 34
777 ± 26
398 ± 48
260 ± 45
A S. meliloti nodC mutant is affected in nod gene expression
nod gene expression in S. meliloti GR4, the tep1 mutant and a nodC mutant.
β-galactosidase activity (Miller U)
387 ± 48
144 ± 24
137 ± 34
Effect of glucosamine and N-acetyl glucosamine in nod gene expression in S. meliloti and on nodulation of alfalfa
nod gene expression in S. meliloti GR4 with different concentrations of N-acetyl glucosamine.
β-galactosidase activity (Miller U)
828 ± 251
425 ± 100
369 ± 112
412 ± 107
Glucosamine and N-acetyl glucosamine activate tep1 transcription
tep1 gene expression in S. meliloti GR4 under different growth conditions.
β-galactosidase activity (Miller U)
1523 ± 140
449 ± 16
652 ± 33
792 ± 29
The results obtained in this work suggest that the tep1 gene encodes a transport protein belonging to the MFS family of permeases able to confer chloramphenicol resistance in S. meliloti by expelling the antibiotic outside the cell. A tep1-linked gene in S. meliloti, fadD, plays a role in swarming motility and in nodule formation efficiency on alfalfa plants. We have demonstrated that tep1 is not involved in swarming motility but like fadD affects the establishment of the S. meliloti-alfalfa symbiosis. A tep1 loss-of-function mutation leads to increased nodule formation efficiency but reduced nod gene expression suggesting that Tep1 transports compounds which influence different steps of the nodule formation process. Whether these effects are caused by the same or different compounds putatively transported by Tep1, still needs to be investigated. Curiously, nod gene expression is reduced in a S. meliloti nodC mutant with the same intensity as in the tep1 mutant. This has implications for nod gene regulation in S. meliloti as it rules out the existence of a feedback regulation as described for B. japonicum. On the other hand, it could indicate that Tep1 is involved in the transport of Nod factors or its precursors. Indeed, increased concentrations of the core Nod factor precursor N-acetyl glucosamine reduced nod gene expression. Moreover, both glucosamine and N-acetyl glucosamine inhibit nodulation at high concentrations. Therefore, this constitutes the first work which attributes a role for core Nod factor precursors as regulators for nodulation of the host plant by S. meliloti. Furthermore, the results suggest that the activity of Tep1 can modulate the nodule formation efficiency of the bacteria by controlling the transport of core Nod factor precursors.
Bacterial strains, plasmids, media and chemicals
Sinorhizobium meliloti QS77  is a fadD::Tn5 insertion mutant derivative of wild-type GR4 . The plasmid pRmM57 (nodC::lacZ fusion)  was used to test the expression of the nodC gene and pGD499 (npt::lacZ fusion)  to test the expression of the constitutive kanamycin resistance gene. The pMPTR4 plasmid is a pMP220  derivative in which an Eco RI fragment of 0.6 kb harbouring the intergenic fadD-tep1 region was cloned to create a tep1::lacZ transcriptional fusion. The pGUS3 plasmid containing an nfeD::gusA fusion was used in competition assays . Triparental bacterial matings were performed using pRK2013 as helper plasmid . E. coli was grown routinely at 37°C in Luria-Bertani medium (LB) . S. meliloti strains were grown at 30°C in TY complex medium  or in defined minimal medium (MM) . Growth was determined regularly in a spectrophotometer measuring the absorbance at 600 nm. Glucosamine and N-acetyl glucosamine were obtained from Sigma-Aldrich.
Construction of a S. meliloti tep1 mutant
A null-mutant in ORF SMc02161 was obtained by allelic exchange. Firstly, a 3.6 kb Sac I fragment containing this ORF was subcloned from the fadD containing cosmid pRmersf442  into pUC18  to give pTrans1. To disrupt the ORF SMc02161 in pTrans1, a 2 kb Sma I fragment containing the streptomycin/spectinomycin resistance cassette from pHP45Ω  was inserted into a unique Eco RV site to give pTrans2. Next, the Sac I fragment containing the disrupted ORF was treated with T4 DNA polymerase (Roche Biochemicals) to make blunt ends and then cloned into the Sma I site of the suicide vector pK18mobsac  to give pTrans3. This vector was then used for allelic exchange by introducing it into the S. meliloti strains GR4, and the fadD mutant QS77 via triparental mating, and selecting putative mutants by streptomycin/spectinomycin resistance and sensitivity to sucrose. The resulting SMc02161 mutant GR4T1, and double fadD, SMc02161 mutant QSTR1 were confirmed by Southern hybridization with a specific probe.
Construction of a S. meliloti nodC mutant
To obtain a nodC mutant in S. meliloti, a fragment was amplified from the chromosomal DNA of S. meliloti GR4 by PCR using 5'-CAGATTC AAGGTCACGAAGTGGCTAAC-3' and 5'-ATAAGCTT GTGACAGCCAGTCGCTATTG-3' as forward and reverse primers respectively. An Eco RI-Pst I fragment of 1.5 kb derived from the PCR product and containing half of the nodB gene and most of the nodC gene was subcloned into pUC18  to obtain pGRC8. To disrupt nodC, pGRC8 was digested with Sal I and treated with Klenow (Roche Biochemicals) to create blunt ends. Next, the 2 kb Sma I fragment containing the streptomycin/spectinomycin resistance cassette from pHP45Ω  was introduced to give pNC150. The 3.5 kb Eco RI-Pst I fragment from pNC150 containing the disrupted nodC gene was inserted into Eco RI-Pst I digested pK18mobsac  to give pNC200. This suicide vector was then used to obtain the S. meliloti nodC mutant GR4C5, which was confirmed by Southern hybridization.
Swarming behaviour assay
Swarming assays were performed as described previously . Briefly, liquid cultures of S. meliloti, initiated from glycerol stocks, were grown at 30°C in TY broth with shaking to late logarithmic phase (optical density at 600 nm = 1–1.2). After incubation, cells were pelleted, washed twice in MM and resuspended in 0.1 volume of the latter medium. 2 μl drops of this suspension were deposited on the surface of plates containing MM with 0.7% agar and allowed to dry for 10 min. The plates were then inverted and incubated overnight (14–16 h) at 30°C and then scored for swarming motility.
Alfalfa (Medicago sativa L.) seeds were sterilized and germinated as described by Olivares et al. . To test the infectivity of the rhizobial strains, 24 individual plants were inoculated with each rhizobial suspension (106 colony forming units (cfu)/plant). To prepare the inoculants, rhizobial strains were previously grown in liquid TY medium up to an OD600 of 0.5 and then diluted accordingly. When addition of Nod factor precursors (glucosamine and N-acetyl glucosamine) was required, these compounds were added at the same moment as the bacterial inoculum. After inoculation, the number of nodulated plants and the number of nodules per plant were recorded daily.
To determine competitive ability, 12 plants were inoculated with GR4 × GR4 (pGUS3) or GR4T1 × GR4 (pGUS3) mixtures at ratios 1:1. The plasmid pGUS3 contains the marker gene coding for β-glucuronidase (GUS). To determine nodule occupancy, roots were collected 12 days after inoculation, briefly washed with water, and incubated overnight in the dark at 37°C in 1 mM X-Gluc (5-bromo-chloro-3-indolyl-β-D-glucuronide, Apollo Scientific, UK) in 50 mM sodium-phosphate buffer (pH 7.5) with 1% SDS. Those nodules occupied by GR4 (pGUS3) stain blue whereby nodule occupancy could be determined by counting blue and white nodules.
Measurement of β-galactosidase activity
S. meliloti cells containing lacZ fusions were grown in liquid MM containing tetracycline to ensure plasmid maintenance. Bacteria were grown in liquid cultures overnight at 30°C to early logarithmic phase (OD600 of 0.2–0.4) in the presence or absence of 5 μM luteolin and different concentrations of glucosamine or N-acetyl glucosamine when required. Samples of 100 μl of the bacterial culture were taken and assayed for β-galactosidase activity by the SDS-chloroform method described by Miller .
This work was supported by grants BMC2001-0253 and BIO2007-62988 from the Spanish Ministerio de Ciencia y Tecnología to MJS.
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