Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3^W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a ‘gate’ for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3^W406A protein may offer improved Cas3-based genetic editing in human cells.

Wastewater treatment plants (WWTPs) receive clinically relevant Acinetobacter species originating from hospitals and municipal sewage, yet their role in enhanced biological phosphorus removal (EBPR) remains unclear. This study evaluated the capacity of Acinetobacter spp. to accumulate orthophosphate (P) under conditions simulating the aerobic phase of EBPR and investigated the physiological and molecular determinants of P accumulation. Clinically relevant A. pittii and A. baumannii exhibited comparable growth to the model P-accumulating species A. junii and successfully proliferated in synthetic wastewater. However, A. junii maintained significantly higher P uptake rates, accumulated more intracellular polyP granules, and continued to remove P after entering the stationary phase, whereas P removal by A. pittii and A. baumannii reached a plateau. While A. junii and A. pittii contained a single ppk1 gene, two distinct ppk1 homologues and the corresponding PPK1 isoforms were identified in A. baumannii. Nevertheless, neither ppk1 copy number, ppk1 expression, nor the abundance of key enzymes involved in canonical polyP metabolism explained the enhanced P accumulation capacity of A. junii. Proteomic analyses suggested that continued P uptake in A. junii was associated with physiological adaptations that support the maintenance of cellular fitness during the stationary phase. Consequently, EBPR systems may favour the persistence and proliferation of clinically relevant Acinetobacter spp. without substantially benefiting from their contribution to P removal.