Identification of a Gene Involved in Polysaccharide Export as a Transcription Target of FruA, an Essential Factor for Myxococcus xanthus Development

Abstract

Fruiting body development in Myxococcus xanthus is a multicellular event that is coordinated by exchanging intercellular signals. FruA is a transcription factor essential for fruiting body development and is thought to play a key role in the C-signal pathway. Here we present the first identification of a gene regulated by FruA. The gene was isolated from a genomic library via in vitro selection in a DNA binding assay by using the DNA-binding domain of FruA tagged with His8 at the C-terminal end (FruA-DBD-H8). The gene, named fdgA (FruA-dependent gene A), encodes a protein homologous to the outer-membrane auxiliary family protein involved in the polysaccharide export system. FruA-DBD-H8 bound the upstream promoter region of the fdgA gene from nucleotide –89 to nucleotide –64 with respect to the transcription initiation site, which was required for the induction of fdgA expression during development. fdgA mRNA induced during development was absent in a fruA deletion strain. The deletion of fdgA resulted in defective fruiting body formation and reduced sporulation efficiency (1% that of the parent strain). Moreover, FruA was required for the developmental expression of sasA, which is also involved in the biosynthesis of the lipopolysaccharide O-antigen and is required for fruiting body development. Furthermore, the expression of both fdgA and sasA was partially dependent on the C-signal. These findings expand our understanding of the signal transduction pathway mediated by FruA during development in M. xanthus. Fruiting body development in Myxococcus xanthus is a multicellular event that is coordinated by exchanging intercellular signals. FruA is a transcription factor essential for fruiting body development and is thought to play a key role in the C-signal pathway. Here we present the first identification of a gene regulated by FruA. The gene was isolated from a genomic library via in vitro selection in a DNA binding assay by using the DNA-binding domain of FruA tagged with His8 at the C-terminal end (FruA-DBD-H8). The gene, named fdgA (FruA-dependent gene A), encodes a protein homologous to the outer-membrane auxiliary family protein involved in the polysaccharide export system. FruA-DBD-H8 bound the upstream promoter region of the fdgA gene from nucleotide –89 to nucleotide –64 with respect to the transcription initiation site, which was required for the induction of fdgA expression during development. fdgA mRNA induced during development was absent in a fruA deletion strain. The deletion of fdgA resulted in defective fruiting body formation and reduced sporulation efficiency (1% that of the parent strain). Moreover, FruA was required for the developmental expression of sasA, which is also involved in the biosynthesis of the lipopolysaccharide O-antigen and is required for fruiting body development. Furthermore, the expression of both fdgA and sasA was partially dependent on the C-signal. These findings expand our understanding of the signal transduction pathway mediated by FruA during development in M. xanthus. Developmental programs in living organisms are guided by a number of different signal transduction systems involved in the integration of environmental and cellular signals. These systems regulate the temporal and spatial expression of genes specific to distinctive stages of development. The developmental progression of a soil bacterium, Myxococcus xanthus, is also controlled by a number of signal transduction systems (1Shimkets L.J. Annu. Rev. Microbiol. 1999; 53: 525-549Crossref PubMed Scopus (179) Google Scholar, 2Kaiser D. Annu. Rev. Microbiol. 2004; 58: 75-98Crossref PubMed Scopus (174) Google Scholar). Upon nutrient limitation, M. xanthus cells begin to coordinately migrate toward aggregation centers by gliding on a solid surface to form a fruiting body that holds 105 cells. Inside fruiting bodies only 10% of the starved cells differentiate to spores. The developmental process is achieved by a series of sophisticated intercellular signaling pathways that regulate the expression of a specific set of genes. Five intercellular signals, the A-, B-, C-, D-, and E-signals, are known to be involved and each is essential for fruiting body development. The C-signal is a cell-surface associated morphogen required for rippling, aggregation, and sporulation, and its transmission occurs by a contact-dependent mechanism that involves end-to-end contact between cells (3Kim S.K. Kaiser D. Cell. 1990; 61: 19-26Abstract Full Text PDF PubMed Scopus (151) Google Scholar, 4Hagen T.J. Shimkets L.J. J. Bacteriol. 1990; 172: 15-23Crossref PubMed Google Scholar, 5Sager B. Kaiser D. Genes Dev. 1994; 8: 2793-2804Crossref PubMed Scopus (104) Google Scholar). Recently, it has been reported that the C-signal (p17) is a proteolytic product of CsgA (p25) and corresponds to the C-terminal part of p25 (6Lobedanz S. Søgaard-Andersen L. Genes Dev. 2003; 17: 2151-2161Crossref PubMed Scopus (101) Google Scholar). The fruA gene encodes a putative transcription factor essential for development and is a member of the FixJ response regulator subfamily of the two-component His-Asp phosphorelay system (7Ogawa M. Fujitani S. Mao X. Inouye S. Komano T. Mol. Microbiol. 1996; 22: 757-767Crossref PubMed Scopus (100) Google Scholar, 8Ellehauge E. Nørregaard-Madsen M. Søgaard-Andersen L. Mol. Microbiol. 1998; 30: 807-817Crossref PubMed Scopus (110) Google Scholar). FruA is required for the execution of aggregation, fruiting body formation, and subsequent sporulation. The expression of fruA is developmentally regulated, dependent on A- and E-signals, and initiates at 6 h after the onset of development. Recently, a fruA promoter-binding protein was isolated from developmental cell extracts by using a DNA binding assay with the DNA fragment containing the upstream region of the fruA promoter (9Ueki T. Inouye S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8782-8787Crossref PubMed Scopus (57) Google Scholar). Based upon its partial sequence, this fruA promoter-binding protein was found to be identical to MrpC, which belongs to the cyclic AMP receptor protein family of transcriptional regulators (9Ueki T. Inouye S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8782-8787Crossref PubMed Scopus (57) Google Scholar). MrpC activates the expression of fruA by binding cis-acting elements in the upstream region of the fruA promoter. mrpC and mrpAB had previously been identified via transposon insertion mutagenesis as essential loci for cellular aggregation and sporulation (10Sun H. Shi W. J. Bacteriol. 2001; 183: 4786-4795Crossref PubMed Scopus (68) Google Scholar). The mrpA and mrpB genes encode a histidine kinase and a NtrC-like response regulator, respectively, of the two-component His-Asp phosphorelay system. It has been proposed that the expression of mrpC depends on the mrpAB operon and that MrpC autoregulates its own gene expression (11Sun H. Shi W. J. Bacteriol. 2001; 183: 6733-6739Crossref PubMed Scopus (41) Google Scholar). Based on the analysis of developmental markers in a fruA::Tc strain, the expression of various developmental genes induced after 5 h of development was severely hampered (7Ogawa M. Fujitani S. Mao X. Inouye S. Komano T. Mol. Microbiol. 1996; 22: 757-767Crossref PubMed Scopus (100) Google Scholar). In addition, genes known to be expressed before 4 h of development were found to be normally expressed during early development but were unable to be repressed later in fruA::Tc (7Ogawa M. Fujitani S. Mao X. Inouye S. Komano T. Mol. Microbiol. 1996; 22: 757-767Crossref PubMed Scopus (100) Google Scholar). Furthermore, among six proteins identified by the analysis of protein expression patterns in the wild-type, fruA::Tc, and ΔcsgA strains during development, the expression of five proteins was found to be C-signal-independent and one to be C-signal-dependent (12Horiuchi T. Taoka M. Isobe T. Komano T. Inouye S. J. Biol. Chem. 2002; 277: 26753-26760Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These results indicate that developmental genes under the control of FruA can be classified into two groups, C-signal-independent and C-signal-dependent (12Horiuchi T. Taoka M. Isobe T. Komano T. Inouye S. J. Biol. Chem. 2002; 277: 26753-26760Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In addition, transposon mutagenesis identified mutants involved in the C-signal pathway that were phenotypically divided into two classes (13Søgaard-Andersen L. Slack F.J. Kimsey H Kaiser D. Genes Dev. 1996; 10: 740-754Crossref PubMed Scopus (86) Google Scholar). Class I mutants are deficient in aggregation but sporulate and produce C-factor at wild-type levels. Class II mutants have deficiencies in all C-factor responses. Because fruA was also identified genetically as the only class II mutant gene (14Søgaard-Andersen L. Kaiser D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2675-2679Crossref PubMed Scopus (72) Google Scholar), FruA has been proposed to play a key role and to be activated by phosphorylation with a C-signal receptor kinase in the C-signal transduction system (15Søgaard-Andersen L. Overgaard M. Lobedanz S. Ellehauge E. Jelsbak L. Rasmussen A.A. Mol. Microbiol. 2003; 48: 1-8Crossref PubMed Scopus (54) Google Scholar). However, such a kinase has not been identified to date. Given that the expression of a number of genes was altered between the wild-type and fruA::Tc strains during development (7Ogawa M. Fujitani S. Mao X. Inouye S. Komano T. Mol. Microbiol. 1996; 22: 757-767Crossref PubMed Scopus (100) Google Scholar, 8Ellehauge E. Nørregaard-Madsen M. Søgaard-Andersen L. Mol. Microbiol. 1998; 30: 807-817Crossref PubMed Scopus (110) Google Scholar, 12Horiuchi T. Taoka M. Isobe T. Komano T. Inouye S. J. Biol. Chem. 2002; 277: 26753-26760Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), it was important to identify which genes are directly activated or repressed by FruA. To identify genes directly regulated by FruA, we performed in vitro selection for a genomic DNA library by using the DNA-binding domain of FruA tagged with eight histidine residues at the C-terminal end (FruA-DBD-H8). 2The abbreviations used are: FruA-DBD-H8, DNA-binding domain of FruA tagged with eight His residues at the C-terminal end; CF, clone-fruiting; nt, nucleotide(s); OMA, outer membrane auxiliary; ORF, open reading frame. We isolated a DNA fragment containing the promoter region and the N-terminal part of a protein homologous to the outer membrane auxiliary (OMA) family protein, which is involved in the polysaccharide export system. This gene was named fdgA (FruA-dependent gene A). cis-Acting elements in the fdgA promoter were identified, and developmental expression of fdgA was not detectable in a ΔfruA strain newly constructed in this study. Moreover, the deletion of fdgA resulted in defective fruiting body formation and reduced sporulation efficiency. In addition, the induction of the sasA locus that is required for the biosynthesis of the lipopolysaccharide O-antigen and development (16Kaplan H.B. Kuspa A. Kaiser D. J. Bacteriol. 1991; 173: 1460-1470Crossref PubMed Google Scholar, 17Guo D. Bowden M.G. Pershad R. Kaplan H.B. J. Bacteriol. 1996; 178: 1631-1639Crossref PubMed Google Scholar) was not detected in the ΔfruA strain during development. Bacterial Strains and Growth Conditions—M. xanthus DZF1 (18Inouye M. Inouye S. Zusman D.R. Dev. Biol. 1979; 68: 579-591Crossref PubMed Scopus (66) Google Scholar) was used as a parent strain and was grown in CYE medium (19Campos J.M. Geisselsoder J. Zusman D.R. J. Mol. Biol. 1978; 119: 167-178Crossref PubMed Scopus (177) Google Scholar) supplemented with kanamycin or streptomycin when necessary. For fruiting body formation, M. xanthus was spotted on clone-fruiting (CF) agar plates (20Hagan D.C. Bretcher A.P. Kaiser D. Dev. Biol. 1978; 64: 284-296Crossref PubMed Scopus (260) Google Scholar). Escherichia coli JM83 (21Vieira J. Messing J. Gene. 1982; 19: 259-268Crossref PubMed Scopus (3785) Google Scholar) was used as a recipient strain for transformation unless otherwise mentioned and was grown in Luria-Bertaini medium (22Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 439Google Scholar) supplemented with antibiotics when necessary. Preparation of FruA-DBD-H8—The DNA fragment encoding FruA-DBD-H8 from Pro at 152 to Leu at 229 tagged with histidine residues at the C-terminal end was amplified by PCR with pMF05 (7Ogawa M. Fujitani S. Mao X. Inouye S. Komano T. Mol. Microbiol. 1996; 22: 757-767Crossref PubMed Scopus (100) Google Scholar) and the oligonucleotide primers 5′-TCCATATGCCGGTGACGTCACCCACC-3′ and 5′-TCGGATCCCTA(GTG)8GAGGTCCGGCGGCGGCCGGA-3′ (NdeI and BamHI sites are underlined). PCR products were digested with NdeI and BamHI and ligated into pET11a (Novagen). The resultant plasmid, pET11a/FDBD-H8, was transformed into E. coli BL21 (DE3) (23Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar). FruA-DBD-H8 was induced by the addition of isopropyl 1-thio-β-d -galactopyranoside at a final concentration of 1 mm in Luria-Bertani medium supplemented with ampicillin at 37 °C for 2 h. Cells were harvested by centrifugation and washed with 10 mm Tris-HCl (pH Cells were in the H mm Tris-HCl (pH mm 1 mm and 10 mm supplemented with and by centrifugation at for the was with at a final concentration of and was to for the was washed with the H containing and with the H containing mm FruA-DBD-H8 was with a containing 10 mm Tris-HCl (pH mm 1 mm and mm The was a of 10 mm Tris-HCl (pH mm 1 mm 1 mm and for the protein were performed on or in a In for a DNA genomic DNA library was constructed by partially with DNA from M. xanthus DNA of were isolated by For in vitro selection a DNA binding assay was performed as with the library as a and FruA-DBD-H8 in a of the DNA binding the were to were isolated from the after To the from the the DNA fragment of containing sites from the promoter was used as a for and S. The containing with to that of promoter were The were from the and with and The DNA were with and in of the DNA was ligated at the in which between the and the of two sites to the The were and were from transformed cells on the The were digested with and the DNA were isolated with The DNA were used for the in vitro selection as a The in vitro selection was as the containing the were DNA and fdgA promoter region from to was amplified by PCR using the containing the fragment in 2 and the oligonucleotide primers and 2 for the the and BamHI sites are underlined). amplified product was digested with and BamHI and in DNA fragment for a was by the with and BamHI and the fragment by The isolated DNA fragment was with by a fragment of DNA For a to the region from to was as with oligonucleotide primers and DNA fragment for a was by the with and BamHI and the fragment by The isolated fragment was with by a fragment of DNA only the in 2 was DNA binding were performed at °C for 10 in 10 the containing mm (pH mm 10 mm 1 mm 10 10% and 1 of The binding patterns were by by For after DNA binding I was to the and the were at °C for The were by the containing mm and of the with the DNA were with in the of The DNA were in and by by fdgA promoter from and to were to in the transcriptional (9Ueki T. Inouye S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8782-8787Crossref PubMed Scopus (57) Google Scholar). was at and h after the initiation of development on agar plates as L. Kuspa A. Kaiser D. Dev. Biol. PubMed Scopus Google Scholar). of ΔfruA strain was constructed by the fragment with a streptomycin The and the sites are at the and from the N-terminal end of FruA, (7Ogawa M. Fujitani S. Mao X. Inouye S. Komano T. Mol. Microbiol. 1996; 22: 757-767Crossref PubMed Scopus (100) Google Scholar). strain was constructed by the fragment with a kanamycin The and sites are in the promoter region and the open reading of the fdgA gene, residues from 1 to of are strain was constructed by the containing the promoter region and the of the fdgA gene into the of the strain. of a by FruA from a DNA identify a gene directly regulated by FruA, we performed in vitro selection for a genomic DNA library in a DNA binding We used the DNA-binding domain of FruA tagged with eight histidine residues at the C-terminal The DNA isolated after each selection were with and were used for a DNA binding assay with the were were bound by FruA-DBD-H8 DNA fragment in the of isolated after the of the selection was found to a putative promoter region and N-terminal part of on a in the for at The DNA fragment gene was from the The isolated gene was gene or of fdgA during Growth and expression was by analysis with from DZF1 and ΔfruA during and fruiting body development and of fdgA mRNA were of was detected in both and developmental and its expression was of fruA In the two were detected only in developmental cells. of was in early and development, and the was in development. These were not detectable in the ΔfruA strain. The of were by DNA not and are in The DNA fragment containing the promoter region from to with respect to the transcription initiation was found to be bound by FruA-DBD-H8 analysis that the two from –89 to and from to at –64 were by FruA-DBD-H8 from I and that the at was to I between the two in were when of FruA-DBD-H8 was it to have binding To the of in fdgA expression in various of the upstream fdgA promoter were to the gene and into the The of was at and h of development The deletion of the region from to not fdgA expression during and development, and the deletion of the region from to reduced fdgA expression during development. In the deletion of between and from promoter resulted in in fdgA and region a in the of the fdgA promoter. The of by promoter during development was to the in expression of fdgA during development of fdgA gene encodes a protein homologous to of that belongs to the family involved in polysaccharide export PubMed Scopus Google Scholar). has a that the by the in and the signal II Escherichia coli and and Molecular for D. Scholar). of DNA that is of However, transcription PCR analysis that was of the promoter not that fdgA is To the of the fdgA gene, a strain was the fdgA gene was expressed during the strain in CYE medium (19Campos J.M. Geisselsoder J. Zusman D.R. J. Mol. Biol. 1978; 119: 167-178Crossref PubMed Scopus (177) Google Scholar) as the parent strain not In during development the strain defective fruiting body formation on agar plates The strain and fruiting which were not as as of the parent strain. In under this the ΔfruA strain fruiting bodies number of were after the fruiting bodies were harvested and with The efficiency of sporulation of the strain was that of the parent strain after 5 of development on agar To that the defective development of the strain was to the of the fdgA gene, the DNA fragment containing the promoter region and the region of the fdgA gene was into the strain. The strain fruiting bodies to of the parent strain Furthermore, the strain in efficiency to that of the parent strain. the fdgA gene was essential for fruiting body formation and sporulation during development. of the sasA at the sasA locus that are involved in lipopolysaccharide O-antigen biosynthesis have been to be essential for development for (16Kaplan H.B. Kuspa A. Kaiser D. J. Bacteriol. 1991; 173: 1460-1470Crossref PubMed Google Scholar, 17Guo D. Bowden M.G. Pershad R. Kaplan H.B. J. Bacteriol. 1996; 178: 1631-1639Crossref PubMed Google Scholar). we the sasA locus is regulated by FruA. analysis that sasA mRNA induced during development was in the ΔfruA strain, but mRNA was expressed in both the parent and the ΔfruA strains the DNA binding assay that FruA-DBD-H8 not the sasA promoter region under in which FruA-DBD-H8 the fdgA promoter region not it that the sasA locus is regulated by of C-signal on of fdgA and FruA is proposed to in the C-signal transduction pathway (14Søgaard-Andersen L. Kaiser D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2675-2679Crossref PubMed Scopus (72) Google Scholar, L. Overgaard M. Lobedanz S. Ellehauge E. Jelsbak L. Rasmussen A.A. Mol. Microbiol. 2003; 48: 1-8Crossref PubMed Scopus (54) Google Scholar), the expression of fdgA and sasA was in a ΔcsgA strain J. J. Bacteriol. PubMed Google Scholar). analysis that fdgA mRNA induced during development in the ΔcsgA strain In addition, sasA mRNA induced during development also in the ΔcsgA strain it that the expression of both fdgA and sasA is partially dependent on the C-signal. We have identified a gene, named encoding family protein involved in the polysaccharide export system as a transcription of FruA from the genomic library of M. xanthus. The expression of fdgA was regulated during and development. The developmental expression of fdgA was dependent on FruA. The deletion of fdgA resulted in defective fruiting body formation and reduced sporulation efficiency during development. fdgA is the first gene identified as a transcription of FruA. To identify genes directly regulated by FruA, we performed in vitro selection for the M. xanthus genomic DNA library by using a DNA binding assay with In the in vitro selection a DNA fragment containing a binding for a specific transcription factor can be isolated from a genomic DNA S. S. M. Scholar). The of the it to the binding identified by in vitro selection is in the promoter region of a In for analysis of gene expression such as and in the identification of genes or proteins regulated by a transcription factor as as that are directly It that FruA activates fdgA expression during development by binding a and identified by the The analysis that have to FruA-DBD-H8 in However, analysis that region has the on the of the fdgA promoter. Because the region to I is between a and it is that binding of FruA-DBD-H8 to the fdgA promoter results in in the fdgA promoter and that this is important for the of the fdgA promoter. the early of development, fdgA expression is from the promoter by binding of FruA to a and the developmental is it is that developmental such as a different transcription factor and a factor fdgA expression at The induction of fdgA at is partially dependent on the that the C-signal be also involved in the of gene expression during development. M. xanthus of to as and a role in fruiting body development M. M. Kaiser D. for D. Scholar). have not been for sporulation during fruiting body development, the in polysaccharide is and polysaccharide for of the of the cell during sporulation induced by D. M. Kaiser D. for D. Scholar). The of the polysaccharide are and the of polysaccharide to be It is not known which are by cells and which are involved in fruiting body development. of polysaccharide by using the strain the fdgA gene encodes family protein involved in the polysaccharide export and fdgA expression is regulated during and development. In addition, at two of were identified from the M. xanthus not The analysis of also to the of polysaccharide in M. xanthus. It has been that both proteins and proteins are required for the polysaccharide export system in PubMed Scopus Google Scholar). The M. xanthus sasA required for lipopolysaccharide O-antigen and (16Kaplan H.B. Kuspa A. Kaiser D. J. Bacteriol. 1991; 173: 1460-1470Crossref PubMed Google Scholar, 17Guo D. Bowden M.G. Pershad R. Kaplan H.B. J. Bacteriol. 1996; 178: 1631-1639Crossref PubMed Google Scholar). The and genes encode D. Bowden M.G. Pershad R. Kaplan H.B. J. Bacteriol. 1996; 178: 1631-1639Crossref PubMed Google Scholar). Furthermore, the fdgA and the sasA mutants in that fruiting body formation is and sporulation efficiency is reduced during development (16Kaplan H.B. Kuspa A. Kaiser D. J. Bacteriol. 1991; 173: 1460-1470Crossref PubMed Google Scholar). it is to that fdgA and gene products in the pathway during development. In addition, the expression of both genes is dependent on fruA during development. However, it that sasA expression is regulated from fdgA FruA-DBD-H8 the fdgA promoter but not the sasA promoter in Because response regulator proteins are known to a with when B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), phosphorylation of FruA be required to the sasA promoter. It is known that the expression of and encoding outer membrane is regulated by the phosphorylation of response regulator in the E. coli system S. J. Inouye M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). It is also that the sasA promoter factor for FruA binding that be sasA expression is partially dependent on the C-signal. The for FruA to both to be The developmental of the fruA mutant are of the fdgA The fruA mutant fruiting body formation and E. Nørregaard-Madsen M. Søgaard-Andersen L. Mol. Microbiol. 1998; 30: 807-817Crossref PubMed Scopus (110) Google Scholar, T. Inouye S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8782-8787Crossref PubMed Scopus (57) Google Scholar). It is that FruA genes fdgA as by analysis (14Søgaard-Andersen L. Kaiser D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2675-2679Crossref PubMed Scopus (72) Google Scholar). This is by the identification of genes using the not We are genes. of genes regulated by FruA the signal transduction pathways it were from The for at We are to L. D. for reading of the We J. for the ΔcsgA strain. We also for DNA and H. for