Spx is a global regulator that is widespread among the low-G؉C-content Gram-positive bacteria. Spx has been extensively studied in Bacillus subtilis, where it acts as an activator and a repressor of transcription in response to disulfide stress. Under nonstress conditions, Spx is rapidly degraded by the ClpXP protease. This degradation is enhanced by the YjbH adaptor protein. Upon disulfide stress, the amount of Spx rapidly increases due to a decrease in degradation. In the opportunistic pathogen Staphylococcus aureus, Spx is a global regulator influencing growth, biofilm formation, and general stress protection, and cells lacking the spx gene exhibit poor growth also under nonstress conditions. To investigate the mechanism by which the activity of Spx is regulated, we identified a homolog in S. aureus of the B. subtilis yjbH gene. The gene encodes a protein that shows approximately 30% sequence identity to YjbH of B. subtilis. Heterologous expression of S. aureus yjbH in a B. subtilis yjbH mutant restored Spx to wild-type levels both under nonstress conditions and under conditions of disulfide stress. From these studies, we conclude that the two YjbH homologues have a conserved physiological function. Accordingly, inactivation of yjbH in S. aureus increased the level of Spx protein and transcription of the Spx-regulated gene trxB. Notably, the yjbH mutant exhibited reduced growth and increased pigmentation, and both phenotypes were reversed by complementation of the yjbH gene.

SummaryBacteria use stress response pathways to activate diverse target genes to react to a variety of stresses. The Bacillus subtilis Spx protein is a global transcriptional regulator that controls expression of more than 140 genes and operons in response to thiol-specific oxidative stress. Under nonstress conditions the concentration of Spx is kept low by proteolysis catalyzed by the ClpXP complex. Spx protein levels increase in response to disulfide stress and decrease when the cells cope with the stress. The cytosolic adaptor protein YjbH is required to target Spx for efficient proteolysis by ClpXP. We demonstrate that YjbH aggregates in response to disulfide stress, that is, the YjbH protein is soluble under nonstressed conditions and destabilized during stress leading to aggregation. Stress conditions (heat and ethanol) that cause severe perturbations in protein stability/folding also induced aggregation of YjbH and led to induction of Spx. By heterologous expression of a less aggregation prone YjbH homolog Spx induction was abolished. Thus we show that moderation of YjbH solubility is an important mechanism of signal transduction and represents a new mechanism of controlling the activity of adaptor proteins.

The role of inorganic pyrophosphate (PPi) as an energy carrier in the central metabolism of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was investigated. In agreement with its annotated genome sequence, cell extracts were shown to exhibit PPi-dependent phosphofructokinase and pyruvate phosphate dikinase activity. In addition, membrane-bound pyrophosphatase activity was demonstrated, while no significant cytosolic pyrophosphatase activity was detected. During the exponential growth phase, high PPi levels (approximately 4 +/- 2 mM) and relatively low ATP levels (0.43 +/- 0.07 mM) were found, and the PPi/ATP ratio decreased 13-fold when the cells entered the stationary phase. Pyruvate kinase activity appeared to be allosterically affected by PPi. Altogether, these findings suggest an important role for PPi in the central energy metabolism of C. saccharolyticus.

Objectives Prostate volume (PV) in combination with prostate specific antigen (PSA) yields PSA density which is an increasingly important biomarker. Calculating PV from MRI is a time-consuming, radiologist-dependent task. The aim of this study was to assess whether a deep learning algorithm can replace PI-RADS 2.1 based ellipsoid formula (EF) for calculating PV. Methods Eight different measures of PV were retrospectively collected for each of 124 patients who underwent radical prostatectomy and preoperative MRI of the prostate (multicenter and multi-scanner MRI’s 1.5 and 3 T). Agreement between volumes obtained from the deep learning algorithm (PVDL) and ellipsoid formula by two radiologists (PVEF1 and PVEF2) was evaluated against the reference standard PV obtained by manual planimetry by an expert radiologist (PVMPE). A sensitivity analysis was performed using a prostatectomy specimen as the reference standard. Inter-reader agreement was evaluated between the radiologists using the ellipsoid formula and between the expert and inexperienced radiologists performing manual planimetry. Results PVDL showed better agreement and precision than PVEF1 and PVEF2 using the reference standard PVMPE (mean difference [95% limits of agreement] PVDL: −0.33 [−10.80; 10.14], PVEF1: −3.83 [−19.55; 11.89], PVEF2: −3.05 [−18.55; 12.45]) or the PV determined based on specimen weight (PVDL: −4.22 [−22.52; 14.07], PVEF1: −7.89 [−30.50; 14.73], PVEF2: −6.97 [−30.13; 16.18]). Inter-reader agreement was excellent between the two experienced radiologists using the ellipsoid formula and was good between expert and inexperienced radiologists performing manual planimetry. Conclusion Deep learning algorithm performs similarly to radiologists in the assessment of prostate volume on MRI. Key Points • A commercially available deep learning algorithm performs similarly to radiologists in the assessment of prostate volume on MRI. • The deep-learning algorithm was previously untrained on this heterogenous multicenter day-to-day practice MRI data set.