(B) Quantification of cell aggregation in S. oneidensis MR-1 wild type and mutants in planktonic culture under minimal medium conditions. The ratio of the optical density measured at 600 nm of wild type and mutant cultures after and before dispersion was used to determine their aggregation phenotypes. These data indicate a possible role for mxdA and mxdB in cell-surface
adhesion when growing in minimal medium. When comparing growth rates in LB to minimal medium, we found no correlation between growth rate and mxd expression, suggesting that a low growth rate, as found under starvation conditions in minimal medium, was most likely not responsible for mxd induction (data not shown). We therefore hypothesized STAT inhibitor that limitation for essential nutrients or accumulation of metabolites might be involved in mxd induction, and specifically tested whether carbon or nitrogen limitation induced mxd expression. For this purpose we constructed a ML323 mouse wild type mxd::lacZ reporter strain (AS832) (see Table 1 and 2). This strain was grown in LB medium to an OD600=0.3. Cells were pelleted, resuspended in minimal medium amended with 50 mM sodium lactate, incubated for 120 minutes at 30°C and subsequently assayed for specific β- galactosidase activity. Similarly, cells were also exposed to minimal
medium without carbon or nitrogen source. As a control, cells were resuspended in the same LB culture medium. As shown in Figure 2 no increase Astemizole in mxd expression was observed when cells were incubated in the LB culture medium for 120 minutes (Figure 2) and compared to the same sample at t=0 minutes. Similarly, cells exposed to minimal medium void of a nitrogen source also did not show any increase in mxd expression. Cells exposed to minimal medium supplemented with lactate led to minor mxd induction. However, shifting cells to minimal medium void of a carbon source led to significant mxd induction (~400 MU). Thus, starvation for carbon appears to be important for mxd expression
in S. oneidensis MR-1. Table 1 Strains used in this study Strain Relevant genotype or description Source or reference E. coli S17-lambda pir thi pro recA hsdR [RP4-2Tc::Mu-Km::tn7]lambda pir Tpr Smr [38] AS259 (BW20767) RP4-2-Tc::Mu-1 Kan::Tn7 find more integrant leu-63::IS10 recA1 zbf-5 creB510 hsdR17 endA1 thi uidA (deltaMluI)::pir + [12] AS262 S17-lambda pir harbouring pUX-BF13 [39] AS392 S17-lambda pir harbouring pGP704-mini-Tn7(Gm) P A1/04/03-GFPmut3* [39] S. oneidensis AS93 S. oneidensis MR-1, wild type, tagged with GFPmut3* in a Tn7 construct, Genr [12] AS536 AS93 harbouring pME6031(Tc)::Pmxd -300+1 lacZ (pJM1) This study AS556 AS93 harbouring pME6031(Tc)::lacZ (promoterless) This study AS579 (MR-1) S.