Synthetic peptides were used to generate specific primary antisera against the M. oxyfera NirS (α-NirS) and pMMO (α-pMmoB1) in rabbits. We additionally cloned and heterologously expressed a fragment of pmoB in E. coli and used the expressed fragment to raise antiserum (α-pMmoB2). All antisera were affinity-purified and their specificity was tested on whole-cell extract of the M. oxyfera enrichment culture using SDS-PAGE and immunoblot analysis. Incubations with the antiserum targeting NirS showed a band of approximately the expected size (58.2 kDa; Fig. 2, lane 6). No bands were detected in blots incubated with blocking
buffer or preimmune serum instead of the antiserum (negative controls; data not shown). For the
antisera against pMMO, both α-pMmoB1 and α-pMmoB2 showed a band of about the expected size (44.2 kDa; Fig. 2, lanes Alpelisib supplier 2 and 4), which were absent when incubated with either blocking buffer or preimmune serum instead of the antiserum (negative controls; data not shown). When using the same antisera dilutions, a stronger signal was observed when using α-pMmoB2 compared to α-pMmoB1. These results showed that the derived antisera were specific for the targeted proteins and provide a reliable basis for immunogold localization of the enzymes in ultrathin sections of M. oxyfera cells. Cells from the M. oxyfera enrichment culture were chemically fixed and cryosectioned. Methylomirabilis oxyfera cells could be distinguished from other cells of the community by their polygonal cell shape (Wu et al., selleck kinase inhibitor 2012). The identity of the polygon-shaped cells to M. oxyfera has been confirmed previously by fluorescence in situ hybridization (FISH) using ‘NC10’; Fossariinae bacteria-specific probes (Wu et al., 2012). As in our previous study, the polygon-shaped M. oxyfera cells lacked ICM and the configuration of the cytoplasmic membrane was predominantly smooth and devoid of invaginations (Fig. 3). Cells from the other community members were morphologically diverse. The negative control where ultrathin sections of M. oxyfera cells were incubated with PAG5 or PAG10 alone showed no background labelling (data not shown). Likewise,
cross-reactivity of the affinity-purified antisera with other cells was not detected. In the incubations with α-pMmoB1 or α-pMmoB2, only M. oxyfera cells were specifically labelled. The gold particles occurred at or close to the cytoplasmic membrane (Fig. 3). As for immunoblot analysis, more labelling was observed when using α-pMmoB2 compared to α-pMmoB1 when using the same antisera dilutions. Ultrathin cryosections of M. oxyfera cells were incubated with α-NirS for the determination of the intracellular location of this enzyme. Labelling was observed only in the polygon-shaped M. oxyfera cells (Fig. 4). The negative control where ultrathin sections of M. oxyfera cells were incubated with PAG5 or PAG10 alone showed no background labelling (data not shown).