Triple mutants uncover three new genes required for social motility in Myxococcus xanthus

Abstract

The bacterium Myxococcus xanthus glides over surfaces using two different locomotive mechanisms, called S (social) and A (adventurous) motility that enable cells to move both as groups and as individuals. Neither mechanism involves flagella. The functions of these two motors are coordinated by the activity of a small Ras-like protein, encoded by the mglA gene. The results of previous studies of a second-site suppressor of the mglA-8 missense mutation masK-815 indicate that MglA interacts with a protein tyrosine kinase, MasK, to control social motility. Sequence analysis of the sites of 12 independent insertions of the transposon magellan-4 that result in the loss of motility in an M. xanthus mglA-8 masK-815 double mutant shows that nine of these 12 insertions are in genes known to be required for S gliding motility. This result confirms that the masK-815 suppressor restores S but not A motility. Three of the 12 insertions define three new genes required for S motility and show that the attachment of heptose to the lipopolysaccharide inner core, an ortholog of the CheR methyltransferase, and a large protein with YD repeat motifs, are required for S motility. When these three insertions are backcrossed into an otherwise wild-type genetic background, their recombinants are found to have defects in S, but not, A motility. The spectrum of magellan-4 insertions that lead to the loss of S motility in the mglA-8 masK-815 double mutant background is different than that resulting from a previous mutant hunt starting with a different (A mutant) genetic background, suggesting that the number of genes required for S motility in M. xanthus is quite large.

Figure 1.—

Colony morphologies of mutants with magellan-4 insertions that abolish S motility in the mglA-8 masK-815 genetic background. Colonies formed by strains (A) DK1622 (wild type), (B) DK4135 (mglA-8), (C), MxH1104 (mglA-8 masK-815), (D) MxH1189 (mglA-8 masK-815 mis-189), (E) MxH1195 (mglA-8 masK-815 mis-195), and (F) MxH1195 (mglA-8 masK-815 mis-198) are shown. The mis-189, mis-195, and mis-189 alleles are magellan-4 insertions in the MXAN_7103, MXAN_6679, and MXAN_4710 genes, respectively (Table 1), predicted to encode a homolog of the CheR methyltransferase named SgnI, a large protein with YD repeat motifs named SgnH and an enzyme that is involved in the LPS biosynthesis pathway named SgnG. Photographs were taken using a Nikon FXA microscope, and are shown at 15× magnification.

Figure 2.—

Magellan-4 insertions in the mglA-8 masK-815 genetic background identify three additional genes required for S motility. The regions of the M. xanthus genome with the MXAN_4710, MXAN_6679, and MXAN_7103 genes are shown. Genes are depicted as boxes, with arrows below the boxes indicating their directions of transcription. Coordinates and gene numbers are those of the M. xanthus genome sequence (http://cmr.tigr.org/tigr-scripts/CMR/GenomePage.cgi?org_search=&org=gmx). The positions of magellan-4 insertions obtained in the current mutant hunt are indicated by open triangles above the genes; those of magellan-4 insertions obtained in our previous mutant hunt (Youderian and Hartzell 2005) as filled triangles below the genes. Adjacent genes are considered to be in the same transcription unit if they are separated by <20 bp. (Top) Genes MXAN_4711 (lpxK) and MXAN_4714 (kdtA), predicted to be upstream of MXAN_4710 in the same operon, are predicted to encode essential functions required for the synthesis of the lipid A-KDO moiety of O-antigen to which heptoses are then attached. MXAN_4710 and MXAN_4709 together encode the two functional domains of the enzyme HldE (RfaE); MXAN_4710 is predicted to encode d-β-d-heptose 7-phosphate kinase, and MXAN4709 is predicted to encodes d-β-d-heptose 1-phosphate adenosyltransferase. MXAN_4708 is predicted to encode a 55 amino acid hypothetical protein. Gene MXAN_4717, which may be the first gene in this operon, is predicted to encode a protein with both DnaJ and response regulator domains. (Middle) The gene MXAN_6672 is predicted to be upstream of sglK (MXAN_6671) (Weimer et al. 1998) in the same operon. It likely encodes a homolog of GrpE, which participates with DnaK in the HSP70 chaperone complex (Szabo et al. 1994). We have yet to obtain magellan-4 insertions in this gene. If this gene is not essential (the M. xanthus MXAN_4331 gene likely encodes a second homolog of GrpE), then we predict that these insertions will also result in an S motility defect, because such insertions should, at the least, be polar on the expression of sglK. The sgnH gene (MXAN_6679) appears to be the only gene in its operon. (Bottom) The MXAN_7103 gene is predicted to encode a homolog of CheR; it also appears to be the only gene in its operon.

Figure 3.—

Magellan-4 insertions in the MXAN_4710 (sgnG), MXAN_6679 (sgnH), and MXAN_7103 (sgnI) genes result in defects in S motility but not A motility. The spreading areas of single (MxH1289, MxH1295, and MxH1298), double (DK6204 and MxH1104), and triple mutants (MxH1189, MxH1195, and MxH1198) are compared with the spreading of the wild-type strain (DK1622) on CTPM medium containing 0.3 and 1.5% agar. The final spreading area is the difference between the colony area at T = 0 and T = 120 hr after incubation at 32°.

Figure 4.—

Gliding motility of isolated cells is evident in the MXAN_4710 (sgnG), MXAN_6679 (sgnH), and MXAN_7103 (sgnI) mutants. The colony edge morphology of M. xanthus strains (A) MxH1289, (B) MxH1295, and (C) MxH1298 are shown. The 15× (final magnification) images were taken with a Nikon FXA microscope.

Figure 5.—

MXAN_6679 is predicted to encode a protein product with 36 YD repeats. The product of MXAN_6679 (sgnH) is predicted to be a 340-kDa protein. The presence of an N-terminal signal sequence, shown in A, suggests that SgnH is secreted. Over half of the protein includes a series of YD/Rhs repeats (solid bar from 1076 to 2720). Rhs proteins contain extended repeat regions that are thought to be involved in ligand binding. The extracellular repeat contains the dipeptide YD. Potential YD repeats in the predicted product of the MXAN_6679 (sgnH) gene were aligned by visual inspection and by SMART (Simple Modular Architecture Research Tool) LeTunic et al. 2002. Repeat regions, shown in B, are numbered by the residue numbers of the predicted protein product of the sgnH gene. The consensus sequence of the YD repeats shared by the vertebrate teneurin-1 protein and the predicted products of the E. coli rhs genes (Minet et al. 1999) is shown in C. Amino acids occurring in the consensus sequence are shaded.