Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-Cho, Kagawa 761-0795, Japan
Correspondence
Yoshio Kimura
kimura{at}ag.kagawa-u.ac.jp
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ABSTRACT |
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The GenBank/DDBJ accession numbers for the sequences reported in this paper are AB111917 (rppA) and AB111918 (mmrA).
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INTRODUCTION |
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Chemotaxis transducers can sense changes in extracellular attractants and various repellents (Le Moual & Koshland, 1996; Parkinson, 1993
). Typical chemotaxis transducers, known as methyl-accepting chemotaxis proteins (MCPs), are integral membrane proteins and have two distinct modules, an N-terminal periplasmic module involved in sensing various stimuli and a C-terminal cytoplasmic module involved in signalling and adaptation. Chemoeffector binding causes a conformational change in the cytoplasmic structure that regulates the activity of a two-component signal transduction system (a histidine kinase CheA and a response regulator CheY). In addition to these proteins, a linker (CheW) between CheA and MCP, a signal terminator (CheZ), a methyltransferase (CheR) and a methylesterase (CheB) are involved in the bacterial chemotaxis signal transduction pathway (Parkinson, 1993
).
The M. xanthus dif locus encodes a set of chemotaxis homologues required for S motility and the biogenesis of fibrils (Yang et al., 1998, 2000
; Bellenger et al., 2002
). The dif mutants are defective in the formation of fruiting bodies, S motility, cellular cohesion and fibril biogenesis. Another chemotaxis locus, the frz locus, which also encodes chemotaxis homologues, is required for directed cell movement but not for S motility (Shi & Zusman, 1994
; Ward & Zusman, 1997
). frz mutants show defects in the regulation of reversal in their gliding direction, and when starved they formed tangled frizzy filaments instead of normal fruiting bodies (Zusman, 1982
; Blackhart & Zusman, 1985
).
To study chemotaxis transducers, we cloned an MCP homologous gene, rppA, from an M. xanthus genomic library. The rppA gene formed an operon together with mmrA, homologous to various multidrug transporter genes. We report here that an rppA-mmrA double mutant of M. xanthus showed a phenotype similar to those of polysaccharide-reduced mutants.
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METHODS |
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Cloning of rppA gene from a genomic DNA library.
Two degenerate oligonucleotides (Hcd1, 5'-GG(C/G)TT(G/T)(G/T)C(C/G)GTGGTGGC(C/G)G(A/G)(C/G)GAG-3', and Hcd2, 5'-GG(C/G)TTC(G/T)C(C/G)ATCGTGGC(C/G)G(A/G)(C/G)GAG-3') used as probes were designed to correspond to the highly conserved domain (HCD) of chemotaxis transducers (Le Moual & Koshland, 1996). The codon usage patterns of M. xanthus were used to minimize the overall degeneracy of each oligonucleotide. The oligonucleotides were labelled with digoxigenin-11-dUTP using an oligonucleotide tailing kit (Roche Diagnostics). The M. xanthus genomic library was constructed in
EMBL3 according to Kimura et al. (2001)
. A clone containing a putative transducer-encoding gene was isolated from the genome library by screening with a mixture of probes Hcd1 and Hcd2. The phage DNA of the clone was digested with SacI and ligated into the SacI site of pBluescript (Alting-Mees & Short, 1989
) and sequenced by automated sequencing with the BigDye terminator kit (Applied Biosystems).
Construction of rppA, mmrA and rppA-mmrA disruption mutants.
A 1·2 kb kanamycin-resistance cassette amplified by PCR from TnV (Furuichi et al., 1985) was ligated into NaeI- and StuI-digested rppA, and into NaeI-digested mmrA (Fig. 1
). The rppA : : Kmr and mmrA : : Kmr constructs were amplified by PCR using a pair of primers: 5'-TCGTGATGCTCGCATTCG-3' (rpp-1) and 5'-GCATCAGCGTGGACATGC-3' (mmr-1), and 5'-TCAACGTCATCGACGACG-3' (rpp-2) and 5'-GCATCAGCGTGGACATGC-3' (mmr-2), respectively. The rppA-mmrA double mutant was generated by insertion of the above kanamycin-resitance cassette into NaeI-digested rppA and mmrA. The rppA-mmrA : : Kmr construct was also amplified by PCR using rpp-1 and mmr-2. The PCR products were used for electroporation, which was performed as described by Plamann et al. (1992)
. Gene replacement was verified by PCR and Southern hybridization analyses.
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Development assay.
Fruiting bodies were formed on CF plates (Hagen et al., 1979) with 0·3 % or 1·5 % agar at 30 °C. The fruiting bodies were photographed under a light microscope. The fruiting bodies were harvested in TM buffer (10 mM Tris/HCl, pH 7·6, 8 mM MgCl2), and sonicated using a Branson sonifier. After incubation for 2 h at 50 °C, the number of viable spores was quantified by plating on CYE agar (Campos et al., 1978
).
Colony spreading and agglutination assays.
For colony spreading assays, 2 µl M. xanthus cells at 1x1010 cells ml-1 was spotted onto the centre of CYE medium with 0·3 % or 1·5 % agar, and incubated at 32 °C for 4 days (Shi & Zusman, 1993). The agglutination assay was performed as described by Kim et al. (1999)
. Cells from exponential cultures were washed twice with 10 mM MOPS (pH 6·8), inoculated into MCM buffer (10 mM MOPS, pH 6·8, 10 mM MgCl2, 1 mM CaCl2) or CYE medium to a density of 2x108 cells ml-1, and incubated statically at 25 °C. At 10 or 30 min intervals, the OD600 was measured in a spectrophotometer.
Congo red binding assay.
The wild-type and mutant cell suspensions, at a density of 1x109 cells ml-1, were placed on TPM agar containing 57 µM Congo red, and incubated at 30 °C for 24 h (Kearns et al., 2002). The colour of the cell aggregates was observed.
Reversal period assay.
This assay was performed by a modification of the method previously described by Kearns et al. (2000). Dilauroyl phosphatidylethanolamine (PE) and dioleoyl PE were dissolved in chloroform and chloroform/methanol (1 : 1, v/v), respectively. Four microlitres of PE solution (0·05 µg µl-1) was applied to TPM agar, and dried for 20 min at 37 °C. Five microlitres of M. xanthus cells diluted to 2x107 cells ml-1 in MOPS buffer (10 mM MOPS, pH 7·6, 8 mM MgSO4) was inoculated on top of the test compound and incubated at 30 °C for 20 min. The cellular reversal frequency was monitored with a digital camera through a Nikon microscope. At least 20 cells from each strain were followed over a 45 min period.
Western blotting analysis of fibrils.
M. xanthus wild-type and mutant cells were incubated in MCM buffer for 5 h at room temperature, harvested and washed with 10 mM MOPS buffer (pH 7·5). Proteins of cells (5x108) were separated by SDS-PAGE (12 % polyacrylamide) and electroblotted onto PVDF membrane (Bio-Rad) using a Trans Blot SD semidry transfer cell (Bio-Rad) according to the manufacturer's instructions. The membranes were blocked with 3 % bovine serum albumin in PBS-T buffer (10 mM sodium phosphate, pH 7·2, 150 mM NaCl, 0·1 % Tween 20) and then developed with a primary antibody mAb 2105 (Behmlander & Dworkin, 1991) and a secondary antibody composed of horseradish-peroxidase-conjugated goat anti-mouse immunoglobulin G. The membranes were washed with PBS-T buffer and detected with ECL Western blotting detection reagents (Amersham Biotech).
Polysaccharide measurement.
M. xanthus polysaccharide was measured by a modification of a method described previously (Kim et al., 1999). M. xanthus cells grown in CYE medium to a density of 5x108 cells ml-1 were collected by centrifugation, and resuspended at 2x109 cells ml-1 in fresh CYE medium. Samples were collected from the cell suspensions at the beginning and end of a 6 h incubation, washed in 0·85 % NaCl, and sonicated. The pelletable carbohydrate was obtained by centrifugation, and used to measure polysaccharide. Protein concentration was determined using the protein assay of Bradford (1976)
.
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RESULTS |
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The nucleotide sequence of rppA (GenBank accession no. AB111917) encodes a polypeptide composed of 718 amino acids corresponding to a molecular mass of 77 682 Da. The C-terminal region of the deduced protein from rppA shows very strong similarity to the chemotaxis transducer proteins, and shares homology with domains in the PilJ protein of Pseudomonas aeruginosa (29 % identity, 54 % similarity) (Darzins, 1994), the NahY protein of Pseudomonas putida (30 % identity, 46 % similarity) (Grimm & Harwood, 1999
) and the DifA protein of M. xanthus (24 % identity, 47 % similarity) (Yang et al., 1998
). The putative RppA protein possesses four transmembrane helices (residues 2239, 4969, 169189, and 199219), a HAMP (linker) domain (residues 221273), at least two methylation domains (residues 385399 and 609624), a signalling domain (residues 372633), and a coiled coil (residues 682718) (Zhulin, 2001
). Four repeated sequences (EQA/TXGSK/EQV/IXXS/AXE/H), partially similar to the methylation domain, were present in the C-terminal region (residues 582679) of RppA.
The mmrA gene (GenBank accession no. AB111918) was located 11 bp downstream from the stop codon of the rppA coding region. The predicted MmrA protein was 425 amino acids in length with a molecular mass of 44 689 Da. The mmrA gene product exhibited similarity to multidrug transporters; it exhibited sequence similarity with the putative multidrug-resistance protein (Mdr) of Aeromonas hydrophila (33 % identity, 54 % similarity) (Zhang et al., 2000), efflux pump protein (MxcK) of Stigmatella aurantiaca (35 % identity, 52 % similarity) (Silakowski et al., 2000
) and chloramphenicol-resistance protein (Cmr) of Rhodococcus fascians (25 % identity, 40 % similarity) (Desomer et al., 1992
). MmrA is predicted to consist of 12 transmembrane
-helices and has several highly homologous amino acid sequence motifs (GXXXDRXGRR in loops 23 and 89, and Dmic1500631mic1500631R in loop 45) (Rouch et al., 1990
; Yamaguchi et al., 1992
) that are conserved in equivalent positions of proton-coupled symporters, antiporters and uniporters. The orf3 gene encodes a Tat translocase homologue (Sargent et al., 1998
), and it was located 53 bp downstream of mmrA.
We examined whether rppA, mmrA and orf3 were co-transcribed in the wild-type strain using RT-PCR. RT-PCR, with primers that amplified a 247 bp fragment of rppA-mmrA, or a 155 bp fragment of mmrA-orf3. The results of these experiments suggested that rppA and mmrA were transcribed as a single transcript, but that orf3 was not co-transcribed with rppA and mmrA (Fig. 2). We also examined whether orf3 was transcribed in the rppA-mmrA mutant. The expected 169 bp product was amplified from total RNA of the double mutant when treated by reverse transcriptase (Fig. 2
), indicating that orf3 was expressed in this mutant. We confirmed that the RT-PCR products were detected at the same positions as PCR products amplified from genomic DNA on agarose gel (data not shown).
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Characterization of mutants
Construction of mutants.
A specific rppA or mmrA deletion mutant was constructed using the gene replacement technique (Fig. 1). In rppA and mmrA mutants, the NaeIStuI fragment of the rppA gene comprising the signalling and R1 domains, and the middle region (NaeI fragment) of the mmrA gene, were removed and replaced with kanamycin-resistance cassettes. An rppA-mmrA double mutant, which lacked the signalling and RI domains of RppA and the amino-terminal half of MmrA, was also constructed and examined with various phenotypic tests.
Developmental phenotype.
The mutants were examined for fruiting body development. The wild-type strain and all mutant strains formed normal fruiting bodies after 23 days of incubation on CF plates with 1·5 % agar. Within the fruiting bodies of the wild-type and mmrA mutant, vegetative cells were converted into spherical myxospores, while the rppA and rppA-mmrA mutants formed many short rod-shaped spores. The double mutant cells formed fruiting bodies about 1 day later than the wild-type strain, and the viable spore yield of the double-mutant strain was approximately 30 % of that of the wild-type strain (Fig. 3a). There were no obvious differences in viable spore yields between wild-type, rppA and mmrA mutants (data not shown). When spotted on CF plates with 0·3 % agar, the wild-type, rppA and mmrA mutants formed fruiting bodies, but the rppA-mmrA mutant did not (Fig. 3b
).
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DISCUSSION |
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The rppA-mmrA mutant showed reduced motility on 1·5 % agar and reduced cellular cohesion in agglutination buffer. In addition, the mutant had a longer basal reversal period, and reduced excitation in the reversal period in response to dilauroyl PE. On the other hand, rppA and mmrA single mutants showed wild-type phenotypes except for the spore shape of the rppA mutant. Since we confirmed, using RT-PCR, that orf3, located downstream of mmrA, was transcribed in the rppA-mmrA mutant, the phenotypes of the double mutant would not be due to polar effects. These results imply that the single deletion of RppA or MmrA may be complemented by other MCPs or multidrug transporters, respectively; however, the double deletion may make complementation with these proteins difficult. We searched the M. xanthus genome sequence database (TIGR database) for genes that encode proteins homologous to RppA or MmrA. The two gene regions with highest homology to RppA and MmrA were located at sequence positions 212732214165 and 28456212846667, respectively; the proteins encoded by these two gene regions showed 26 % identity and 45 % similarity to RppA and 35 % identity and 55 % similarity to MmrA.
The phenotypes of the double mutant show some similarity to those of fibril-deficient mutants (Kearns et al., 2000, 2002
). M. xanthus fibrils are used to attach to other cells, and are required for cellcell cohesion and S motility. Since fibril-deficient mutants (tglA/stk, dsp and difA) or a fibril protein A deficient mutant (fibA) lack excitation in response to dilauroyl PE, it has been thought that fibrils contain the dilauroyl PE chemoreceptors. The difA mutant, completely lacking fibril protein and polysaccharide, showed no obvious signs of agglutination, and was defective in S motility (Yang et al., 2000
). However, the rppA-mmrA mutant was not completely deficient in swarming and cohesion, and had A and S motility cells at the colony edge, suggesting that the double-mutant cells can form fibrils but that the amount of fibril may be lower than that in wild-type cells. We next compared the amount of polysaccharide and fibril protein in the wild-type and the mutant cells. Immunoblot analysis showed no obvious difference in fibril-specific protein between wild-type cells and the mutant cells. On the other hand, the double-mutant cells had reduced amounts of polysaccharide compared with wild-type cells. This result was confirmed by the fact that the double-mutant cells were not well stained with Congo red. The phenotypes of the double mutant may be due to the reduction in fibril polysaccharide. This phenotype is similar to that of the calcofluor white binding deficient mutants (Cds) of M. xanthus (Ramaswamy et al., 1997
). Nine Cds mutants generated by transposon mutagenesis showed significantly lower levels of stationary-phase polysaccharide than the wild-type strain. The Cds mutants also had decreased cell cohesion, fibril production and fruiting body formation. There is a possibility that the transposon insertions in one of or some Cds mutants may have occurred in the rppA gene. In recent years, it has become clear that there is a relation between cell motility, colony morphology and polysaccharide production in Pseudomonas aeruginosa, Pseudomonas fluorescens and Vibrio parahaemolyticus (D'Argenio et al., 2002
; Guvener & McCarter, 2003
; Spiers et al., 2003
).
Sequence analyses suggest that MmrA is a potential member of the major facilitator superfamily (MFS) of transporters. Several transporters involved with bacterial chemotaxis have been reported. The M. xanthus ATP-binding cassette (ABC) transporter, AbcA, was found to interact with FrzZ, a CheY homologue protein, using the yeast two-hybrid system (Ward et al., 1998). Ward et al. (1998)
speculated that AbcA might be involved in the export of a molecule required for the autochemotactic process. M. xanthus pilH encodes an ABC transporter homologue that is required for type IV pilus biogenesis, and a pilH mutant lacks S-motility (Wu et al., 1998
). Tol proteins, known to be involved in macromolecule transport, were recently shown to be required for M. xanthus A motility (Youderian et al., 2003
). Pseudomonas putida PcaK is an MFS transporter and chemoreceptor protein that functions in aromatic acid chemotaxis (Harwood et al., 1994
). Although a single mutation in the mmrA gene did not affect the phenotype, the rppA-mmrA double mutation appeared to decrease the amount of polysaccharide in stationary-phase cells. MmrA and RppA may interact and function in the biogenesis and/or assembly of polysaccharide. In contrast to dif mutants, the rppA-mmrA mutant was not completely deficient in polysaccharide. It is also possible that RppA and MmrA proteins have other functions. We are currently investigating whether these proteins possess other functions in M. xanthus.
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ACKNOWLEDGEMENTS |
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Received 23 September 2003;
revised 5 December 2003;
accepted 10 December 2003.
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