Structure of the WYL-domain containing transcription activator, DriD, in complex with ssDNA effector and DNA target site.

Transcription regulators play central roles in orchestrating responses to changing environmental conditions. Recently the Caulobacter crescentus transcription activator DriD, which belongs to the newly defined WYL-domain family, was shown to regulate DNA damage responses independent of the canonical SOS pathway. However, the molecular mechanisms by which DriD and other WYL-regulators sense environmental signals and recognize DNA are not well understood. We showed DriD DNA-binding is triggered by its interaction with ssDNA, which is produced during DNA damage. Here we describe the structure of the full-length C. crescentus DriD bound to both target DNA and effector ssDNA. DriD consists of an N-terminal winged-HTH (wHTH) domain, linker region, three-helix bundle, WYL-domain and C-terminal WCX-dimer domain. Strikingly, DriD binds DNA using a novel, asymmetric DNA-binding mechanism that results from different conformations adopted by the linker. Although the linker does not touch DNA, our data show that contacts it makes with the wHTH are key for specific DNA binding. The structure indicates how ssDNA-effector binding to the WYL-domain impacts wHTH DNA binding. In conclusion, we present the first structure of a WYL-activator bound to both effector and target DNA. The structure unveils a unique, asymmetric DNA binding mode that is likely conserved among WYL-activators.

Pulcherrimin protects Bacillus subtilis against oxidative stress during biofilm development.

Pulcherrimin is an iron-binding reddish pigment produced by various bacterial and yeast species. In the soil bacterium Bacillus subtilis, this pigment is synthesized intracellularly as the colorless pulcherriminic acid by using two molecules of tRNA-charged leucine as the substrate; pulcherriminic acid molecules are then secreted and bind to ferric iron extracellularly to form the red-colored pigment pulcherrimin. The biological importance of pulcherrimin is not well understood. A previous study showed that secretion of pulcherrimin caused iron depletion in the surroundings and growth arrest on cells located at the edge of a B. subtilis colony biofilm. In this study, we identified that pulcherrimin is primarily produced under biofilm conditions and provides protection to cells in the biofilm against oxidative stress. We presented molecular evidence on how pulcherrimin lowers the level of reactive oxygen species (ROS) and alleviates oxidative stress and DNA damage caused by ROS accumulation in a mature biofilm. We also performed global transcriptome profiling to identify differentially expressed genes in the pulcherrimin-deficient mutant compared with the wild type, and further characterized the regulation of genes by pulcherrimin that are related to iron homeostasis, DNA damage response (DDR), and oxidative stress response. Based on our findings, we propose pulcherrimin as an important antioxidant that modulates B. subtilis biofilm development.

Prophage-like gene transfer agents promote Caulobacter crescentus survival and DNA repair during stationary phase.

Gene transfer agents (GTAs) are prophage-like entities found in many bacterial genomes that cannot propagate themselves and instead package approximately 5 to 15 kbp fragments of the host genome that can then be transferred to related recipient cells. Although suggested to facilitate horizontal gene transfer (HGT) in the wild, no clear physiological role for GTAs has been elucidated. Here, we demonstrate that the α-proteobacterium Caulobacter crescentus produces bona fide GTAs. The production of Caulobacter GTAs is tightly regulated by a newly identified transcription factor, RogA, that represses gafYZ, the direct activators of GTA synthesis. Cells lacking rogA or expressing gafYZ produce GTAs harboring approximately 8.3 kbp fragment of the genome that can, after cell lysis, be transferred into recipient cells. Notably, we find that GTAs promote the survival of Caulobacter in stationary phase and following DNA damage by providing recipient cells a template for homologous recombination-based repair. This function may be broadly conserved in other GTA-producing organisms and explain the prevalence of this unusual HGT mechanism.

ssDNA is an allosteric regulator of the C. crescentus SOS-independent DNA damage response transcription activator, DriD.

DNA damage repair systems are critical for genomic integrity. However, they must be coordinated with DNA replication and cell division to ensure accurate genomic transmission. In most bacteria, this coordination is mediated by the SOS response through LexA, which triggers a halt in cell division until repair is completed. Recently, an SOS-independent damage response system was revealed in Caulobacter crescentus. This pathway is controlled by the transcription activator, DriD, but how DriD senses and signals DNA damage is unknown. To address this question, we performed biochemical, cellular, and structural studies. We show that DriD binds a specific promoter DNA site via its N-terminal HTH domain to activate transcription of genes, including the cell division inhibitor didA A structure of the C-terminal portion of DriD revealed a WYL motif domain linked to a WCX dimerization domain. Strikingly, we found that DriD binds ssDNA between the WYL and WCX domains. Comparison of apo and ssDNA-bound DriD structures reveals that ssDNA binding orders and orients the DriD domains, indicating a mechanism for ssDNA-mediated operator DNA binding activation. Biochemical and in vivo studies support the structural model. Our data thus reveal the molecular mechanism underpinning an SOS-independent DNA damage repair pathway.

Escherichia coli SymE is a DNA-binding protein that can condense the nucleoid.

Type I toxin-antitoxin (TA) systems typically consist of a protein toxin that imbeds in the inner membrane where it can oligomerize and form pores that change membrane permeability, and an RNA antitoxin that interacts directly with toxin mRNA to inhibit its translation. In Escherichia coli, symE/symR is annotated as a type I TA system with a non-canonical toxin. SymE was initially suggested to be an endoribonuclease, but has predicted structural similarity to DNA binding proteins. To better understand SymE function, we used RNA-seq to examine cells ectopically producing it. Although SymE drives major changes in gene expression, we do not find strong evidence of endoribonucleolytic activity. Instead, our biochemical and cell biological studies indicate that SymE binds DNA. We demonstrate that the toxicity of symE overexpression likely stems from its ability to drive severe nucleoid condensation, which disrupts DNA and RNA synthesis and leads to DNA damage, similar to the effects of overproducing the nucleoid-associated protein H-NS. Collectively, our results suggest that SymE represents a new class of nucleoid-associated proteins that is widely distributed in bacteria.

Characterizing the portability of phage-encoded homologous recombination proteins.

Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded single-stranded DNA annealing proteins (SSAPs) improve HR 1,000-fold above endogenous levels. However, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus and Caulobacter crescentus, we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host's single-stranded DNA-binding protein (SSB) and are portable between species only if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with SSBs can significantly improve genome editing efficiency, in some species enabling SSAP functionality even without host compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes that are inaccessible through sequential nucleotide conversions.

A Decrease in Serine Levels during Growth Transition Triggers Biofilm Formation in Bacillus subtilis.

Biofilm development in Bacillus subtilis is regulated at multiple levels. While a number of known signals that trigger biofilm formation do so through the activation of one or more sensory histidine kinases, it was discovered that biofilm activation is also coordinated by sensing intracellular metabolic signals, including serine starvation. Serine starvation causes ribosomes to pause on specific serine codons, leading to a decrease in the translation rate of sinR, which encodes a master repressor for biofilm matrix genes and ultimately triggers biofilm induction. How serine levels change in different growth stages, how B. subtilis regulates intracellular serine levels, and how serine starvation triggers ribosomes to pause on selective serine codons remain unknown. Here, we show that serine levels decrease as cells enter stationary phase and that unlike most other amino acid biosynthesis genes, expression of serine biosynthesis genes decreases upon the transition into stationary phase. The deletion of the gene for a serine deaminase responsible for converting serine to pyruvate led to a delay in biofilm formation, further supporting the idea that serine levels are a critical intracellular signal for biofilm activation. Finally, we show that levels of all five serine tRNA isoacceptors are decreased in stationary phase compared with exponential phase. However, the three isoacceptors recognizing UCN serine codons are reduced to a much greater extent than the two that recognize AGC and AGU serine codons. Our findings provide evidence for a link between serine homeostasis and biofilm development in B. subtilisIMPORTANCE In Bacillus subtilis, biofilm formation is triggered in response to environmental and cellular signals. It was proposed that serine limitation acts as a proxy for nutrient status and triggers biofilm formation at the onset of biofilm entry through a novel signaling mechanism caused by global ribosome pausing on selective serine codons. In this study, we reveal that serine levels decrease at the biofilm entry due to catabolite control and a serine shunt mechanism. We also show that levels of five serine tRNA isoacceptors are differentially decreased in stationary phase compared with exponential phase; three isoacceptors recognizing UCN serine codons are reduced much more than the two recognizing AGC and AGU codons. This finding indicates a possible mechanism for selective ribosome pausing.

Mechanisms of Resistance to the Contact-Dependent Bacteriocin CdzC/D in Caulobacter crescentus.

The Cdz bacteriocin system allows the aquatic oligotrophic bacterium Caulobacter crescentus to kill closely related species in a contact-dependent manner. The toxin, which aggregates on the surfaces of producer cells, is composed of two small hydrophobic proteins, CdzC and CdzD, each bearing an extended glycine-zipper motif, that together induce inner membrane depolarization and kill target cells. To further characterize the mechanism of Cdz delivery and toxicity, we screened for mutations that render a target strain resistant to Cdz-mediated killing. These mutations mapped to four loci, including a TonB-dependent receptor, a three-gene operon (named zerRAB for zipper envelope resistance), and perA (for pentapeptide envelope resistance). Mutations in the zerRAB locus led to its overproduction and to potential changes in cell envelope composition, which may diminish the susceptibility of cells to Cdz toxins. The perA gene is also required to maintain a normal cell envelope, but our screen identified mutations that confer resistance to Cdz toxins without substantially affecting the cell envelope functions of PerA. We demonstrate that PerA, which encodes a pentapeptide repeat protein predicted to form a quadrilateral ß-helix, localizes primarily to the outer membrane of cells, where it may serve as a receptor for the Cdz toxins. Collectively, these results provide new insights into the function and mechanisms of an atypical, contact-dependent bacteriocin system.IMPORTANCE Bacteriocins are commonly used by bacteria to kill neighboring cells that compete for resources. Although most bacteriocins are secreted, the aquatic, oligotrophic bacterium Caulobacter crescentus produces a two-peptide bacteriocin, CdzC/D, that remains attached to the outer membranes of cells, enabling contact-dependent killing of cells lacking the immunity protein CdzI. The receptor for CdzC/D has not previously been reported. Here, we describe a genetic screen for mutations that confer resistance to CdzC/D. One locus identified, perA, encodes a pentapeptide repeat protein that resides in the outer membrane of target cells, where it may act as the direct receptor for CdzC/D. Collectively, our results provide new insight into bacteriocin function and diversity.

Protein lysine acetylation plays a regulatory role in Bacillus subtilis multicellularity.

Protein lysine acetylation is a post-translational modification that alters the charge, conformation, and stability of proteins. A number of genome-wide characterizations of lysine-acetylated proteins, or acetylomes, in bacteria have demonstrated that lysine acetylation occurs on proteins with a wide diversity of functions, including central metabolism, transcription, chemotaxis, and cell size regulation. Bacillus subtilis is a model organism for studies of sporulation, motility, cell signaling, and multicellular development (or biofilm formation). In this work, we investigated the role of global protein lysine acetylation in multicellular development in B. subtilis. We analyzed the B. subtilis acetylome under biofilm-inducing conditions and identified acetylated proteins involved in multicellularity, specifically, swarming and biofilm formation. We constructed various single and double mutants of genes known to encode enzymes involved in global protein lysine acetylation in B. subtilis. Some of those mutants showed a defect in swarming motility while others demonstrated altered biofilm phenotypes. Lastly, we picked two acetylated proteins known to be important for biofilm formation, YmcA (also known as RicA), a regulatory protein critical for biofilm induction, and GtaB, an UTP-glucose-1-phosphate uridylyltransferase that synthesizes a nucleotide sugar precursor for biosynthesis of exopolysaccharide, a key biofilm matrix component. We performed site-directed mutagenesis on the acetylated lysine codons in ymcA and gtaB, respectively, and assayed cells bearing those point mutants for biofilm formation. The mutant alleles of ymcA(K64R), gtaB(K89R), and gtaB(K191R) all demonstrated a severe biofilm defect. These results indicate the importance of acetylated lysine residues in both YmcA and GtaB. In summary, we propose that protein lysine acetylation plays a global regulatory role in B. subtilis multicellularity.

Bacillus subtilis utilizes the DNA damage response to manage multicellular development.

Bacteria switch between free-living and a multicellular state, known as biofilms, in response to cellular and environmental cues. It is important to understand how these cues influence biofilm development as biofilms are not only ubiquitous in nature but are also causative agents of infectious diseases. It is often believed that any stress triggers biofilm formation as a means of bacterial protection. In this study, we propose a new mechanism for how cellular and environmental DNA damage may influence biofilm formation. We demonstrate that Bacillus subtilis prevents biofilm formation and cell differentiation when stressed by oxidative DNA damage. We show that during B. subtilis biofilm development, a subpopulation of cells accumulates reactive oxygen species, which triggers the DNA damage response. Surprisingly, DNA damage response induction shuts off matrix genes whose products permit individual cells to stick together within a biofilm. We further revealed that DDRON cells and matrix producers are mutually exclusive and spatially separated within the biofilm, and that a developmental checkpoint protein, Sda, mediates the exclusiveness. We believe this represents an alternative survival strategy, ultimately allowing cells to escape the multicellular community when in danger.

Characterization of the regulation of a plant polysaccharide utilization operon and its role in biofilm formation in Bacillus subtilis.

The soil bacterium Bacillus subtilis is often found in association with plants in the rhizosphere. Previously, plant polysaccharides have been shown to stimulate formation of root-associated multicellular communities, or biofilms, in this bacterium, yet the underlying mechanism is not fully understood. A five-gene gan operon (ganSPQAB) in B. subtilis has recently been shown to be involved in utilization of the plant-derived polysaccharide galactan. Despite these findings, molecular details about the regulation of the operon and the role of the operon in biofilm formation remain elusive. In this study, we performed comprehensive genetic analyses on the regulation of the gan operon. We show that this operon is regulated both by a LacI-like transcription repressor (GanR), which directly binds to pairs of inverted DNA repeats in the promoter region of the operon, and by the catabolite control protein A (CcpA). Derepression can be triggered by the presence of the inducer ß-1,4-galactobiose, a hydrolysis product of galactan, or in situ when B. subtilis cells are associated with plant roots. In addition to the transcriptional regulation, the encoded ß-galactosidase GanA (by ganA), which hydrolyzes ß-1,4-galactobiose into galactose, is inhibited at the enzymatic level by the catalytic product galactose. Thus, the galactan utilization pathway is under complex regulation involving both positive and negative feedback mechanisms in B. subtilis. We discuss about the biological significance of such complex regulation as well as a hypothesis of biofilm induction by galactan via multiple mechanisms.

Genome-Wide Investigation of Biofilm Formation in Bacillus cereus.

Bacillus cereus is a soil-dwelling Gram-positive bacterium capable of forming structured multicellular communities, or biofilms. However, the regulatory pathways controlling biofilm formation are less well understood in B. cereus In this work, we developed a method to study B. cereus biofilms formed at the air-liquid interface. We applied two genome-wide approaches, random transposon insertion mutagenesis to identify genes that are potentially important for biofilm formation, and transcriptome analyses by RNA sequencing (RNA-seq) to characterize genes that are differentially expressed in B. cereus when cells were grown in a biofilm-inducing medium. For the first approach, we identified 23 genes whose disruption by transposon insertion led to altered biofilm phenotypes. Based on the predicted function, they included genes involved in processes such as nucleotide biosynthesis, iron salvage, and antibiotic production, as well as genes encoding an ATP-dependent protease and transcription regulators. Transcriptome analyses identified about 500 genes that were differentially expressed in cells grown under biofilm-inducing conditions. One particular set of those genes may contribute to major metabolic shifts, leading to elevated production of small volatile molecules. Selected volatile molecules were shown to stimulate robust biofilm formation in B. cereus Our studies represent a genome-wide investigation of B. cereus biofilm formation.IMPORTANCE In this work, we established a robust method for B. cereus biofilm studies and applied two genome-wide approaches, transposon insertion mutagenesis and transcriptome analyses by RNA-seq, to identify genes and pathways that are potentially important for biofilm formation in B. cereus We discovered dozens of genes and two major metabolic shifts that seem to be important for biofilm formation in B. cereus Our study represents a genome-wide investigation on B. cereus biofilm formation.

RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation.

In the nosocomial opportunistic pathogen Acinetobacter baumannii, RecA-dependent mutagenesis, which causes antibiotic resistance acquisition, is linked to the DNA damage response (DDR). Notably, unlike the Escherichia coli paradigm, recA and DDR gene expression in A. baumannii is bimodal. Namely, there is phenotypic variation upon DNA damage, which may provide a bet-hedging strategy for survival. Thus, understanding recA gene regulation is key to elucidate the yet unknown DDR regulation in A. baumannii Here, we identify a structured 5' untranslated region (UTR) in the recA transcript which serves as a cis-regulatory element. We show that a predicted stem-loop structure in this 5' UTR affects mRNA half-life and underlies bimodal gene expression and thus phenotypic variation in response to ciprofloxacin treatment. We furthermore show that the stem-loop structure of the recA 5' UTR influences intracellular RecA protein levels and, in vivo, impairing the formation of the stem-loop structure of the recA 5' UTR lowers cell survival of UV treatment and decreases rifampin resistance acquisition from DNA damage-induced mutagenesis. We hypothesize that the 5' UTR allows for stable recA transcripts during stress, including antibiotic treatment, enabling cells to maintain suitable RecA levels for survival. This innovative strategy to regulate the DDR in A. baumannii may contribute to its success as a pathogen.IMPORTANCEAcinetobacter baumannii is an opportunistic pathogen quickly gaining antibiotic resistances. Mutagenesis and antibiotic resistance acquisition are linked to the DNA damage response (DDR). However, how the DDR is regulated in A. baumannii remains unknown, since unlike most bacteria, A. baumannii does not follow the regulation of the Escherichia coli paradigm. In this study, we have started to uncover the mechanisms regulating the novel A. baumannii DDR. We have found that a cis-acting 5' UTR regulates recA transcript stability, RecA protein levels, and DNA damage-induced phenotypic variation. Though 5' UTRs are known to provide stability to transcripts in bacteria, this is the first example in which it regulates a bimodal DDR response through recA transcript stabilization, potentially enabling cells to have enough RecA for survival and genetic variability.

High throughput microencapsulation of Bacillus subtilis in semi-permeable biodegradable polymersomes for selenium remediation.

Encapsulating bacteria within constrained microenvironments can promote the manifestation of specialized behaviors. Using double-emulsion droplet-generating microfluidic synthesis, live Bacillus subtilis bacteria were encapsulated in a semi-permeable membrane composed of poly(ethylene glycol)-b-poly(D,L-lactic acid) (mPEG-PDLLA). This polymer membrane was sufficiently permeable to permit exponential bacterial growth, metabolite-induced gene expression, and rapid biofilm growth. The biodegradable microparticles retained structural integrity for several days and could be successfully degraded with time or sustained bacterial activity. Microencapsulated B. subtilis successfully captured and contained sodium selenite added outside the polymersomes, converting the selenite into elemental selenium nanoparticles that were selectively retained inside the polymer membrane. This remediation of selenium using polymersomes has high potential for reducing the toxicity of environmental selenium contamination, as well as allowing selenium to be harvested from areas not amenable to conventional waste or water treatment.

Acetic Acid Acts as a Volatile Signal To Stimulate Bacterial Biofilm Formation.

UNLABELLED: Volatiles are small air-transmittable chemicals with diverse biological activities. In this study, we showed that volatiles produced by the bacterium Bacillus subtilis had a profound effect on biofilm formation of neighboring B. subtilis cells that grew in proximity but were physically separated. We further demonstrated that one such volatile, acetic acid, is particularly potent in stimulating biofilm formation. Multiple lines of genetic evidence based on B. subtilis mutants that are defective in either acetic acid production or transportation suggest that B. subtilis uses acetic acid as a metabolic signal to coordinate the timing of biofilm formation. Lastly, we investigated how B. subtilis cells sense and respond to acetic acid in regulating biofilm formation. We showed the possible involvement of three sets of genes (ywbHG, ysbAB, and yxaKC), all encoding putative holin-antiholin-like proteins, in cells responding to acetic acid and stimulating biofilm formation. All three sets of genes were induced by acetate. A mutant with a triple mutation of those genes showed a severe delay in biofilm formation, whereas a strain overexpressing ywbHG showed early and robust biofilm formation. Results of our studies suggest that B. subtilis and possibly other bacteria use acetic acid as a metabolic signal to regulate biofilm formation as well as a quorum-sensing-like airborne signal to coordinate the timing of biofilm formation by physically separated cells in the community. IMPORTANCE: Volatiles are small, air-transmittable molecules produced by all kingdoms of organisms including bacteria. Volatiles possess diverse biological activities and play important roles in bacteria-bacteria and bacteria-host interactions. Although volatiles can be used as a novel and important way of cell-cell communication due to their air-transmittable nature, little is known about how the volatile-mediated signaling mechanism works. In this study, we demonstrate that the bacterium Bacillus subtilis uses one such volatile, acetic acid, as a quorum-sensing-like signal to coordinate the timing of the formation of structurally complex cell communities, also known as biofilms. We further characterized the molecular mechanisms of how B. subtilis responds to acetic acid in stimulating biofilm formation. Our study also suggests that acetic acid may be used as a volatile signal for cross-species communication.

A bacterial volatile signal for biofilm formation.

Bacteria constantly monitor the environment they reside in and respond to potential changes in the environment through a variety of signal sensing and transduction mechanisms in a timely fashion. Those signaling mechanisms often involve application of small, diffusible chemical molecules. Volatiles are a group of small air-transmittable chemicals that are produced universally by all kingdoms of organisms. Past studies have shown that volatiles can function as cell-cell communication signals not only within species, but also cross-species. However, little is known about how the volatile-mediated signaling mechanism works. In our recent study (Chen, et al. mBio (2015), 6: e00392-15), we demonstrated that the soil bacterium Bacillus subtilis uses acetic acid as a volatile signal to coordinate the timing of biofilm formation within physically separated cells in the community. We also showed that the bacterium possesses an intertwined gene network to produce, secrete, sense, and respond to acetic acid, in stimulating biofilm formation. Interestingly, many of those genes are highly conserved in other bacterial species, raising the possibility that acetic acid may act as a volatile signal for cross-species communication.