Search

Nine compounds and three modes of applications were tested to improve the control of two Capnodis spp., C. tenebrionis L. and C. carbonaria Klug (Coleoptera: Buprestidae), in local stone-fruit plantations. The best result was obtained by azinphos-methyl applied as a foliar spray. Suitable chemicals for dusting were methiocarb, azinphos-methyl and carbosulfan applied to the stem in late spring or prior to fruit harvest. Soil application of imidacloprid as a systemic insecticide is successful for protection of young plantations and nurseries. C. tenebrionis was less susceptible than C. carbonaria to the tested insecticides. [ABSTRACT FROM AUTHOR]

Conjugation-mediated DNA delivery is the primary mode for antibiotic resistance spread in bacteria; yet, molecular mechanisms regulating the conjugation process remain largely unexplored. While conjugative plasmids typically require bacterial attachment to solid surfaces for facilitation of donor-to-recipient proximity, the pLS20 conjugative plasmid, prevalent among Gram-positive Bacillus spp., uniquely requires fluid environments to enhance its transfer. Here, we show that pLS20, carried by Bacillus subtilis, induces multicellular clustering, which can accommodate various species, hence offering a stable platform for DNA delivery in a liquid milieu. We further discovered that induction of pLS20 promoters, governing crucial conjugative genes, is dependent on the presence of donor cell flagella, the major bacterial motility organelle. Moreover, the pLS20 regulatory circuit is controlled by a mechanosensing signal transduction pathway responsive to flagella rotation, thus activating conjugation gene expression exclusively during the host motile phase. This flagella-conjugation coupling strategy may allow the dissemination of the plasmid to remote destinations, allowing infiltration into new niches., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

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 preserve cellular dormancy for years, but still hold the remarkable ability to revive and recommence life. This cellular awakening begins with a rapid and irreversible event termed germination; however, the metabolic determinants required for its success have been hardly explored. Here, we show that at the onset of the process of sporulation, the metabolic enzyme RocG catabolizes glutamate, facilitating ATP production in the spore progenitor cell, and subsequently influencing the eventual spore ATP reservoir. Mutants displaying low RocG levels generate low ATP-containing spores that exhibit severe germination deficiency. Importantly, this phenotype could be complemented by expressing RocG at a specific window of time during the initiation of sporulation. Thus, we propose that despite its low abundance in dormant spores, ATP energizes spore germination, and its production, fueled by RocG, is coupled with the initial developmental phase of spore formation., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

The cell envelope of Gram-negative bacteria is a complex structure, essential for bacterial survival and for resistance to many antibiotics. Channels that cross the bacterial envelope and the host cell membrane form secretion systems that are activated upon attachment to host, enabling bacteria to inject effector molecules into the host cell, required for bacterium-host interaction. The type III secretion system (T3SS) is critical for the virulence of several pathogenic bacteria, including enteropathogenic Escherichia coli (EPEC). EPEC T3SS activation is associated with repression of carbon storage regulator (CsrA), resulting in gene expression remodeling, which is known to affect EPEC central carbon metabolism and contributes to the adaptation to a cell-adherent lifestyle in a poorly understood manner. We reasoned that the changes in the bacterial envelope upon attachment to the host and the activation of a secretion system may involve a modification of the lipid composition of bacterial envelope. Accordingly, we performed a lipidomics analysis on mutant strains that simulate T3SS activation. We saw a shift in glycerophospholipid metabolism toward the formation of lysophospholipids, attributed to corresponding upregulation of the phospholipase gene pldA and the acyltransferase gene ygiH upon T3SS activation in EPEC. We also detected a shift from menaquinones and ubiquinones to undecaprenyl lipids, concomitant with abnormal synthesis of O antigen. The remodeling of lipid metabolism is mediated by CsrA and associated with increased bacterial cell size and zeta potential and a corresponding alteration in EPEC permeability to vancomycin, increasing the sensitivity of T3SS-activated strains and of adherent wild-type EPEC to the antibiotic. IMPORTANCE The characterization of EPEC membrane lipid metabolism upon attachment to the host is an important step toward a better understanding the shift of EPEC, a notable human pathogen, from a planktonic to adherent lifestyle. It may also apply to other pathogenic bacteria that use this secretion system. We predict that upon attachment to host cells, the lipid remodeling upon T3SS activation contributes to bacterial fitness and promotes host colonization, and we show that it is associated with increased cell permeability and higher sensitivity to vancomycin. To the best of our knowledge, this is the first demonstration of a bacterial lipid remodeling due to activation of a secretion system.

The Gram positive bacterium Bacillus subtilis and its relatives are capable of forming a durable dormant long-lasting spore. Although spores can remain dormant for years, they possess the remarkable capacity to rapidly resume life and convert into actively growing cells. This cellular transition initiates with a most enigmatic irreversible event, termed germination, lasting only for a few minutes. Germination is typified by a morphological conversion that culminates in loss of spore resilient properties. Yet, the molecular events occurring during this brief critical phase are largely unknown. The current widely accepted view considers germination to occur without the need for any macromolecule synthesis; however, accumulating data from our laboratory and others, highlighted here, provide evidence that both transcription and translation occur during germination and are required for its execution. We further underline numerous overlooked studies, conducted mainly during the 1960s-1970s, reinforcing this notion. We propose to revisit the fascinating process of spore germination and redefine it as a pathway involving macromolecule synthesis. We expect our perspective to shed new light on the awakening process of a variety of spore-forming environmental, commensal, and pathogenic bacteria and possibly be applicable to additional organisms displaying a quiescent life form., Competing Interests: None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Appearance of plaques on a bacterial lawn is a sign of successive rounds of bacteriophage infection. Yet, mechanisms evolved by bacteria to limit plaque spread have been hardly explored. Here, we investigated the dynamics of plaque development by lytic phages infecting the bacterium Bacillus subtilis. We report that plaque expansion is followed by a constriction phase owing to bacterial growth into the plaque zone. This phenomenon exposed an adaptive process, herein termed "phage tolerance response", elicited by non-infected bacteria upon sensing infection of their neighbors. The temporary phage tolerance is executed by the stress-response RNA polymerase sigma factor σ X (SigX). Artificial expression of SigX prior to phage attack largely eliminates infection. SigX tolerance is primarily conferred by activation of the dlt operon, encoding enzymes that catalyze D-alanylation of cell wall teichoic acid polymers, the major attachment sites for phages infecting Gram-positive bacteria. D-alanylation impedes phage binding and hence infection, thus enabling the uninfected bacteria to form a protective shield opposing phage spread., (© 2021 The Authors.)

Bacteria can produce membranous nanotubes that mediate contact-dependent exchange of molecules among bacterial cells. However, it is unclear how nanotubes cross the cell wall to emerge from the donor or to penetrate into the recipient cell. Here, we report that Bacillus subtilis utilizes cell wall remodeling enzymes, the LytC amidase and its enhancer LytB, for efficient nanotube extrusion and penetration. Nanotube production is reduced in a lytBC mutant, and the few nanotubes formed appear deficient in penetrating into target cells. Donor-derived LytB molecules localize along nanotubes and on the surface of nanotube-connected neighbouring cells, primarily at sites of nanotube penetration. Furthermore, LytB from donor B. subtilis can activate LytC of recipient bacteria from diverse species, facilitating cell wall hydrolysis to establish nanotube connection. Our data provide a mechanistic view of how intercellular connecting devices can be formed among neighbouring bacteria.

This study evaluated the efficacy of two entomopathogenic Hypocrealean fungi, Metarhizium brunneum (laboratory isolate) and Beauveria bassiana (the commercial product Botanigard), for preventative control of the false codling moth, Thaumatotibia leucotreta. The mortality of eggs and first instar larvae was studied in three different assays. First, fungal virulence was examined under optimal laboratory conditions (25 °C, 85% RH) by placing T. leucotreta eggs on conidia-impregnated filter paper. One-day-old eggs and first instar larvae were susceptible to both fungi. In contrast, 5-day-old eggs (advanced embryo development) were susceptible to M. brunneum, but not to B. bassiana. The activity of both fungi against eggs was assessed under two humidity regimes: 85% RH-optimal for fungal germination, and 60% RH-the average humidity in the laboratory. Pieces of parchment paper serving as oviposition surfaces were treated with each of the fungi and introduced to gravid females at different time points after inoculation (0, 2, 7 and 14 days). Although the tested fungal species differed in their virulence to T. leucotreta eggs, both reduced hatching rate under both humidity regimes to 8.3-58.3%, compared to 71.7-83.3% in the control treatments. To evaluate reduction of T. leucotreta infestation of fruit, 'Ori' citrus fruit (easy peeler Citrus sinensis) were treated with each of the fungi. Eggs were placed on the fruit peels and the fruit were maintained under room conditions (25 °C, 60% RH). Between 41.7% and 54.1% of fruit in control groups were infested by the T. leucotreta larvae. Treatments with either of the fungi resulted in about 16% infestation of the fruit with larvae, a marked (3.3-fold) reduction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Bacteria in nature are known to survive for long periods under restricting conditions, mainly by reducing their growth rate and metabolic activity. Here, we uncover a novel strategy utilized by bacterial cells to resist aging by propagating rather than halting division. Bacterial aging was monitored by inspecting colonies of the Gram-positive soil bacterium Bacillus subtilis , which is capable of differentiating into various cell types under nutrient exhaustion. We revealed that after days of incubation, rejuvenating subpopulations, arrayed over the mother colony, emerged. These subpopulations were found to harbor mutations in a variety of genes, restricting the ability of the cells to differentiate. Surprisingly, even mutations that are not classically designated to developmental pathways, concluded in differentiation deficiency, indicating that multiple paths can reach this same outcome. We provide evidence that the evolved mutants continue to divide under conditions that favor entry into quiescence, hence becoming abundant within the aging population. The occurrence of such nondifferentiating mutants could impact bacterial population dynamics in natural niches. IMPORTANCE Until now, bacterial cells facing nutrient deprivation were shown to enter dormancy as a strategy to survive prolonged stress, with the most established examples being sporulation, stationary phase, and persistence. Here, we uncovered an opposing strategy for long-term bacterial survival, in which mutant subpopulations cope with a challenging niche by proliferating rather than by stalling division. We show that this feature stems from mutations in genes disturbing the capability of the cells to differentiate into a quiescent state, enabling them to divide under restrictive conditions. Our study challenges the dogma of bacterial aging by highlighting an additional survival strategy resembling that of cancerous cells in animal organs., (Copyright © 2019 Hashuel and Ben-Yehuda.)

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.

Bacillus subtilis diadenylate cyclase DisA converts two ATPs into c-di-AMP, but this activity is suppressed upon interaction with sites of DNA damage. DisA forms a rapid moving focus that pauses upon induction of DNA damage during spore development. We report that DisA pausing, however, was not observed in the absence of the RecO mediator or of the RecA recombinase, suggesting that DisA binds to recombination intermediates formed by RecA in concert with RecO. DisA, which physically interacts with RecA, was found to reduce its ATPase activity without competing for nucleotides or ssDNA. Furthermore, increasing DisA concentrations inhibit RecA-mediated DNA strand exchange, but this inhibition failed to occur when RecA was added prior to DisA, and was independent of RecA-mediated nucleotide hydrolysis or increasing concentrations of c-di-AMP. We propose that DisA may preserve genome integrity by downregulating RecA activities at several steps of the DNA damage tolerance pathway, allowing time for the repair machineries to restore genome stability. DisA might reduce RecA-mediated template switching by binding to a stalled or reversed fork., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacteriophages (phages) are the most abundant entities in nature, yet little is known about their capacity to acquire new hosts and invade new niches. By exploiting the Gram-positive soil bacterium Bacillus subtilis (B. subtilis) and its lytic phage SPO1 as a model, we followed the coevolution of bacteria and phages. After infection, phage-resistant bacteria were readily isolated. These bacteria were defective in production of glycosylated wall teichoic acid (WTA) polymers that served as SPO1 receptor. Subsequently, a SPO1 mutant phage that could infect the resistant bacteria evolved. The emerging phage contained mutations in two genes, encoding the baseplate and fibers required for host attachment. Remarkably, the mutant phage gained the capacity to infect non-host Bacillus species that are not infected by the wild-type phage. We provide evidence that the evolved phage lost its dependency on the species-specific glycosylation pattern of WTA polymers. Instead, the mutant phage gained the capacity to directly adhere to the WTA backbone, conserved among different species, thereby crossing the species barrier., (© 2019 John Wiley & Sons Ltd.)

Microbiota and intestinal epithelium restrict pathogen growth by rapid nutrient consumption. We investigated how pathogens circumvent this obstacle to colonize the host. Utilizing enteropathogenic E. coli (EPEC), we show that host-attached bacteria obtain nutrients from infected host cell in a process we termed host nutrient extraction (HNE). We identified an inner-membrane protein complex, henceforth termed CORE, as necessary and sufficient for HNE. The CORE is a key component of the EPEC injectisome, however, here we show that it supports the formation of an alternative structure, composed of membranous nanotubes, protruding from the EPEC surface to directly contact the host. The injectisome and flagellum are evolutionarily related, both containing conserved COREs. Remarkably, CORE complexes of diverse ancestries, including distant flagellar COREs, could rescue HNE capacity of EPEC lacking its native CORE. Our results support the notion that HNE is a widespread virulence strategy, enabling pathogens to thrive in competitive niches., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We have previously described the existence of membranous nanotubes, bridging adjacent bacteria, facilitating intercellular trafficking of nutrients, cytoplasmic proteins, and even plasmids, yet components enabling their biogenesis remain elusive. Here we reveal the identity of a molecular apparatus providing a platform for nanotube biogenesis. Using Bacillus subtilis (Bs), we demonstrate that conserved components of the flagellar export apparatus (FliO, FliP, FliQ, FliR, FlhB, and FlhA), designated CORE, dually serve for flagellum and nanotube assembly. Mutants lacking CORE genes, but not other flagellar components, are deficient in both nanotube production and the associated intercellular molecular trafficking. In accord, CORE components are located at sites of nanotube emergence. Deleting COREs of distinct species established that CORE-mediated nanotube formation is widespread. Furthermore, exogenous COREs from diverse species could restore nanotube generation and functionality in Bs lacking endogenous CORE. Our results demonstrate that the CORE-derived nanotube is a ubiquitous organelle that facilitates intercellular molecular trade across the bacterial kingdom., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bacteria use elaborate molecular machines for intercellular contact-dependent interactions. We discuss a relatively less explored type of intercellular connections mediated by tubular membranous bridges, termed nanotubes. Increasing evidence suggests that nanotube structures mediate cytoplasmic molecular trade among neighboring cells of the same and different species. Further, nanotubes were found to facilitate both antagonistic and cooperative interspecies interactions, thereby allowing the emergence of new non-heritable phenotypes in multicellular bacterial communities. We propose that nanotube-mediated cytoplasmic sharing represents a widespread form of bacterial interactions in nature, providing an enormous potential for the emergence of new features. Here we review the current knowledge on bacterial nanotubes, and highlight the gaps in our current understanding of their operation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Fumarase is distributed between two compartments of the eukaryotic cell. The enzyme catalyses the reversible conversion of fumaric to L-malic acid in mitochondria as part of the tricarboxylic acid (TCA) cycle, and in the cytosol/nucleus as part of the DNA damage response (DDR). Here, we show that fumarase of the model prokaryote Bacillus subtilis (Fum-bc) is induced upon DNA damage, co-localized with the bacterial DNA and is required for the DDR. Fum-bc can substitute for both eukaryotic functions in yeast. Furthermore, we found that the fumarase-dependent intracellular signaling of the B. subtilis DDR is achieved via production of L-malic acid, which affects the translation of RecN, the first protein recruited to DNA damage sites. This study provides a different evolutionary scenario in which the dual function of the ancient prokaryotic fumarase, led to its subsequent distribution into different cellular compartments in eukaryotes.

Colonies are an abundant form of bacterial multicellularity; however, relatively little is known about the initial stages of their construction. We have previously described that colony development of the soil bacterium Bacillus subtilis is a highly ordered process, typically initiating with the formation of extending cell chains arranged in a Y shape structure. Furthermore, we demonstrated that Y arm extension is a key for defining the size of the future colony. Here we conducted a genetic screen surveying for mutants deficient in these early developmental stages, and revealed LtaS, the major lipoteichoic acid (LTA) synthase, to be crucial for execution of these events. We found that the ltaS mutant fails to produce proper Y shape structures, forming extremely elongated chains of cells with no evidence of chain breakage, necessary for Y shape formation. Furthermore, we show that frequent cell death at the tips of the cell chains is a major cause in limiting arm extension. Collectively, these perturbations lead to the production of a small sized colony by the mutant. Thus, deficiency in LTA synthesis causes a mechanical failure in executing the colony developmental program.

Bacteria have developed various mechanisms by which they sense, interact, and kill other bacteria, in an attempt to outcompete one another and survive. Here we show that Bacillus subtilis can kill and prey on Bacillus megaterium. We find that Bacillus subtilis rapidly inhibits Bacillus megaterium growth by delivering the tRNase toxin WapA. Furthermore, utilizing the methionine analogue L-azidohomoalanine as a nutrient reporter, we provide evidence of nutrient extraction from Bacillus megaterium by Bacillus subtilis. Toxin delivery and nutrient extraction occur in a contact-dependent manner, and both activities are abolished in the absence of the phosphodiestrase YmdB, shown previously to mediate intercellular nanotube formation. Furthermore, we detect the localization of WapA molecules to nanotubes. Thus, we propose that Bacillus subtilis utilizes the same nanotube apparatus in a bidirectional manner, delivering toxin and acquiring beneficial cargo, thereby maximally exploiting potential niche resources.Bacteria can exchange nutrients and macromolecules through tubular membranous structures called nanotubes. Here, the authors show that Bacillus subtilis can kill and prey on Bacillus megaterium by delivering a toxin and extracting nutrients in a nanotube-dependent manner.

Bacteriophages (phages) typically exhibit a narrow host range, yet they tremendously impact horizontal gene transfer (HGT). Here, we investigate phage dynamics in communities harboring phage-resistant (R) and sensitive (S) bacteria, a common scenario in nature. Using Bacillus subtilis and its lytic phage SPP1, we demonstrate that R cells, lacking SPP1 receptor, can be lysed by SPP1 when co-cultured with S cells. This unanticipated lysis was triggered in part by phage lytic enzymes released from nearby infected cells. Strikingly, we discovered that occasionally phages can invade R cells, a phenomenon we termed acquisition of sensitivity (ASEN). We found that ASEN is mediated by R cells transiently gaining phage attachment molecules from neighboring S cells and provide evidence that this molecular exchange is driven by membrane vesicles. Exchange of phage attachment molecules could even occur in an interspecies fashion, enabling phage adsorption to non-host species, providing an unexplored route for HGT. VIDEO ABSTRACT., (Copyright © 2017 Elsevier Inc. All rights reserved.)

When grown on a solid surface, bacteria form highly organized colonies, yet little is known about the earliest stages of colony establishment. Following Bacillus subtilis colony development from a single progenitor cell, a sequence of highly ordered spatiotemporal events was revealed. Colony was initiated by the formation of leading-cell chains, deriving from the colony center and extending in multiple directions, typically in a "Y-shaped" structure. By eradicating particular cells during these early stages, we could influence the shape of the resulting colony and demonstrate that Y-arm extension defines colony size. A mutant in ymdB encoding a phosphodiesterase displayed unordered developmental patterns, indicating a role in guiding these initial events. Finally, we provide evidence that intercellular nanotubes contribute to proper colony formation. In summary, we reveal a "construction plan" for building a colony and provide the initial molecular basis for this process., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacteria display an array of contact-dependent interaction systems that have evolved to facilitate direct cell-to-cell communication. We have previously identified a mode of bacterial communication mediated by nanotubes bridging neighboring cells. Here, we elucidate nanotube architecture, dynamics, and molecular components. Utilizing Bacillus subtilis as a model organism, we found that at low cell density, nanotubes exhibit remarkable complexity, existing as both intercellular tubes and extending tubes, with the latter frequently surrounding the cells in a "root-like" fashion. Observing nanotube formation in real time showed that these structures are formed in the course of minutes, displaying rapid movements. Utilizing a combination of super-resolution, light, and electron microscopy, we revealed that nanotubes are composed of chains of membranous segments harboring a continuous lumen. Furthermore, we discovered that a conserved calcineurin-like protein, YmdB, presents in nanotubes and is required for both nanotube production and intercellular molecular trade., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Bacterial spores can remain dormant for decades, yet harbor the exceptional capacity to rapidly resume metabolic activity and recommence life. Although germinants and their corresponding receptors have been known for more than 30 years, the molecular events underlying this remarkable cellular transition from dormancy to full metabolic activity are only partially defined., Results: Here, we examined whether protein phospho-modifications occur during germination, the first step of exiting dormancy, thereby facilitating spore revival. Utilizing Bacillus subtilis as a model organism, we performed phosphoproteomic analysis to define the Ser/Thr/Tyr phosphoproteome of a reviving spore. The phosphoproteome was found to chiefly comprise newly identified phosphorylation sites located within proteins involved in basic biological functions, such as transcription, translation, carbon metabolism, and spore-specific determinants. Quantitative comparison of dormant and germinating spore phosphoproteomes revealed phosphorylation dynamics, indicating that phospho-modifications could modulate protein activity during this cellular transition. Furthermore, by mutating select phosphorylation sites located within proteins representative of key biological processes, we established a functional connection between phosphorylation and the progression of spore revival., Conclusions: Herein, we provide, for the first time, a phosphoproteomic view of a germinating bacterial spore. We further show that the spore phosphoproteome is dynamic and present evidence that phosphorylation events play an integral role in facilitating spore revival.

Unlabelled: Chromosomal DNA is a constant source of information, essential for any given cell to respond and adapt to changing conditions. Here, we investigated the fate of exponentially growing bacterial cells experiencing a sudden and rapid loss of their entire chromosome. Utilizing Bacillus subtilis cells harboring an inducible copy of the endogenous toxin yqcG, which encodes an endonuclease, we induced the formation of a population of cells that lost their genetic information simultaneously. Surprisingly, these DNA-less cells, termed DLCs, did not lyse immediately and exhibited normal cellular morphology for a period of at least 5 h after DNA loss. This cellular integrity was manifested by their capacity to maintain an intact membrane and membrane potential and cell wall architecture similar to those of wild-type cells. Unlike growing cells that exhibit a dynamic profile of macromolecules, DLCs displayed steady protein and RNA reservoirs. Remarkably, following DLCs by time lapse microscopy revealed that they succeeded in synthesizing proteins, elongating, and dividing, apparently forming de novo Z rings at the midcell position. Taken together, the persistence of key cellular events in DLCs indicates that the information to carry out lengthy processes is harbored within the remaining molecular components., Importance: Perturbing bacterial growth by the use of antibiotics targeting replication, transcription, or translation has been a subject of study for many years; however, the consequences of a more dramatic event, in which the entire bacterial chromosome is lost, have not been described. Here, we followed the fate of bacterial cells encountering an abrupt loss of their entire genome. Surprisingly, the cells preserved an intact envelope and functioning macromolecules. Furthermore, cells lacking their genome could still elongate and divide hours after the loss of DNA. Our data suggest that the information stored in the transient reservoir of macromolecules is sufficient to carry out complex and lengthy processes even in the absence of the chromosome. Based on our study, the formation of DNA-less bacteria could serve as a novel vaccination strategy, enabling an efficient induction of the immune system without the risk of bacterial propagation within the host., (Copyright © 2015 Elbaz and Ben-Yehuda.)

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.)

Protein kinases are regulated by a large number of mechanisms that vary from one kinase to another. However, a fundamental activation mechanism shared by all protein kinases is phosphorylation of a conserved activation loop threonine residue. This is achieved in many cases via autophosphorylation. The mechanism and structural basis for autophosphorylation are not clear and are in fact enigmatic because this phosphorylation occurs when the kinase is in its inactive conformation. Unlike most protein kinases, MAP kinases are not commonly activated by autophosphorylation but rather by MEK-dependent phosphorylation. Here we show that p38β, a p38 isoform that is almost identical to p38α, is exceptional and spontaneously autoactivates by autophosphorylation. We identified a 13-residue-long region composed of part of the αG-helix and the MAPK insert that triggers the intrinsic autophosphorylation activity of p38β. When inserted into p38α, this fragment renders it spontaneously active in vitro and in mammalian cells. We further found that an interaction between the N terminus and a particular region of the C-terminal extension suppresses the intrinsic autophosphorylation of p38β in mammalian cells. Thus, this study identified the structural motif responsible for the unique autophosphorylation capability of p38β and the motif inhibiting this activity in living cells. It shows that the MAPK insert and C-terminal extension, structural motifs that are unique to MAPKs, play a critical role in controlling autophosphorylation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.

Finding bacterial cellular targets for developing novel antibiotics has become a major challenge in fighting resistant pathogenic bacteria. We present a novel compound, Relacin, designed to inhibit (p)ppGpp production by the ubiquitous bacterial enzyme RelA that triggers the Stringent Response. Relacin inhibits RelA in vitro and reduces (p)ppGpp production in vivo. Moreover, Relacin affects entry into stationary phase in Gram positive bacteria, leading to a dramatic reduction in cell viability. When Relacin is added to sporulating Bacillus subtilis cells, it strongly perturbs spore formation regardless of the time of addition. Spore formation is also impeded in the pathogenic bacterium Bacillus anthracis that causes the acute anthrax disease. Finally, the formation of multicellular biofilms is markedly disrupted by Relacin. Thus, we establish that Relacin, a novel ppGpp analogue, interferes with bacterial long term survival strategies, placing it as an attractive new antibacterial agent.

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 ability of bacteria to responsively regulate the expression of translation components is crucial for rapid adaptation to fluctuating environments. Utilizing Bacillus subtilis (B. subtilis) as a model organism, we followed the dynamics of the translational machinery at a single cell resolution during growth and differentiation. By comprehensive monitoring the activity of the major rrn promoters and ribosomal protein production, we revealed diverse dynamics between cells grown in rich and poor medium, with the most prominent dissimilarities exhibited during deep stationary phase. Further, the variability pattern of translational activity varied among the cells, being affected by nutrient availability. We have monitored for the first time translational dynamics during the developmental process of sporulation within the two distinct cellular compartments of forespore and mother-cell. Our study uncovers a transient forespore specific increase in expression of translational components. Finally, the contribution of each rrn promoter throughout the bacterium life cycle was found to be relatively constant, implying that differential expression is not the main purpose for the existence of multiple rrn genes. Instead, we propose that coordination of the rrn operons serves as a strategy to rapidly fine tune translational activities in a synchronized fashion to achieve an optimal translation level for a given condition.

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.

Understanding the organization of a bacterial cell requires the elucidation of the mechanisms by which proteins localize to particular subcellular sites. Thus far, such mechanisms have been suggested to rely on embedded features of the localized proteins. Here, we report that certain messenger RNAs (mRNAs) in Escherichia coli are targeted to the future destination of their encoded proteins, cytoplasm, poles, or inner membrane in a translation-independent manner. Cis-acting sequences within the transmembrane-coding sequence of the membrane proteins are necessary and sufficient for mRNA targeting to the membrane. In contrast to the view that transcription and translation are coupled in bacteria, our results show that, subsequent to their synthesis, certain mRNAs are capable of migrating to particular domains in the cell where their future protein products are required.

Bacteria are known to communicate primarily via secreted extracellular factors. Here we identify a previously uncharacterized type of bacterial communication mediated by nanotubes that bridge neighboring cells. Using Bacillus subtilis as a model organism, we visualized transfer of cytoplasmic fluorescent molecules between adjacent cells. Additionally, by coculturing strains harboring different antibiotic resistance genes, we demonstrated that molecular exchange enables cells to transiently acquire nonhereditary resistance. Furthermore, nonconjugative plasmids could be transferred from one cell to another, thereby conferring hereditary features to recipient cells. Electron microscopy revealed the existence of variously sized tubular extensions bridging neighboring cells, serving as a route for exchange of intracellular molecules. These nanotubes also formed in an interspecies manner, between B. subtilis and Staphylococcus aureus, and even between B. subtilis and the evolutionary distant bacterium Escherichia coli. We propose that nanotubes represent a major form of bacterial communication in nature, providing a network for exchange of cellular molecules within and between species., (Copyright © 2011 Elsevier Inc. All rights reserved.)