Cells generate purine nucleotides through de novo purine biosynthesis (DNPB) and purine salvage. Purine salvage represses DNPB to prevent excessive purine nucleotide synthesis through mechanisms that are incompletely understood. We identified Nudix hydrolase 5 (NUDT5) as a DNPB regulator. During purine salvage, NUDT5 suppresses DNPB independently of its catalytic function but rather through interaction with phosphoribosyl pyrophosphate amidotransferase (PPAT), the rate-limiting enzyme in the DNPB pathway. The NUDT5-PPAT interaction promoted PPAT oligomerization, suppressed PPAT’s enzymatic activity, and facilitated disassembly of the purinosome, a metabolon that functions in DNPB. Disrupting the NUDT5-PPAT interaction overcame DNPB suppression during purine salvage, permitting excessive DNPB and inducing thiopurine resistance. Therefore, NUDT5 governs the balance between DNPB and salvage to maintain appropriate cellular purine nucleotide concentrations.

Homologous recombination (HR) is an error-free DNA double-strand break (DSB) repair pathway, which safeguards genome integrity and cell viability. Human C-terminal binding protein (CtBP)—interacting protein (CtIP) is a central regulator of the pathway which initiates the DNA end resection in HR. Ubiquitination modification of CtIP is known in some cases to control DNA resection and promote HR. However, it remains unclear how cells restrain CtIP activity in unstressed cells. We show that the ubiquitin E3 ligase PPIL2 is recruited to DNA damage sites through interactions with an HR-related protein ZNF830, implying PPIL2’s involvement in DNA repair. We found that PPIL2 interacts with and ubiquitinates CtIP at the K426 site, representing a hereunto unknown ubiquitination site. Ubiquitination of CtIP by PPIL2 suppresses HR and DNA resection. This inhibition of PPIL2 is also modulated by phosphorylation at multiple sites by PLK1, which reduces PPIL2 ubiquitination of CtIP. Our findings reveal new regulatory complexity in CtIP ubiquitination in DSB repair. We propose that the PPIL2-dependent CtIP ubiquitination prevents CtIP from interacting with DNA, thereby inhibiting HR.

Bacterial cryptic prophage (defective prophage) genes are known to drastically influence host physiology, such as causing cell growth arrest or lysis, upon expression. Many phages encode lytic proteins to destroy the cell envelope. As natural antibiotics, only a few lysis target proteins were identified. ydfD is a lytic gene from the Qin cryptic prophage that encodes a 63-amino-acid protein, the ectopic expression of which in Escherichia coli can cause nearly complete cell lysis rapidly. The bacterial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is responsible for synthesizing the isoprenoids uniquely required for sustaining bacterial growth. In this study, we provide evidence that YdfD can interact with IspG, a key enzyme involved in the MEP pathway, both in vivo and in vitro. We show that intact YdfD is required for the interaction with IspG to perform its lysis function and that the mRNA levels of ydfD increase significantly under certain stress conditions. Crucially, the cell lysis induced by YdfD can be abolished by the overexpression of ispG or the complementation of the IspG enzyme catalysis product methylerythritol 2,4-cyclodiphosphate. We propose that YdfD from the Qin cryptic prophage inhibits IspG to block the MEP pathway, leading to a compromised cell membrane and cell wall biosynthesis and eventual cell lysis.

CGA-N46 is a small antifungal-derived peptide and consists of the 31st–76th amino acids of the N-terminus of human chromogranin A. Polycistronic expression of recombinant CGA-N46 in Bacillus subtilis DB1342 was used to improve its production, but the yield of CGA-N46 was still low. In the present study, response surface methodology (RSM) was used to optimize culture medium composition and growth conditions of the engineered strain B. subtilis DB1342(p-3N46) for the further increase in CGA-N46 yield. The results of two-level factorial experiments indicated that dextrin and tryptone were significant factors affecting CGA-N46 expression. Central composite design (CCD) was used to determine the ideal conditions of each significant factors. From the results of CCD, the optimal medium composition was predicted to be dextrin 16.6 g/L, tryptone 19.2 g/L, KH₂PO₄·H₂O 6 g/L, pH 6.5. And the optimal culture process indicated inoculation of B. subtilis DB1342(p-3N46) seed culture into fresh culture medium at 5 % (v/v), followed by expression of CGA-N46 for 56 hours at 30 °C induced by 2 % (v/v) sucrose after one hour of shaking culture. To test optimal CGA-N46 peptide expression, the yeast growth inhibition assay was employed and it was found that under optimal culture conditions, CGA-N46 inhibited the growth of Candida albican by 42.17, 30.86 % more than that in the pre-optimization conditions. In summary, RSM can be used to optimize expression conditions of CGA-N46 in engineered strains B. subtilis DB1342(p-3N46).