Reply to Lovley, “Untangling Geobacter sulfurreducens Nanowires”

Research on Geobacter sulfurreducens nanowires is a hot topic that is full of controversies and unknowns. Cytochrome OmcS and OmcZ nanowires have been detected on G. sulfurreducens growing on an anode by cryo-electron microscopy and are well documented in the literature (1–3). In contrast, due to the lack of structural information for pili, the identification and function of pili remain controversial (4–6). Particularly, a recent study suggested the identity of pili was akin to type II secretion pseudopili in the periplasm, and consequently, the nonexistence of pili on the cell was proposed (5). However, based on our studies (7, 8) and referencing current literature (3, 9–11), we prefer a model having those three nanowires on the surface of G. sulfurreducens growing on an anode. In a recent study (12), we investigated the functions of those three nanowires in Geobacter sulfurreducens anode biofilms. To inhibit the expression of an individual nanowire, gene deletions were performed by strictly following the literature. Deletion of omcS (13) and omcZ (Fig. 1A) was verified by Western blotting, and the deletion of pilB was verified by PCR (13). We found a structural role for pili and OmcZ nanowires and conductive roles for OmcS and OmcZ nanowires. In his Letter to the Editor, Lovley questions these conclusions as he alleges our G. sulfurreducens PCA strain only expressed pili. His primary concern lies in the assumption that no filaments were able to be identified in the electron microscopy (EM) figures of our DPilB strain. The PilB ATPase is a pilus assembly protein. It has been well documented that a deletion of pilB was able to inhibit the expression of pili on the cell but did not affect the secretion of cytochromes (13, 14). Therefore, cytochrome nanowires are expected to be expressed in a DpilB strain. However, as previous studies have reported, the successful identification of nanowires by EM in G. sulfurreducens was affected by culture conditions and determined by the growth stage (1, 6, 15). In addition, other filaments but not nanowires could also occasionally be identified on G. sulfurreducens (16). Thus, EM examining nanowires is a sophisticated process and deemed not to be a good method to judge the expression and type of nanowires. Instead, atomic force microscopy (AFM) is thought to be a good technique to examine nanowires considering that different nanowires have different surface structures and are assumed to have tiny diameter differences. However, be that as it may, Lovley also barely detected pili on the quintuple cytochrome gene deletion strain DBESTZ (Fig. 2A) where he insisted that they were being expressed (17). This recent study also suggested that compared to pili, OmcS nanowires were much less expressed on the cell (17) and OmcZ nanowire expression was stimulated by an electric field (2). Considering our EM samples of a DpilB strain were collected from an NBAF culture growing at a suboptimal temperature of 25°C which is suitable for the expression of pili, it was foreseeable that the identification of cytochrome nanowires by EM on those cells is only by luck. Therefore, no pili were expressed, and it is not surprising that no cytochrome nanowires have been identified on the randomly selected DPilB cells. We further tested DpilB cells collected from the anode (Fig. 1B). As indicated, Editor Nancy E. Freitag, University of Illinois at Chicago Copyright © 2022 Liu et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Christopher Rensing, rensing@iue.ac. cn. The authors declare no conflict of interest. This is a response to a letter by Lovley (https:// doi.org/10.1128/mBio.00850-22).