INTRODUCTION

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The gliding bacterium Myxococcus xanthus has been suggested to utilize autochemotactic and autochemokinetic motility behaviors during its complex life cycle, which involves vegetative swarming to search for food and, in the absence of nutrients, aggregation to form raised multicellular fruiting bodies (39, 40). Both behaviors appear to be dependent on the Frz signal transduction pathway, components of which are highly homologous to the chemotaxis (Che) proteins of the enteric bacteria (25, 27). The Frz system has also been proposed to play a role similar to that of the Che system in that it regulates a mechanism for directional change, modulating the frequency of cell reversals during gliding. Null mutations in most of the frz genes result in cells that rarely reverse direction (6). The Frz proteins have also been shown to possess biochemical properties similar to those of the Che proteins. Methylation is required for activity of the proposed receptor, FrzCD (26), while FrzE has been shown to both autophosphorylate on the CheA-like domain and transfer this phosphate to the CheY-like domain (1). In the enteric bacteria, the response regulator protein CheY has a well-established interaction with the flagella motor apparatus: CheY-P associates with the motor switch complex, which results in motor reversals and reorienting tumbles (3). However, in M. xanthus, no corresponding motility apparatus which could interact with the Frz system has been identified. Additionally, M. xanthus has three CheY homologues within the Frz system. The FrzE protein is a fusion of two of the enteric Che proteins, the N-terminal portion showing homology to CheA, the histidine protein kinase, and the C-terminal portion having homology to CheY (27). Similarly, the FrzZ protein is composed of two domains, linked by an alanine-proline rich region, although both of these domains share homology with CheY (37).

The role of multiple CheY-like response regulators in motility of M. xanthus is unknown. A phenotypic analysis of frzZ mutants has shown the cells to have defects in both aggregation and vegetative swarming. During development in a DZF1 background (which contains a leaky sglA mutation), the cells show a frizzy aggregation phenotype, producing wild-type levels of spores, as is characteristic of other frz mutants in this genetic background (42). Likewise, in the fully motile DZ2 background, vegetative swarming was shown to be reduced, particularly on low-percentage agars. However, the cells were still able to move toward attractant stimuli and away from repellents in a spatial chemotaxis assay (37). Thus, the FrzZ protein has been suggested to be a modulator of chemotactic responses in M. xanthus, rather than being part of the central pathway. Interestingly, the two domains of FrzZ, although both showing homology to CheY, are only 27.5% identical to each other, suggesting that they could play alternative roles or interact with different proteins. While both domains retain the equivalent of CheY Asp-57, the conserved phosphorylation site, and other residues associated with the active site in enteric bacteria (Asp-12, Asp-13, and Lys-109) (36), neither domain shows conservation of the residues suggested to be associated with interactions at the motor switch complex (34). To explore the roles of the CheY-like domains of FrzZ, we used the yeast two-hybrid system to detect protein-protein interactions (13, 31). Since the two domains of FrzZ (FrzZ1 and FrzZ2) are distinct, each was cloned separately into the system as bait to be screened against a library. We present the results of using this system with M. xanthus proteins and suggest that FrzZ may interact with a developmentally important ATP-binding cassette (ABC) transporter, thereby proposing a novel role for a CheY-like protein.

	MATERIALS AND METHODS

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Strains and culture conditions. The strains used in this study are listed in Table 1. Plasmids are listed in Table 2. M. xanthus strains were grown in CYE medium (8), and developmental assays were performed on CF starvation media (16). Escherichia coli strains were grown in LB media. Yeast were grown in YPAD rich media or minimal SD media containing appropriate dropout solutions and 2% glucose (Stratagene).

HybriZAP two-hybrid system. DNA-binding domain fusion constructs were prepared by PCR amplification of the DNA encoding the bait proteins. PCR was performed with the high-fidelity polymerase Pfu. Primers were designed with 5' EcoRI restriction sites (underlined in the sequences below) to simplify cloning into the EcoRI site of pBD-GAL4. The following primer pairs were used: FrzZ1 (pBDZ1), forward 5'-ATGAATTCACGATGTCGCGCGTACTGGTCATTGATGACAGCCCG and reverse 5'-ATGAATTCGATGCTCAGGGCGGGGGGGCCAATGAGACCCATGAC; FrzZ2 (pBDZ2), forward 5'-ATGAATTCAAGCCGCGCATCCTCATCGTGGATGAC and reverse 5'-ATGAATTCCTACTCGTTACCGGTGGGCATCAGCTC; and FrzE (pBDE), forward 5'-ATGAATTCCGTACGCCGGCCATGGACACCGAGGCTCTC and reverse 5'-ATGAATTCTCAGGTCAGCCGGTCGATGGCCTGCGCGAG. The insertions within the resultant constructs (pBDZ1, pBDZ2, and pBDE) were sequenced to ensure error-proof amplifications, and then the plasmids were transformed into the Saccharomyces cerevisiae host strain YRG-2. The transformants containing just the binding domain constructs were tested for reporter gene expression prior to retransformation with the activation domain library or test activation domain fusion constructs.

DNA manipulations and PCR. All plasmids used in this study were prepared by using a QIAprep spin miniprep kit (Qiagen). Chromosomal DNA was prepared by the following miniprep protocol. A 1.5-ml sample of saturated culture was pelleted and then resuspended in 567 µl of Tris-EDTA (TE); 30 µl of 10% sodium dodecyl sulfate and 3 µl of proteinase K (20 mg/ml) were added, and the mix was incubated for 1 h at 37°C. A 100-µl volume of 5 M NaCl was added, followed by 80 µl of CTAB (cetyltrimethylammonium bromide)-NaCl solution; 50 ml of CTAB-NaCl solution was prepared by mixing 2.05 g of NaCl with 5 g of CTAB in distilled H₂O and heating at 65°C until dissolved before use. The mix was then incubated at 65°C for 10 min and then extracted with an equal volume of chloroform-isoamyl alcohol. The aqueous phase was removed and extracted again with an equal volume of phenol-chloroform-isoamyl alcohol. DNA was precipitated by the addition of 0.6 volume of isopropanol, washed in 70% ethanol, dried, and resuspended in TE buffer. Restriction enzyme digests and modifying enzyme protocols were performed as specified by the manufacturers. PCR optimizations and cycling parameters were identified by the protocol of Kramer and Coen (21). In general, glycerol concentrations of 20% were required for high yields of pure products. Taq polymerase (Promega) and Pfu polymerase (Stratagene) were used in amplifications requiring low and high fidelity, respectively.

Cloning and sequencing. Sequencing upstream of the 0.7-kb fragment cloned in pADZ165a was performed on a 3.5-kb SacI fragment (pABCS3.5) which overlapped the original 0.7-kb insert. Sequencing downstream was performed on two SacI fragments of 1.8 and 1.3 kb, cloned as pABCS1.8 (which also overlapped the original insert) and pABCS1.3 (which lies downstream of the 1.8-kb SacI fragment), respectively. The junction between these two downstream SacI fragments and the final region downstream of the 1.3-kb SacI fragment were sequenced on a 2.1-kb BamHI-KpnI clone (pABCBK2.1). The above pABC clones were all constructed in pUC18, allowing initial sequencing to be performed with the universal forward and reverse primers. Further sequence was obtained by primer walking or construction of further subclones (not listed). All sequencing was performed by the UC Davis Sequencing Facility.

Production of mutants. Mutants were constructed by cloning internal fragments of the specified genes into the 3.3-kb vector pZErO-2 (Invitrogen). These constructs were then electroporated into M. xanthus strains, and selection on CYE containing kanamycin (25 µg/ml) was used to identify mutants which had the entire vector inserted into the chromosome by homologous recombination.

Phenotypic analyses. Developmental defects were screened for on CF starvation agar by plating 5 µl of cells at 10⁹ CFU/ml directly onto the plate and incubating for 96 h. Spore counts were performed on these aggregates after 4 days of incubation. Cells were removed from the agar and resuspended in TM buffer (10 mM Tris-HCl, 8 mM MgSO₄ [pH 7.6]). Spore clumps were dispersed by sonication, and appropriate dilutions were placed in a Petroff-Hausser counting chamber for counting under magnification. Cell mixing experiments were performed on CF agar after cells were concentrated in morpholinepropanesulfonic acid (MOPS)-Mg²⁺ buffer (10 mM MOPS [pH 7.6], 8 mM MgCl₂) and mixed in various ratios of the two cell types. Aggregates were photographed at 24-h intervals for 4 days.

Computer analysis. Genetic analyses, including GC coding predictions, identification of open reading frames (ORFs), translations, sequence alignments, and the identification of membrane-buried regions, were performed with the program Gene Inspector (Textco Inc.).

Nucleotide sequence accession number. The DNA sequence presented here has been submitted to the EMBL, GenBank, and DDBJ nucleotide sequence data libraries under accession no. AF047554.

	RESULTS

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Identification of an activation domain fusion clone that interacts with the FrzZ1 bait. S. cerevisiae YRG-2 cells carrying the GAL4-binding domain-FrzZ1 fusion construct (pBDZ1) were transformed with a library of randomly sheared M. xanthus DNA activation domain fusion constructs (pADLIB). The resultant transformants were selected for HIS3 reporter gene activity by plating on minimal medium (SD-Leu-Trp-His). Cells growing under these restrictive conditions should contain binding and activation domain constructs which express interacting protein fusions. A second reporter gene assay, for expression of -galactosidase, was performed on all stably growing transformants. One transformant, which gave positive signals for both reporter gene assays, was studied further to ascertain the specificity of the interaction between the activation domain fusion protein and FrzZ1. The activation domain construct (pADZ165a) was purified from yeast cells and then retransformed into fresh YRG-2 cells containing test or control binding domain constructs (Table 3). The combination of pBDZ1 plus pADZ165a again produced a positive signal in both reporter gene assays. However, the expressed fusion from pADZ165a did not interact with those of the binding domain constructs expressing other M. xanthus response regulator proteins (including the second domain of FrzZ [FrzZ2], FrzE, and AsgA [33]). The control combination of p53 plus pADZ165a also resulted in negative reporter gene assays, although the leaky nature of the HIS3 reporter gene produced low-level growth on the selective media assay in all cases.

View larger version (8K): [in this window] [in a new window]   FIG. 1.   Restriction map of the ORFs identified by sequencing upstream and downstream of the 0.7-kb insert in pADZ165a. The original 0.7-kb EcoRI-XhoI insert cloned in pADZ165a is shown as the resultant GAL4 activation domain protein fusion. Sequencing constructs are shown below the main map. Restriction enzymes: B, BamHI; E, EcoRI; K, KpnI; S, SacI; X, XhoI.

Interacting protein shows homology to a group of bacterial ABC exporter systems. Gapped BLAST analyses (2) were performed on translations of the three complete and two incomplete ORFs identified within the 7-kb sequenced region. Neither of the translated proteins from the first (orf1) or last (orf5) partial ORF showed any significant homologies with protein sequences within the GenBank-EMBL data bank, and they were not studied further.

View larger version (105K): [in this window] [in a new window]   FIG. 2.   Alignment of the B. pertussis (B.p) CyaB protein (15) with the M. xanthus (M.x) Orf2 translation (AbcA). The two proteins show 30% identity and 53% similarity. The activation domain fusion created in pADZ165a starts at Orf2 amino acid E572. Double blocks above the aligned sequences indicate conserved amino acids, while single blocks define gaps in the alignment.

Mutational analyses. A 532-bp, PCR-derived internal fragment of the abcA gene was cloned into pZErO-2 to provide a kanamycin-resistant (Km^r) construct (pZABC) that could be integrated into the M. xanthus chromosome by homologous recombination at the abcA site, since pZErO-2 cannot replicate in M. xanthus. Using this method, a single crossover event would result in the targeted gene being divided into two truncated parts separated by the vector. The construct pZABC was introduced into the FB strain DZF1 by electroporation, and the resultant Km^r electroporants were screened by Southern blotting to ensure that the insertion had occurred at the correct site. Southern analysis showed that the 3.5-kb SacI genomic fragment, which contains the abcA gene, was missing in all mutants tested and replaced by two bands at approximately 3.1 and 4.1 kb, due to the insertion of pZABC (which contains a single internal SacI site). M. xanthus cells (DZF4198) with the correct insertion mutation were assayed for developmental defects by plating on starvation agar and were shown to display the frizzy aggregation phenotype (Fig. 3b), which is distinct from the parent DZF1 aggregation phenotype (Fig. 3a). Spore counts were performed on the parent DZF1, and frizzy aggregates and spore numbers were shown to be similar, although slightly reduced in the AbcA mutant (data not shown). A preliminary analysis of the reversal frequency of cell gliding, performed to ascertain that the frizzy phenotype of the AbcA mutant was associated with modified motility behavior, showed that the AbcA mutant does display a reduced frequency of cell reversals during gliding (8.2 ± 2.5 reversals/h), as has been observed in other Frz mutants (6), compared to the parent DZF1 reversal frequency (14.9 ± 4.7 reversals/h). While this reduction in reversal frequency is not as dramatic as those observed previously in frz mutants, it does suggest that mutations in the abcA locus have an effect on single cell movements.

View larger version (128K): [in this window] [in a new window]   FIG. 3.   Developmental aggregation and extracellular complementation by cell mixing in M. xanthus DZF1 (FB parent) (a), DZF4198 (frizzy) (b), DK1300 (nonfruiting) (c), and DZF4198 plus DK1300 (9:1 ratio).

Complementation of the Frz phenotype in DZF4198. If the ABC transporter does play a role in developmental aggregation by exporting a specific molecule into the extracellular environment, it would be predicted that a developmentally wild-type cell might rescue the DZF4198 AbcA mutant phenotype when presented in a mixed culture. Accordingly, AbcA mutant cells were mixed with DK1300, which is unable to aggregate due to a social motility defect (Fig. 3c) (19), and the mixture was spotted onto starvation plates in order to screen for rescue of development by extracellular complementation. Figure 3d shows rescue of the frizzy phenotype of the DZF4198 cells by DK1300 at a 9:1 ratio, supporting the hypothesis that AbcA could be involved in the export of a developmentally important aggregation-associated molecule. These results were confirmed by using the A S mglA motility mutant in a submerged culture assay (22). Again, rescue of the AbcA frizzy phenotype was observed when the same ratio (9:1) of cells was used (not shown).

	DISCUSSION

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In this study, use of the two-hybrid system has identified a potential interaction between the first domain of the FrzZ response regulator and the cytoplasmic domain of an ABC transporter (AbcA) bearing strong similarity to the bacterial exporters. Although more experimental work is required to fully confirm this interaction, additional evidence that the AbcA transporter is connected to the Frz signal transduction pathway was provided by an analysis of DZF1 cells with insertion mutations at the abcA locus. The frizzy phenotype displayed by these cells indicates that the AbcA protein is required for developmental aggregation and provides secondary evidence for a direct interaction between FrzZ and AbcA. Currently, we have no evidence that the two genes lying downstream of AbcA, suggested to be part of the same operon, have any role in the proposed FrzZ-AbcA interaction or, indeed, in the overall developmental process.

While sequence analysis of the AbcA protein suggested it to be a member of the RTX family of protein exporters, such as CyaB of B. pertussis and HlyB of E. coli, the potential absence of an associated accessory factor indicates that it is more likely to be a member of the nonprotein exporters. Such nonprotein exporters, which include ChvA in A. tumefaciens and NdvA in Rhizobium meliloti (both of which transport -1,2-glucan), do not utilize accessory factors or specific outer membrane components, since they do not secrete their products directly into the extracellular environment. The ability to extracellularly complement the DZF4198 AbcA frizzy phenotype in a cell mixing experiment provides preliminary evidence that the ABC transporter may indeed perform an exporter role and that the transported molecule could be required for developmental aggregation. Currently, we are exploring methods for purification of the transported molecule, using complementation of the DZF4198 aggregation phenotype as an assay, in order to clarify the role of AbcA as an exporter, analyze the export pathway of the molecule, and facilitate behavioral studies of cells exposed to what could potentially be a developmental aggregation signal.

While analyzing the DZF4198 (abcA sglA) mutant, we noted a vegetative swarming phenotype under nutrient-rich conditions. During growth on CYE agar, DZF4198 appeared to swarm more efficiently than the parent DZF1. However, since the DZF1 strain has a mutation in the sglA gene which results in reduced motility, the abcA mutation was transferred into the fully motile, wild-type strain DZ2. The lack of either a developmental or vegetative motility-associated phenotype in this DZ2 background (DZ24200) may suggest that the SglA protein can complement the abcA mutation, since a major difference between the DZF1 and DZ2 strains is the presence of the sglA1 mutation in DZF1. Previous work by Kashefi and Hartzell (20) has indicated that SglA may activate a function which can substitute for the Frz chemotaxis system during development. However, DZF1 contains other, undefined motility defects with respect to DZ2 (38a) which could play Frz-associated roles. Therefore, we are currently analyzing the potential SglA-AbcA association further.

The identification of a potential interaction between a component of the Frz signal transduction system and an ABC transporter involved in developmental aggregation supports the hypothesis that M. xanthus may export specific motility-associated molecules into the extracellular environment during starvation-induced development, in agreement with the autochemotactic model (40). Such autochemotactic situations are unusual, but not unknown, in bacteria. For example, both E. coli and Salmonella typhimurium have been shown to secrete a strong attractant (aspartate) when grown on components of the tricarboxylic acid cycle, which are themselves poor attractants (4, 7). The bacteria are then attracted to the secreted aspartate, resulting in the cells accumulating into aggregates with complex and intricate patterns. While the rationale for the enteric bacteria forming these patterns is unknown, the myxobacteria clearly use the aggregates in the construction of fruiting bodies.

M. xanthus has for many years been known to utilize extracellular signaling during starvation-induced development (23). Previous studies have also suggested that M. xanthus could generate signaling molecules involved in directed motility functions (24, 28). The work presented in this study complements such suggestions and provides a potential mechanism for export of the signals, as well as providing a potentially useful assay for identification of a potential signaling molecule. While the Frz signal transduction pathway has many similarities to the Che system of the enteric bacteria, our results suggest that Frz regulation of motility behavior is more complex than the enteric paradigm. In particular, we suggest that the FrzZ protein may be involved in a novel role for a CheY homologue, not interacting at the gliding motor but regulating the export of a developmentally active molecule. Future work will be aimed at further characterization of this system and an analysis of the motility-associated signaling molecule(s) used by M. xanthus during its complex life cycle.