Zhang et al. reported stable MglAQ82L expressed from the att site, however our constructed mutants (which integrated at the chromosomal site) failed to accumulate SU5402 order stable MglA protein [18]. Time-lapse microscopy failed to detect any movement on 1.5% agarose for Quisinostat cost either strain; motility in MC was nearly identical with the parent. Loss of transcript did not appear to account for the problem because, as shown in Figure 4, the levels of mglA transcript for both the Q82A and Q82R were found to be elevated. The apparent increase in mRNA level by qRT-PCR and, paradoxically, the decreased expression of MglA may be due to alterations in the predicted secondary structure of mgl RNA resulting

from codon 82 modifications. All activating mutation strains were assayed for their localization. We did not detect MglA in the Q82 mutants, consistent with the Western blot showed in Figure 6D. In the G21V and L22V, we observed localization as previously seen in Figure 3D, which depicts the L22V localization pattern. The localization pattern for P80A was indistinguishable from the WT (WT shown in Figure 3A). Mutations that are predicted to affect surface residues alter or decrease MglA function and may affect protein-protein interactions Based on the three-dimensional model of MglA (Figure

1), we predicted that residues Asp52, Thr54, Leu117, Leu120 and Leu124 might be surface exposed. Asp52 and Thr54 lie within a region that corresponds with a GAP (GTPase Activating Protein) effector-binding region of eukaryotic GTPases [36]. Leu117, Leu120 and Leu124 are three of the leucines that comprise a short stretch between Sotrastaurin Leu117 and Leu145 that resembles a leucine repeat (Lx6L) [37] that are likely to reside on a single face of an α-helix. These hydrophobic residues Fenbendazole and their neighbors would either be buried in the interior of the protein or would indicate a potential binding site for

an interacting protein with a similar hydrophobic face. The residues in this leucine-rich repeat (LRR) were indicated in orange in Figure 1 and are highlighted in Figure 7A. The role of each of these residues in gliding and development was investigated. Figure 7 Mutations predicted to alter surface residues abolish function of MglA. Residues predicted to exist on the surface of MglA either failed to complement the deletion phenotype or partially restored the activity of both motility systems. Strains in this panel include MxH2408 (D52A), MxH2406 (T54A), MxH2339 (L117/120A) and MxH2279 (L124K). See Figure 2 legend. Residues D52 and T54 were found to be critical for the function of MglA. Both mutants produced stable MglA protein that had significantly reduced function. Although some gliding flares (including isolated cells) were apparent at the colony edge of each mutant strain (Figure 7C), swarming was abolished (Figure 7B).