Plasmid Construction. Escherichia coli TOP10 (Invitrogen) was used as host for plasmids and grown in LB medium as described (1). Recombinant DNA techniques were performed according to standard protocols (1). PCR amplifications were performed by using Pfx DNA polymerase (Invitrogen). Plasmid constructs were verified by sequencing at the Stanford University Protein and Nucleic Acid Facility.

For pJEL4885.8 construction, an internal fragment of the Mx4885 ORF-containing part of the conserved central region was amplified by PCR with the primer pair 4885fwd1 (5'-ggtcgtggactgcaacctc-3') and 4885rev1 (5'-tggggcgtgagggtgatgc-3'). The fragment covers 429 bp from +567 to +995 relative to the first nucleotide (+1) in the start codon of Mx4885. The PCR fragment was cloned into pCR2.1-TOPO (Invitrogen), which confers kanamycin resistance, resulting in pJEL4885.8.

To construct pJEL4885.6 and pJEL4885.7, a fragment that covers –529 to +142 of the Mx4885 region was amplified by PCR with primers 4885plas1 (5'-cggaattccgcctcatggaggcgc-3') and 4885plas3 (5'-ttaactgcagggaccagtccgtcctgcg). The PCR product was digested with EcoRI and PstI and cloned into pBluescript II KS(+) (Stratagene), generating pJEL4885.1. Next, a fragment from +370 to +1,030 was amplified by PCR with primers 4885plas4 (5'-caactgcagaggtggatgttgcgggggc-3') and 4885plas2 (5'-cgggatcccggccactcatagc-3'). The PCR product was digested with PstI and BamHI and cloned into pJEL4885.1, resulting in pJEL4885.4. A third fragment from +1,279 to +1,881 was PCR-amplified with primers 4885plas5 (5'-caactgcagagcagcgcaccggcctgtcc-3') and 4885plas6 (5'-cgggatccgctggtggtgtgctcg-3'). The PCR product was digested with PstI and BamHI and cloned into pJEL4885.1, generating pJEL4885.5. Next, the EcoRI-BamHI fragments containing the in-frame deletions from pJEL4885.4 and pJEL4885.5 were subcloned into the galactose-based counterselectable plasmid pBJ114 (2) to generate pJEL4885.6 and pJEL4885.7, respectively.

pJEL4885.11 was constructed as follows. ORF Mx4885 was PCR-amplified with primers 4885cf (5'-gggatatccatggataggcccgagg-3') (EcoRV site underlined; start codon in Mx4885 in boldface) and 4485cr (5'-gggctgcagttacttctccaggccctc-3') (PstI site underlined; stop codon in Mx4885 in boldface). The resulting PCR product was digested with EcoRV and PstI and cloned into pBluescript II KS(+), resulting in pJEL4885.9. The pilA promoter (3), including its native leader sequence, was PCR-amplified by using primers pilAcf (5'-ggcccaagcttgatggcaccgtcatgttggacg-3') (HindIII site underlined) and pilArf (5'-gggatatcctcagagaaggttgcaacgggg-3') (EcoRV site underlined). The fragment was digested with HindIII and EcoRV and cloned into pJEL4885.9, resulting in pJEL4885.10. A HindIII–BamHI fragment containing the pilA promoter-Mx4885 gene construct was subcloned into the pSWU30 (4), resulting in pJEL4885.11. pSWU30 contains the attP site and confers oxytetracycline resistance.

pJEL4885.8 contains a 429-bp fragment from +567 to +995 relative to the first nucleotide (+1) in the start codon of Mx4885. Plasmid insertion in Mx4885 was verified by PCR by using plasmid-specific primers TOPOfwd1 (5'-ttggtaccgagctcggatcc-3') and TOPOrev1 (5'-cctctagatgcatgctgagc-3') together with the gene-specific primers 4885fwd2 (5'-accggcagccgcaacgg-3') and 4885rev2 (5'-tggcctcgtggtagggc) that anneal to Myxococcus xanthus DNA flanking the insertion. Disruptions of Mx1288 and Mx3725 were created in a similar manner. Generalized transduction with myxophage Mx4 propagated on DK12702 was used to introduce the Mx4885::pJEL4885.8 mutation into other strains. Resulting strains were verified by PCR. Strains DK12703 (D Mx4885^48-426) and DK12704 (D Mx4885^48-123) are markerless in-frame deletion strains generated by integration of pJEL4885.7 and pJEL4885.6, respectively, followed by excision of the wild-type gene as described in ref. 2. pJEL4885.6 and pJEL4885.7 contain a D Mx4885 allele from which codons 48–123 and codons 48–426 have been deleted, respectively. Loss of the wild-type Mx4885 gene in DK12703 and DK12704 was verified by PCR with primers that anneal to M. xanthus DNA flanking the deletions 4885seq1 (5'-atgcgactcgcacctaaagagg-3') and 4885seq2 (5'-agtccacgaccttgaaggggc-3') for DK12703 and 4885seq1 and 4885seq3 (5'-ttacttctccaggccctc-3') for DK12704. DK12705 was constructed by integration of plasmid pJEL4885.11 at the chromosomal Mx8 phage attachment site attB of DK12702. pJEL4885.11 contains the Mx4885 gene expressed from the pilA promoter. DK12706 was constructed by integration of plasmid pSWU30 at the attB site of DK12702. Correct plasmid integration at the attB site by site-specific recombination was verified by PCR with primers att1 (5'-cagttggttagaacgccggc-3'), att2 (5'-ctgattctcccgagccccac-3'), att3 (5'-cagcaccttgcgctcacgtc-3'), and att4 (5'-gggcaaaacccgagcctctc-3') specific for the 5' and 3' ends of the attP and attB regions.

1. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY).

2. Julien, B., Kaiser, A. D. & Garza, A. (2000) Proc. Natl. Acad. Sci. USA 97, 9098–9103.

3. Wu, S. S. & Kaiser, D. (1997) J Bacteriol. 179, 7748–7758.

4. Wu, S. S. & Kaiser, D. (1995) Mol Microbiol. 18, 547–558.