Collateral effects of deletion of nlpD on rpoS and rpoS-dependent genes

A seminal article, titled “Active bacterial modification of the host environment through RNA polymerase II inhibition,” was published in the JCI in February 2021 (1). The article depicts a novel bacterial phenomenon mediated by the NlpD protein, which was demonstrated using an nlpD deletion mutant, a recombinant NlpD protein (rNlpD), and an nlpD deletion mutant complemented with the nlpD-rpoS operon. The idea in this article is impressive and has a potential impact on bacteriology, especially for studies on NlpD. However, since nlpD is complicated, as marginally referred to in Supplemental Figure 5 in the article by Ambite et al., we here include detailed information about nlpD. nlpD is positioned upstream of rpoS; rpoS encodes the RNA polymerase sigma factor σ³⁸ (RpoS) that regulates many genes, as shown in a recent study that identified differential expression of 1044 genes between the wild-type and rpoS mutant (2). Importantly, nlpD includes rpoS promoters, including the P2 promoter, which is critical for rpoS expression (2–4). The nlpD deletion mutant lacks the rpoS promoter, resulting in no expression of both rpoS and nlpD. Whether the phenotypes observed in the nlpD deletion mutant depend on the NlpD functions should be confirmed using nlpD and not the nlpD-rpoS operon, and we have reviewed the article by Ambite et al. with this view in mind. An nlpD SNP observed in SN25 was mapped to the critical rpoS promoter P2 “TATAAT” (5). SN25 showed low or no expression of RpoS (Supplemental Figure 5B in the article by Ambite et al.), appearing to be a mutant with substantial RpoS deficiency. No nlpD deletion mutant complemented solely with nlpD was tested; however, the mutant complemented with the nlpD-rpoS operon that expressed RpoS in addition to NlpD was studied. Furthermore, since no experiment using rNlpD SNP to confirm the phenotypes of SN25 was performed, whether these phenotypes depend on a loss of function of NlpD remains to be determined. These approaches raise the possibility that the phenotypes of SN25 observed can be attributed to the effects of rpoS/rpoS-dependent genes. We believe that this information will be useful for future studies on host-microbe interactions, especially those focusing on nlpD.