Reply to Losick, "Concerns about Continuing Claims that a Protein Complex Interacts with the Phosphorelay"

David Dubnau
2020 mBio  
KEYWORDS bacterial development, Fe-S clusters, Ric proteins O ur recent paper in mBio (1) prompted Richard Losick to criticize our hypothesis for Ric protein function (2, 3). Although his concerns are not with this recent paper, R. Losick prefers a different hypothesis for Ric protein function (4, 5). However, our respective models are not mutually exclusive. There is no evidence showing that our model is wrong, and there is evidence supporting our model as well as the compatible one proposed
more » ... ible one proposed by R. Losick. Our starting point was with the observations that inactivation of ricA (ymcA), ricF (ylbF), or ricT (yaaT) prevented biofilm formation, genetic competence, and sporulation (6-8), all processes requiring the phosphorylation of Spo0A. The use of reporter constructs confirmed that the activation of Spo0A was dependent on the Ric proteins, while alleles of spo0A that suppress defects in the phosphorelay bypassed the ric requirements for transcription of Spo0A-P-dependent genes (2, 9). Similar results had already been independently reported for RicT (7). Importantly, a purified complex of the three Ric proteins stimulated the phosphorelay two-to threefold in vitro (2), so we proposed this direct action as the role of the Ric proteins. The Losick lab then published papers proposing that these proteins were directly involved in RNA processing (4, 5). Since then we have acknowledged repeatedly that the Ric proteins are required in vivo for RNA processing events (1, 3, 9), and we have ourselves confirmed and extended this observation (1). Furthermore, we showed explicitly that the phosphorelay effect was only part of the Ric story, and we even proposed that the Losick hypothesis could explain the non-phosphorelay-related phenotypes (3, 9) . In what follows we will maintain that neither model has been rigorously proven. Although neither our in vivo data nor R. Losick's data prove that Ric proteins play direct roles in the phosphorelay or in RNA processing, in each case additional evidence has been claimed to support such direct roles. How convincing is this evidence? We will address this critically, first for our model and then for that of R. Losick. Our demonstration that the Fe-S-containing Ric complex stimulates the phosphorelay in vitro (2, 3) is strong evidence that this is a direct role for the complex. R. Losick criticizes our experiments, claiming that "the stimulation experiments lacked the control of testing other proteins". This criticism is mistaken. Four controls were reported (2, 3) as follows. In control 1, the addition of bovine serum albumen had no stimulatory effect. In control 2, the holo-complex was exposed to the atmosphere, leading to modification of the oxygen-sensitive Fe-S clusters, which prevented stimulation without inhibiting the phosphorelay. In control 3, the addition of glutathione S-transferase (GST) alone had no stimulatory effect, and in control 4, the addition of RicF-GST fusion protein, either alone or in complex with RicA and RicT, dramatically inhibited the phosphorelay reaction, supporting the notion that RicF interacted with one or another of the phosphorelay proteins. R. Losick comments that "only a twofold stimulation" was observed, but he has himself characterized the phosphorelay as "a highly sensitive, self-reinforcing switch"
doi:10.1128/mbio.00154-20 pmid:32156813 fatcat:gwvml5dy2jb33gyc2dapnzx7l4