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Sporulating bacteria can retreat into long-lasting dormant spores that preserve the capacity to germinate when propitious. However, how the revival transcriptional program is memorized for years remains elusive. We revealed that in dormant spores, core RNA polymerase (RNAP) resides in a central chromosomal domain, where it remains bound to a subset of intergenic promoter regions. These regions regulate genes encoding for most essential cellular functions, such as rRNAs and tRNAs. Upon awakening, RNAP recruits key transcriptional components, including sigma factor, and progresses to express the adjacent downstream genes. Mutants devoid of spore DNA-compacting proteins exhibit scattered RNAP localization and subsequently disordered firing of gene expression during germination. Accordingly, we propose that the spore chromosome is structured to preserve the transcriptional program by halting RNAP, prepared to execute transcription at the auspicious time. Such a mechanism may sustain long-term transcriptional programs in diverse organisms displaying a quiescent life form., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Bacterial spores can remain dormant for years but possess the remarkable ability to germinate, within minutes, once nutrients become available. However, it still remains elusive how such instant awakening of cellular machineries is achieved. Utilizing Bacillus subtilis as a model, we show that YwlE arginine (Arg) phosphatase is crucial for spore germination. Accordingly, the absence of the Arg kinase McsB accelerated the process. Arg phosphoproteome of dormant spores uncovered a unique set of Arg-phosphorylated proteins involved in key biological functions, including translation and transcription. Consequently, we demonstrate that during germination, YwlE dephosphorylates an Arg site on the ribosome-associated chaperone Tig, enabling its association with the ribosome to reestablish translation. Moreover, we show that Arg dephosphorylation of the housekeeping σ factor A (SigA), mediated by YwlE, facilitates germination by activating the transcriptional machinery. Subsequently, we reveal that transcription is reinitiated at the onset of germination and its recommencement precedes that of translation. Thus, Arg dephosphorylation elicits the most critical stages of spore molecular resumption, placing this unusual post-translational modification as a major regulator of a developmental process in bacteria., Competing Interests: The authors declare no conflict of interest.

The bacterial spore can rapidly convert from a dormant to a fully active cell. Here we study this remarkable cellular transition in Bacillus subtilis and reveal the identity of the newly synthesized proteins throughout spore revival. Our analysis uncovers a highly ordered developmental program that correlates with the spore morphological changes and reveals the spatial and temporal molecular events fundamental to reconstruct a cell. As opposed to current knowledge, we found that translation takes place during the earliest revival event, termed germination, a process hitherto considered to occur without the need for any macromolecule synthesis. Furthermore, we demonstrate that translation is required for execution of germination and relies on the bona fide translational factors RpmE and Tig. Our study sheds light on the spore revival process and on the vital building blocks underlying cellular awakening, thereby paving the way for designing new antimicrobial agents to eradicate spore-forming pathogens., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacterial spores can remain dormant for years, yet they possess a remarkable potential to rapidly resume a vegetative life form. Here, we identified a distinct phase at the onset of spore outgrowth, designated the ripening period. This transition phase is exploited by the germinating spore for molecular reorganization toward elongation and subsequent cell division. We have previously shown that spores of different ages, kept under various temperatures, harbor dissimilar molecular reservoirs (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139-149, 2012). Utilizing this phenomenon, we observed that the length of the ripening period can vary according to the spore molecular content. Importantly, the duration of the ripening period was found to correlate with the initial spore rRNA content and the kinetics of rRNA accumulation upon exiting dormancy. Further, the synthesis of the ribosomal protein RplA and the degradation of the spore-specific protein SspA also correlated with the duration of the ripening period. Our data suggest that the spore molecular cargo determines the extent of the ripening period, a potentially crucial phase for a germinating spore in obtaining limited resources during revival.

Upon nutrient deprivation, Bacillus subtilis initiates the developmental process of sporulation by integrating environmental and extracellular signals. These signals are channeled into a phosphorelay ultimately activating the key transcriptional regulator of sporulation, Spo0A. Subsequently, phosphorylated Spo0A regulates the expression of genes required for sporulation to initiate. Here we identified a group of genes whose transcription levels are controlled by Spo0A during exponential growth. Among them, three upregulated genes, termed sivA, sivB (bslA), and sivC, encode factors found to inhibit Spo0A activation. We furthermore show that the Siv factors operate by reducing the activity of histidine kinases located at the top of the sporulation phosphorelay, thereby decreasing Spo0A phosphorylation. Thus, we demonstrate the existence of modulators, positively controlled by Spo0A, which inhibit inappropriate entry into the costly process of sporulation, when conditions are favorable for exponential growth.

Upon starvation, the bacterium Bacillus subtilis enters the process of sporulation, lasting several hours and culminating in formation of a spore, the most resilient cell type known. We show that a few days following sporulation, the RNA profile of spores is highly dynamic. In aging spores incubated at high temperatures, RNA content is globally decreased by degradation over several days. This degradation might be a strategy utilized by the spore to facilitate its dormancy. However, spores kept at low temperature exhibit a different RNA profile with evidence supporting transcription. Further, we demonstrate that germination is affected by spore age, incubation temperature, and RNA state, implying that spores can acquire dissimilar characteristics at a time they are considered dormant. We propose that, in contrast to current thinking, entering dormancy lasts a few days, during which spores are affected by the environment and undergo corresponding molecular changes influencing their emergence from quiescence., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The bacterium Bacillus subtilis produces the DNA integrity scanning protein (DisA), a checkpoint protein that delays sporulation in response to DNA damage. DisA scans the chromosome and pauses at sites of DNA lesions. Structural analysis showed that DisA synthesizes the small molecule cyclic diadenosine monophosphate (c-di-AMP). Here, we demonstrate that the intracellular concentration of c-di-AMP rises markedly at the onset of sporulation in a DisA-dependent manner. Furthermore, exposing sporulating cells to DNA-damaging agents leads to a global decrease in the level of this molecule. This drop was associated with stalled DisA complexes that halt c-di-AMP production and with increased levels of the c-di-AMP-degrading enzyme YybT. Reduced c-di-AMP levels cause a delay in sporulation that can be reversed by external supplementation of the molecule. Thus, c-di-AMP acts as a secondary messenger, coupling DNA integrity with progression of sporulation.

SpoIIE is a dual-function protein in Bacillus subtilis that contributes to the switch from medial to polar cell division during sporulation and is responsible for activating the cell-specific transcription factor sigma(F). SpoIIE consists of an N-terminal domain with 10 membrane-spanning segments (region I), a C-terminal phosphatase domain (region III), and a central domain (region II) of uncertain function. To investigate the role of SpoIIE in polar division, we took advantage of a system for efficiently producing polar septa during growth in a SpoIIE-dependent manner using cells engineered to produce the sporulation protein in response to an inducer. The results show that regions II and III play a critical role in polar septum formation and that specific amino acid substitutions in those regions affect the abilities of SpoIIE both to promote polar division and to localize to the division machinery. Additionally, we show that neither the phosphatase function of SpoIIE nor the N-terminal, membrane-spanning region is needed for the switch to asymmetric division.

Sporulation in Bacillus subtilis is an attractive system in which to study the translocation of a chromosome across a membrane. Sporulating cells contain two sister chromosomes that are condensed in an elongated axial filament with the origins of replication anchored at opposite poles of the sporangium. The subsequent formation of a septum near one pole divides the sporangium unequally into a forespore (the smaller compartment) and a mother cell. The septum forms around the filament, trapping the origin-proximal region of one chromosome in the forespore. As a consequence, the trapped chromosome transverses the septum with the remainder being left in the mother cell. Next, SpoIIIE assembles at the middle of the septum to create a translocase that pumps the origin-distal, two-thirds of the chromosome into the forespore. Here, we address the question of how the DNA translocase assembles and how it localizes to the septal midpoint. We present evidence that DNA transversing the septum is an anchor that nucleates the formation of the DNA translocase. We propose that DNA anchoring is responsible for the assembly of other SpoIIIE-like DNA translocases, such as those that remove trapped chromosomes from the division septum of cells undergoing binary fission.

We report the identification and characterization on a genome-wide basis of genes under the control of the developmental transcription factor sigma(E) in Bacillus subtilis. The sigma(E) factor governs gene expression in the larger of the two cellular compartments (the mother cell) created by polar division during the developmental process of sporulation. Using transcriptional profiling and bioinformatics we show that 253 genes (organized in 157 operons) appear to be controlled by sigma(E). Among these, 181 genes (organized in 121 operons) had not been previously described as members of this regulon. Promoters for many of the newly identified genes were located by transcription start site mapping. To assess the role of these genes in sporulation, we created null mutations in 98 of the newly identified genes and operons. Of the resulting mutants, 12 (in prkA, ybaN, yhbH, ykvV, ylbJ, ypjB, yqfC, yqfD, ytrH, ytrI, ytvI and yunB) exhibited defects in spore formation. In addition, subcellular localization studies were carried out using in-frame fusions of several of the genes to the coding sequence for GFP. A majority of the fusion proteins localized either to the membrane surrounding the developing spore or to specific layers of the spore coat, although some fusions showed a uniform distribution in the mother cell cytoplasm. Finally, we used comparative genomics to determine that 46 of the sigma(E)-controlled genes in B.subtilis were present in all of the Gram-positive endospore-forming bacteria whose genome has been sequenced, but absent from the genome of the closely related but not endospore-forming bacterium Listeria monocytogenes, thereby defining a core of conserved sporulation genes of probable common ancestral origin. Our findings set the stage for a comprehensive understanding of the contribution of a cell-specific transcription factor to development and morphogenesis.