Sachla, A. | Search bioRxiv

bioRxiv2024

Structural basis for transcription activation through cooperative recruitment of MntR

The manganese transport regulator (MntR) from B. subtilis is a dual regulatory protein that responds to heightened Mn2+ availability in the cell by both repressing the expression of uptake transporters and activating the expression of efflux proteins. Recent work indicates that, in its role as an activator, MntR binds several sites upstream of the genes encoding Mn2+ exporters, leading to a cooperative response to manganese. Here, we use cryo-EM to explore the molecular basis of gene activation by MntR and report a structure of four MntR dimers bound to four 18-base pair sites across an 84-base pair regulatory region of the mneP promoter. Our structures, along with solution studies including mass photometry and in vivo transcription assays, reveal that MntR dimers employ polar and non-polar contacts to bind cooperatively to an array of low-affinity DNA-binding sites. These results reveal the molecular basis for cooperativity in the activation of manganese efflux.

biochemistry↗

bioRxiv2023

TerC Proteins Function During Protein Secretion to Metalate Exoenzymes

Cytosolic metalloenzymes acquire metals from buffered intracellular pools. How exported metalloenzymes are appropriately metalated is less clear. We provide evidence that TerC family proteins function in metalation of enzymes during export through the general secretion (Sec-dependent) pathway. Bacillus subtilis strains lacking MeeF(YceF) and MeeY(YkoY) have a reduced capacity for protein export and a greatly reduced level of manganese (Mn) in the secreted proteome. MeeF and MeeY copurify with proteins of the general secretory pathway, and in their absence the FtsH membrane protease is essential for viability. MeeF and MeeY are also required for efficient function of the Mn2+-dependent lipoteichoic acid synthase (LtaS), a membrane-localized enzyme with an extracytoplasmic active site. Thus, MeeF and MeeY, representative of the widely conserved TerC family of membrane transporters, function in the co-translocational metalation of Mn2+-dependent membrane and extracellular enzymes.

microbiology↗

bioRxiv2019

Mutations of the YidC Insertase alleviate stress from σM-dependent membrane protein overproduction in Bacillus subtilis

In Bacillus subtilis, the extracytoplasmic function {sigma} factor {sigma}M regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-{sigma} factors YhdL and YhdK form a complex that restricts the basal activity of {sigma}M, and the absence of YhdL leads to runaway expression of the {sigma}M regulon and cell death. Here, we report that this lethality can be suppressed by gain-of-function mutations in spoIIIJ, which encodes the major YidC membrane protein insertase in B. subtilis. B. subtilis PY79 SpoIIIJ contains a single amino acid substitution in the substrate-binding channel (Q140K), and this allele suppresses the lethality of high SigM. Analysis of a library of YidC variants reveals that increased charge (+2 or +3) in the substrate-binding channel can compensate for high expression of the {sigma}M regulon. Derepression of the {sigma}M regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidCQ140K substitution. We further show that the fitness defect caused by high {sigma}M activity is exacerbated in the absence of SecDF protein translocase or {sigma}M-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific {sigma}M-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the {sigma}M regulon has evolved to up-regulate membrane-localized complexes involved in cell wall synthesis, and to simultaneously counter the resulting stresses imposed by regulon induction.\n\nAuthor SummaryBacteria frequently produce antibiotics that inhibit the growth of competitors, and many naturally occurring antibiotics target cell wall synthesis. In Bacillus subtilis, the alternative {sigma} factor {sigma}M is induced by cell wall antibiotics, and upregulates genes for peptidoglycan and cell envelope synthesis. However, dysregulation of the {sigma}M regulon, resulting from loss of the YhdL anti-{sigma}M protein, is lethal. We here identify charge variants of the SpoIIIJ(YidC) membrane protein insertase that suppress the lethal effects of high {sigma}M activity. Further analyses reveal that induction of the {sigma}M regulon leads to high level expression of membrane proteins that trigger envelope stress, and this stress is countered by specific genes in the {sigma}M regulon.

microbiology↗