Biology subjects

Herman, J. K.

Publications and source records attributed to Herman, J. K..

4 recordsLinked to original sources

bioRxiv2023

Bacillus subtilis YisK possesses oxaloacetate decarboxylase activity and exhibits Mbl-dependent localization

YisK is an uncharacterized protein in Bacillus subtilis previously shown to interact genetically with the elongasome protein Mbl. YisK overexpression leads to cell widening and lysis, phenotypes that are dependent on mbl and suppressed by mbl mutations. In the present work we characterize YisKs localization, structure, and enzymatic activity. We show that YisK localizes in a punctate and/or punctate-helical pattern that depends on Mbl, and that YisK interacts directly with another elongasome protein, FtsE. YisK belongs to the fumarylacetoacetate hydrolase (FAH) superfamily and crystal structures revealed close structural similarity to two oxaloacetate (OAA) decarboxylases: human mitochondrial FAHD1 and Corynebacterium glutamicum Cg1458. We demonstrate that YisK can also catalyze the decarboxylation of OAA (Km = 134 {micro}M, Kcat = 31 min-1). A catalytic dead variant (YisK E148A, E150A) retains wild-type localization and still widens cells following overexpression, indicating these activities are not dependent on YisK catalysis. Conversely, a non-localizing variant (YisK E30A) retains wild-type enzymatic activity in vitro, but no longer widens cells following overexpression. Together these results suggest YisK may be subject to spatial regulation that depends on the cell envelope synthesis machinery. IMPORTANCEThe elongasome is a protein complex that guides lengthwise growth in some bacteria. We previously showed that in B. subtilis, overexpression of an uncharacterized enzyme (YisK), perturbed function of the actin-like elongasome protein Mbl. Here we show that YisK exhibits Mbl-dependent localization and interacts directly with another component of the elongasome, FtsE. Through biochemical and structural characterization, we demonstrate that like its mitochondrial homolog FAHD1, YisK can catalyze the decarboxylation of the oxaloacetate to pyruvate and CO2. YisK is the first example of an enzyme implicated in central carbon metabolism with subcellular localization that depends on Mbl.

microbiology↗

bioRxiv2022

Magnesium modulates Bacillus subtilis cell division frequency

By chance, we discovered a window of extracellular magnesium (Mg2+) availability that modulates Bacillus subtilis division frequency without affecting growth rate. In this window, cells grown with excess Mg2+ produce shorter cells than those grown in unsupplemented medium. The Mg2+-responsive adjustment in cell length occurs in both rich and minimal media and in domesticated and undomesticated strains. Of other divalent cations tested, manganese (Mn2+) and zinc (Zn2+) also resulted in cell shortening, but only at concentrations that affected growth. Cell length decreased proportionally with increasing Mg2+ from 0.2 mM to 2.0 mM, with little or no detectable change in labile, intracellular Mg2+ based on a riboswitch reporter. Cells grown in excess Mg2+ had fewer nucleoids and possessed more FtsZ-rings per unit cell length, consistent with increased division frequency. Remarkably, when shifting cells from unsupplemented to supplemented medium, more than half of the cell length decrease occurred in the first 10 min, consistent with rapid division onset. Relative to unsupplemented cells, cells growing at steady-state with excess Mg2+ showed enhanced expression of a large number of SigB-regulated genes and activation of the Fur, MntR, and Zur regulons. Thus, by manipulating the availability of one nutrient, we were able to uncouple growth rate from division frequency and identify transcriptional changes suggesting cell division is accompanied by oxidative stress and an enhanced demand to sequester and/or increase uptake of iron, Mn2+, and Zn2+. IMPORTANCEThe signals cells use to trigger cell division are unknown. Although division is often considered intrinsic to the cell-cycle, microorganisms can continue to grow and repeat rounds of DNA replication without dividing, indicating cycles of division can be skipped. Here we show that by manipulating a single nutrient, Mg2+, cell division can be uncoupled from growth rate. This finding can be applied to investigate the nature of the cell division signal(s).

microbiology↗

bioRxiv2022

RefZ and Noc act synthetically to prevent aberrant divisions during Bacillus subtilis sporulation

During sporulation, Bacillus subtilis undergoes an atypical cell division that requires overriding mechanisms which protect chromosomes from damage and ensure inheritance by daughter cells. Instead of assembling between segregated chromosomes at midcell, the FtsZ-ring (Z-ring) coalesces polarly, directing division over one chromosome. The DNA-binding protein RefZ facilitates the timely assembly of polar Z-rings and partially defines the region of chromosome initially captured in the forespore. RefZ binds to motifs (RBMs) located proximal to the origin of replication (oriC). Although refZ and the RBMs are conserved across the Bacillus genus, a refZ deletion mutant sporulates with wildtype efficiency, so the functional significance of RefZ during sporulation remains unclear. To further investigate RefZ function, we performed a candidate-based screen for synthetic sporulation defects by combining {Delta}refZ with deletions of genes previously implicated in FtsZ regulation and/or chromosome capture. Combining {Delta}refZ with deletions of ezrA, sepF, parA, or minD did not detectably affect sporulation. In contrast, a {Delta}refZ {Delta}noc mutant exhibited a sporulation defect, revealing a genetic interaction between RefZ and Noc. Using reporters of sporulation progression, we determined the {Delta}refZ {Delta}noc mutant exhibited sporulation delays after Spo0A activation but prior to late sporulation, with a subset of cells failing to divide polarly or activate the first forespore-specific sigma factor, SigF. The {Delta}refZ {Delta}noc mutant also exhibited extensive dysregulation of cell division, producing cells with extra, misplaced, or otherwise aberrant septa. Our results reveal a previously unknown epistatic relationship that suggests refZ and noc contribute synthetically to regulating cell division and supporting spore development. IMPORTANCEThe DNA-binding protein RefZ and its binding sites (RBMs) are conserved in sequence and location on the chromosome across the Bacillus genus and contribute to the timing of polar FtsZ-ring assembly during sporulation. Only a small number of non-coding and non-regulatory DNA motifs are known to be conserved in chromosomal position in bacteria, suggesting there is strong selective pressure for their maintenance; however a refZ deletion mutant sporulates efficiently, providing no clues as to their functional significance. Here we find that in the absence of the nucleoid occlusion factor Noc, deletion of refZ results in a sporulation defect characterized by developmental delays and aberrant divisions.

microbiology↗

bioRxiv2018

A DNA-binding protein tunes septum placement during Bacillus subtilis sporulation

Bacillus subtilis is a soil bacterium capable of differentiating into a spore form resistant to desiccation, UV radiation, and heat. Early in spore development the cell possesses two copies of a circular chromosome, anchored to opposite cell poles via DNA proximal to the origin of replication (oriC). As sporulation progresses an FtsZ ring (Z-ring) assembles close to one pole and directs septation over one chromosome. The polar division generates two cell compartments with differing chromosomal contents. The smaller \"forespore\" compartment initially contains only 25-30% of one chromosome and this transient genetic asymmetry is required for differentiation. At the population level, the timely assembly of polar Z-rings and the precise capture of the chromosome in the forespore both require RefZ, a DNA-binding protein synthesized early in sporulation. To mediate precise capture of the chromosome RefZ must bind to specific DNA motifs (RBMs) that are localized near the poles around the time of septation, suggesting RefZ binds to the RBMs to affect positioning of the septum relative to the chromosome. RefZs mechanism of action is unknown, however, cells artificially induced to express RefZ during vegetative growth cannot assemble Z-rings or divide, leading to the hypothesis that RefZ-RBM complexes mediate precise chromosome capture by modulating FtsZ function. To investigate this possibility, we isolated 10 RefZ loss-of-function (rLOF) variants unable to inhibit cell division when expressed during vegetative growth, yet were still capable of binding RBM-containing DNA. Sporulating cells expressing the rLOF variants in place of wild-type RefZ phenocopy a {Delta}refZ mutant, suggesting that RefZ mediates chromosome capture through an FtsZ-dependent mechanism. To better understand the molecular basis of RefZs activity, the crystal structure of RefZ was solved and wild-type RefZ and the rLOF variants were further characterized. Our data suggest that RefZs oligomerization state and specificity for the RBMs are critical determinants influencing RefZs ability to affect FtsZ dynamics in vivo. We propose that RBM-bound RefZ complexes function as a developmentally regulated nucleoid occlusion system for fine-tuning the position of the septum relative to the chromosome during sporulation.\n\nAuthor SummaryThe Gram-positive bacterium B. subtilis can differentiate into a dormant cell type called a spore. Early in sporulation the cell divides near one pole, generating two compartments: a larger mother cell and a smaller forespore (future spore). Only approximately 30 percent of one chromosome is initially captured in the forespore compartment at the time of division and this genetic asymmetry is critical for sporulation to progress. Precise chromosome capture requires RefZ, a sporulation protein that binds to specific DNA motifs (RBMs) positioned at the pole near the site of cell division. How RefZ functions at the molecular level is not fully understood. Here we show that RefZ-RBM complexes facilitate chromosome capture by acting through the major cell division protein FtsZ.

microbiology↗