We investigated the Acinetobacter baylyi gene ACIAD1960, known from previous work to be expressed during long-term stationary phase. The protein encoded by this gene had been annotated as a Conserved Hypothetical Protein, surrounded by putative tellurite resistance ("Ter") proteins. Sequence analysis suggested that the protein belongs to the DUF1796 putative papain-like protease family. Here, we show that the purified protein, subsequently named StiP, has cysteine protease activity. Deletion of stiP causes hypersensitivity to tellurite, altered population dynamics during long-term batch culture, and most strikingly, dramatic alteration of normal cell morphology. StiP and associated Ter proteins (the StiP-Ter cluster) are therefore important for regulating cell morphology, likely in response to oxidative damage or depletion of intracellular thiol pools, triggered artificially by tellurite exposure. Our finding has broad significance because while tellurite is an extremely rare compound in nature, oxidative damage, the need to maintain a particular balance of intracellular thiols, and the need to regulate cell morphology are ubiquitous.

We have investigated the gene ACIAD 1960 from Acinetobacter baylyi. A. baylyi is a soil bacterium that is of interest due to its ability to utilize a diverse set of nutrients as a source of carbon and nitrogen, and to its high level of competence with regards to natural transformation. Medically, it is becoming of interest as a potential source of hospital‐acquired infections. Previous work has shown that gene ACIAD1960 is expressed during long‐term stationary phase. We identified that the purified protein expressed by ACIAD 1960 encodes a cysteine protease activity. We named the cysteine protease “StiP” to reflect that it is a long‐term stationary phase induced protease. We present data from a deletion‐insertion mutation which demonstrates that lack of the protein causes alteration of normal cell morphology, smaller twitching zone size, and hypersensitivity to the oxyanion tellurite. The mutant may also have a small growth defect. We show that the presence of divalent metal ions differentially affect its activity. The presence of Ca2+, Mg2+ and Mn2+ all increase activity, with Mn2+ showing the greatest effect. The presence of Zn2+ significantly decreases activity. Additionally, we show that StiP activity decreases in the presence of reducing agents, suggesting that a disulfide bond maycontribute to its overall conformational stability and function. Grant Funding Source: Supported by Colorado College Natural Sciences Executive Committee grants to M.A.D., K.M.L., and C.