Characterization of the Exopolysaccharide Biosynthesis Pathway in Myxococcus xanthus

The secreted polysaccharide referred to as exopolysaccharide (EPS) has important functions in the social life cycle of M. xanthus; however, little is known about how EPS is synthesized. Here, we characterized the EPS biosynthetic machinery and showed that it makes up a Wzx/Wzy-dependent pathway for polysaccharide biosynthesis. Mutants lacking a component of this pathway had reduced type IV pilus-dependent motility and a conditional defect in development. These analyses also suggest that EPS and/or the EPS biosynthetic machinery is important for type IV pilus formation.


Supplemental Figures & Legends
. Characterization of MXAN_7415. (A) Sequence alignment of EpsZ (MXAN_715) and WbaP Se (Accession number: NP_461027.1) showing the Pro and Asp (orange) residues in the motif DX 12 P and the conserved amino acids essential for catalytic activity (red). (B) Complementation of colanic acid synthesis in E. coli K-12 W3110 (∆wcaJ Ec ) by plasmids encoding the indicated PHPT proteins as in Fig. 5C. Scale bar: 2 cm.

Supplementary Experimental Procedures
Plasmid construction. All oligonucleotides used are listed in Table S3. All constructed plasmids were verified by DNA sequencing.
pMP001 (for generation of in-frame deletion of MXAN_7415): up-and downstream fragments were amplified from genomic DNA of DK1622 using the primer pairs 7415_A/7415_B and 7415_C/7415_D respectively, as described in (1). Subsequently, the AB and CD fragments were used as templates to perform an overlapping PCR with the primer pair 7415_A/7415_D to generate the AD fragment. The AD fragment was digested with KpnI/XbaI and cloned in pBJ114.
pMP012 (for generation of in-frame deletion of MXAN_7421): up-and downstream fragments were amplified from genomic DNA of DK1622 using the primer pairs 7421_A/7421_B and 7421_C/7421_D respectively. Subsequently, the AB and CD fragments were used as templates to perform an overlapping PCR with the primer pair 7421_A/7421_D to generate the AD fragment. The AD fragment was digested with KpnI/XbaI and cloned in pBJ114.
pMP015 (for generation of in-frame deletion of MXAN_7442): up-and downstream fragments were amplified from genomic DNA of DK1622 using the primer pairs 7442_A/7442_B and 7442_C/7442_D respectively. Subsequently, the AB and CD fragments were used as templates to perform an overlapping PCR with the primer pair 7442_A/7442_D to generate the AD fragment. The AD fragment was digested with KpnI/XbaI and cloned in pBJ114.
pMP016 (for generation of in-frame deletion of MXAN_7416): up-and downstream fragments were amplified from genomic DNA of DK1622 using the primer pairs 7416_A/7416_B and 7416_C/7416_D respectively. Subsequently, the AB and CD fragments were used as templates to perform an overlapping PCR with the primer pair 7416_A/7416_D to generate the AD fragment. The AD fragment was digested with KpnI/XbaI and cloned in pBJ114.
pMP018 (for generation of in-frame deletion of MXAN_7417): up-and downstream fragments were amplified from genomic DNA of DK1622 using the primer pairs 7417_A/7417_B and 7417_C/7417_D respectively. Subsequently, the AB and CD fragments were used as templates to perform an overlapping PCR with the primer pair 7417_A/7417_D to generate the AD fragment. The AD fragment was digested with EcoRI/XbaI and cloned in pBJ114. pMP124 (for generation of in-frame deletion of MXAN_1043): up-and downstream fragments were amplified from genomic DNA of DK1622 using the primer pairs 1043_A/1043_B and 1043_C/1043_D respectively. Subsequently, the AB and CD fragments were used as templates to perform an overlapping PCR with the primer pair 1043_A/1043_D to generate the AD fragment. The AD fragment was digested with EcoRI/HindIII and cloned in pBJ114. pJJ1 (for generation of in-frame deletion of MXAN_1035): up-and downstream fragments were amplified from genomic DNA of DK1622 using the primer pairs 1035_A/1035_B and 1035_C/1035_D respectively. Subsequently, the AB and CD fragments were used as templates to perform an overlapping PCR with the primer pair 1035_A/1035_D to generate the AD fragment. The AD fragment was digested with KpnI/XbaI and cloned in pBJ114.
pJJ2 (for generation of in-frame deletion of MXAN_1025): up-and downstream fragments were amplified from genomic DNA of DK1622 using the primer pairs 1025_A/1025_B and 1025_C/1025_D respectively. Subsequently, the AB and CD fragments were used as templates to perform an overlapping PCR with the primer pair 1025_A/1025_D to generate the AD fragment. The AD fragment was digested with KpnI/XbaI and cloned in pBJ114.
pJJ3 (for generation of in-frame deletion of MXAN_1052): up-and downstream fragments were amplified from genomic DNA of DK1622 using the primer pairs 1025_A/1025_B and 1052_C/1052_D respectively. Subsequently, the AB and CD fragments were used as templates to perform an overlapping PCR with the primer pair 1052_A/1052_D to generate the AD fragment. The AD fragment was digested with KpnI/BamHI and cloned in pBJ114.