Sporulation-specific Transcription Factor Research Articles

Human-Gut Phages Harbor Sporulation Genes.

Spore-forming bacteria are prevalent in mammalian guts and have implications for host health and nutrition. The production of dormant spores is thought to play an important role in the colonization, persistence, and transmission of these bacteria. Spore formation also modifies interactions among microorganisms such as infection by phages. Recent studies suggest that phages may counter dormancy-mediated defense through the expression of phage-carried sporulation genes during infection, which can alter the transitions between active and inactive states. By mining genomes and gut-derived metagenomes, we identified sporulation genes that are preferentially carried by phages that infect spore-forming bacteria. These included genes involved in chromosome partitioning, DNA damage repair, and cell wall-associated functions. In addition, phages contained homologs of sporulation-specific transcription factors, notably spo0A, the master regulator of sporulation, which could allow phages to control the complex genetic network responsible for spore development. Our findings suggest that phages could influence the formation of bacterial spores with implications for the health of the human gut microbiome, as well as bacterial communities in other environments. IMPORTANCE Phages acquire bacterial genes and use them to alter host metabolism in ways that enhance phage fitness. To date, most auxiliary genes replace or modulate enzymes that are used by the host for nutrition or energy production. However, phage fitness is affected by all aspects of host physiology, including decisions that reduce the metabolic activity of the cell. Here, we focus on endosporulation, a complex and ancient form of dormancy found among the Bacillota that involves hundreds of genes. By coupling homology searches with host classification, we identified 31 phage-carried homologs of sporulation genes that are mostly limited to phages infecting spore-forming bacteria. Nearly one-third of the homologs recovered were regulatory genes, suggesting that phages may manipulate host genetic networks by tapping into their control elements. Our findings also suggest a mechanism by which phages can overcome the defensive strategy of dormancy, which may be involved in coevolutionary dynamics of spore-forming bacteria.

Developmentally regulated volatiles geosmin and 2-methylisoborneol attract a soil arthropod to Streptomyces bacteria promoting spore dispersal

Volatile compounds emitted by bacteria are often sensed by other organisms as odours, but their ecological roles are poorly understood1,2. Well-known examples are the soil-smelling terpenoids geosmin and 2-methylisoborneol (2-MIB)3,4, which humans and various animals sense at extremely low concentrations5,6. The conservation of geosmin biosynthesis genes among virtually all species of Streptomyces bacteria (and genes for the biosynthesis of 2-MIB in about 50%)7,8, suggests that the volatiles provide a selective advantage for these soil microbes. We show, in the present study, that these volatiles mediate interactions of apparent mutual benefit between streptomycetes and springtails (Collembola). In field experiments, springtails were attracted to odours emitted by Streptomyces colonies. Geosmin and 2-MIB in these odours induce electrophysiological responses in the antennae of the model springtail Folsomia candida, which is also attracted to both compounds. Moreover, the genes for geosmin and 2-MIB synthases are under the direct control of sporulation-specific transcription factors, constraining emission of the odorants to sporulating colonies. F. candida feeds on the Streptomyces colonies and disseminates spores both via faecal pellets and through adherence to its hydrophobic cuticle. The results indicate that geosmin and 2-MIB production is an integral part of the sporulation process, completing the Streptomyces life cycle by facilitating dispersal of spores by soil arthropods.

Metabolic engineering of Bacillus subtilis for l-valine overproduction.

Bacillus subtilis has been commonly applied to industrial enzyme production due to its genetic tractability, "generally recognized as safe (GRAS)" status, and robust growth characteristics. In spite of its ideal attributes as a biomanufacturing platform, B. subtilis has seen limited use in the production of other value-added biochemicals. Here, we report the derivation of engineered strains of B. subtilis for l-valine overproduction using our recently developed CRISPR (clustered regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated [protein] 9) toolkit. We first manipulate the native l-valine biosynthetic pathway by relieving transcriptional and allosteric regulation, resulting in a >14-fold increase in the l-valine titer, compared to the wild-type strain. We subsequently identify and eliminate factors limiting l-valine overproduction, specifically increasing pyruvate availability and blocking the competing l-leucine and l-isoleucine biosynthetic pathways. By inactivating (a) pdhA, encoding the E1α subunit of the pyruvate dehydrogenase complex, to increase the intracellular pyruvate pool, and (b) leuA and ilvA, respectively encoding 2-isopropylmalate synthase and l-threonine dehydratase, to abolish the competing pathways, the l-valine titer reached 4.61 g/L in shake flask cultures. Our engineered l-valine-overproducing strains of B. subtilis are devoid of plasmids and do not sporulate due to the inactivation of sigF, encoding the sporulation-specific transcription factor σ F , making them attractive for large-scale l-valine production. However, acetate dissimilation was identified as limiting l-valine overproduction in ΔpdhA B. subtilis strains, and improving acetate dissimilation or identifying alternate modes of increasing pyruvate pools to enhance l-valine-overproduction should be explored.

The Clostridium sporulation programs: diversity and preservation of endospore differentiation.

Bacillus and Clostridium organisms initiate the sporulation process when unfavorable conditions are detected. The sporulation process is a carefully orchestrated cascade of events at both the transcriptional and posttranslational levels involving a multitude of sigma factors, transcription factors, proteases, and phosphatases. Like Bacillus genomes, sequenced Clostridium genomes contain genes for all major sporulation-specific transcription and sigma factors (spo0A, sigH, sigF, sigE, sigG, and sigK) that orchestrate the sporulation program. However, recent studies have shown that there are substantial differences in the sporulation programs between the two genera as well as among different Clostridium species. First, in the absence of a Bacillus-like phosphorelay system, activation of Spo0A in Clostridium organisms is carried out by a number of orphan histidine kinases. Second, downstream of Spo0A, the transcriptional and posttranslational regulation of the canonical set of four sporulation-specific sigma factors (σ(F), σ(E), σ(G), and σ(K)) display different patterns, not only compared to Bacillus but also among Clostridium organisms. Finally, recent studies demonstrated that σ(K), the last sigma factor to be activated according to the Bacillus subtilis model, is involved in the very early stages of sporulation in Clostridium acetobutylicum, C. perfringens, and C. botulinum as well as in the very late stages of spore maturation in C. acetobutylicum. Despite profound differences in initiation, propagation, and orchestration of expression of spore morphogenetic components, these findings demonstrate not only the robustness of the endospore sporulation program but also the plasticity of the program to generate different complex phenotypes, some apparently regulated at the epigenetic level.

Co-ordination between BrlA regulation and secretion of the oxidoreductase FmqD directs selective accumulation of fumiquinazoline C to conidial tissues inAspergillus fumigatus

Aerial spores, crucial for propagation and dispersal of the Kingdom Fungi, are commonly the initial inoculum of pathogenic fungi. Natural products (secondary metabolites) have been correlated with fungal spore development and enhanced virulence in the human pathogen Aspergillus fumigatus but mechanisms for metabolite deposition in the spore are unknown. Metabolomic profiling of A. fumigatus deletion mutants of fumiquinazoline (Fq) cluster genes reveal that the first two products of the Fq cluster, FqF and FqA, are produced to comparable levels in all fungal tissues but the final enzymatically derived product, FqC, predominantly accumulates in the fungal spore. Loss of the sporulation-specific transcription factor, BrlA, yields a strain unable to produce FqA or FqC. Fluorescence microscopy showed FmqD, the oxidoreductase required to generate FqC, was secreted via the Golgi apparatus to the cell wall in an actin-dependent manner. In contrast, all other members of the Fq pathway including the putative transporter, FmqE - which had no effect on Fq biosynthesis - were internal to the hyphae. The co-ordination of BrlA-mediated tissue specificity with FmqD secretion to the cell wall presents a previously undescribed mechanism to direct localization of specific secondary metabolites to spores of the differentiating fungus.

Gene Conservation among Endospore-Forming Bacteria Reveals Additional Sporulation Genes in Bacillus subtilis

The capacity to form endospores is unique to certain members of the low-G+C group of Gram-positive bacteria (Firmicutes) and requires signature sporulation genes that are highly conserved across members of distantly related genera, such as Clostridium and Bacillus. Using gene conservation among endospore-forming bacteria, we identified eight previously uncharacterized genes that are enriched among endospore-forming species. The expression of five of these genes was dependent on sporulation-specific transcription factors. Mutants of none of the genes exhibited a conspicuous defect in sporulation, but mutants of two, ylxY and ylyA, were outcompeted by a wild-type strain under sporulation-inducing conditions, but not during growth. In contrast, a ylmC mutant displayed a slight competitive advantage over the wild type specific to sporulation-inducing conditions. The phenotype of a ylyA mutant was ascribed to a defect in spore germination efficiency. This work demonstrates the power of combining phylogenetic profiling with reverse genetics and gene-regulatory studies to identify unrecognized genes that contribute to a conserved developmental process.

Small proteins link coat and cortex assembly during sporulation in Bacillus subtilis

Mature spores of the bacterium Bacillus subtilis are encased by two concentric shells: an inner shell (the 'cortex'), made of peptidoglycan; and an outer proteinaceous shell (the 'coat'), whose basement layer is anchored to the surface of the developing spore via a 26-amino-acid-long protein called SpoVM. During sporulation, initiation of cortex assembly depends on the successful initiation of coat assembly, but the mechanisms that co-ordinate the morphogenesis of both structures are largely unknown. Here, we describe a sporulation pathway involving SpoVM and a 37-amino-acid-long protein named 'CmpA' that is encoded by a previously un-annotated gene and is expressed under control of two sporulation-specific transcription factors (σ(E) and SpoIIID). CmpA localized to the surface of the developing spore and deletion of cmpA resulted in cells progressing through the sporulation programme more quickly. Overproduction of CmpA did not affect normal growth or cell division, but delayed entry into sporulation and abrogated cortex assembly. In those cells that had successfully initiated coat assembly, CmpA was removed by a post-translational mechanism, presumably in order to overcome the sporulation inhibition it imposed. We propose a model in which CmpA participates in a developmental checkpoint that ensures the proper orchestration of coat and cortex morphogenesis by repressing cortex assembly until coat assembly successfully initiates.

An orthologue of the Myelin-gene Regulatory Transcription Factor regulates Dictyostelium prestalk differentiation

The prestalk region of the Dictyostelium slug is comprised of an anterior population of pstA cells and a posterior population of pstO cells. They are distinguished by their ability to utilize different parts of the promoter of the ecmA gene. We identify, by mutational analysis and DNA transformation, CA-rich sequence elements within the ecmA promoter that are essential for pstA-specific expression and sufficient to direct pstA-specific expression when multimerised. The CA-rich region was used in affinity chromatography with nuclear extracts and bound proteins were identified by mass spectrometry. The CA-rich elements purify MrfA, a protein with extensive sequence similarity to animal Myelin-gene Regulatory Factor (MRF)-like proteins. The MRF-like proteins and MrfA also display more spatially limited but significant sequence similarity with the DNA binding domain of the yeast Ndt80 sporulation-specific transcription factor. Furthermore, the ecmA CA-rich elements show sequence similarity to the core consensus Ndt80 binding site (the MSE) and point mutation of highly conserved arginine residues in MrfA, that in Ndt80 make critical contacts with the MSE, ablate binding of MrfA to its sites within the ecmA promoter. MrfA null strains are delayed in multicellular development and highly defective in pstA-specific gene expression. These results provide a first insight into the intracellular signaling pathway that directs pstA differentiation and identify a non-metazoan orthologue of a family of molecularly uncharacterised transcription factors.

Expression and localization of SpoIISA toxin during the life cycle of Bacillus subtilis

The previously identified spoIIS locus encodes a toxin–antitoxin system in Bacillus subtilis. It comprises two genes, spoIISA encoding a toxin and spoIISB encoding an antitoxin, which lies adjacent to each other on the chromosome. Each of the spoIIS coding sequences is preceded by a promoter region and the two genes together constitute an operon. The function of SpoIISA is unknown, although it has been shown that the absence of SpoIISB or loss of its function leads to a block in sporulation at stage II. The cytoplasmic membrane has been proposed as the target of the SpoIISA toxin. Heterologously expressed SpoIISA–SpoIISB was shown to be functional in Escherichia coli, where again the cytoplasmic membrane was the most probable target for SpoIISA toxicity. Here we analyzed the effects of SpoIISA production during vegetative growth of B. subtilis and during sporulation by following the levels of SpoIISA. SpoIISA levels increase at the point of entry into stationary phase of cell cultures grown in sporulation-inducing medium. However, SpoIISA expression appears to be unrelated to the sporulation process, since it is independent of the major early sporulation-specific transcription factor, Spo0A.We also investigated SpoIISA localization within the cell. We confirmed the predicted localization of SpoIISA at the B. subtilis cytoplasmic membrane. In addition, we observed localization of SpoIISA in higher level structures in a cell-wall-dependent manner.

Ectopic Overproduction of a Sporulation-Specific Transcription Factor Induces Assembly of Prespore-Like Membranous Compartments in Vegetative Cells of Fission Yeast

Mei4 is a key sporulation-specific transcription factor in fission yeast. Ectopic expression of Mei4 in vegetative cells caused formation of nucleated membranous compartments, which shared common features with normal forespore membranes, thereby perturbing nuclear division. These results suggest why expression of development-specific transcription factors must be strictly controlled.

Structural and Genetic Analysis of X-Ray Scattering by Spores of Bacillus subtilis

Dormant spores of Bacillus subtilis exhibit two prominent X-ray scattering peaks. These peaks persisted in spores lacking most alpha/beta-type small, acid-soluble protein or the CotE protein responsible for assembly of much spore coat protein, but they were absent from spores of strains lacking the late sporulation-specific transcription factor GerE.

SKPDT is a signaling peptide that stimulates sporulation and cry1Aa expression in Bacillus thuringiensis but not in Bacillus subtilis

We have identified and characterized in the supernatant of the transition phase of Bacillus thuringiensis var. kurstaki the peptide SKPDT. This peptide was previously identified by in silico analysis by Pottathil and Lazazzera (Front Biosci 8:32-45 2003) as a putative signaling peptide (NprRB) of the Phr family in B. thuringiensis. The chemically synthesized NprRB did not affect the growth kinetics of B. thuringiensis var. kurstaki but stimulated the sporulation, spore release, and transcription of cry1Aa when added to cultures during the transition phase. In fact, when the peptide (100 nM) was added to a culture in transition phase, the transcription of cry1Aa was stimulated almost threefold, mainly from the late promoter BtII, which requires the late-stage sporulation-specific transcription factor sigma (K). On the other hand, NprRB did not have any effect on B. subtilis. Thus, SKPDT seems to be a signaling peptide specific for B. thuringiensis.

Genetic analysis of the Bacillus subtilis sigG promoter, which controls the sporulation-specific transcription factor sigma G.

At the onset of sporulation in Bacillus subtilis, an asymmetric cell division gives rise to two unequal-sized compartments with distinct developmental fates. The smaller compartment, or prespore, becomes the spore, whilst the larger compartment, or mother cell, eventually lyses after contributing to spore maturation. The fate of each compartment is determined by differential gene expression, controlled by the activation of four compartment-specific sigma-factors. The expression and activity of all four sigma-factors are tightly regulated to ensure the correct sequence of morphological events. Prespore-specific genes are transcribed by two sigma-factors, sigma(F) followed by sigma(G). The gene encoding sigma(G) (sigG) is transcribed by sigma(F), but also requires the activity of one of the mother-cell-specific sigma-factors, sigma(E), for its expression. The minimal promoter required for dependence on sigma(E) was found to stretch to just upstream of the -35 site. Analysis of mutant sigG promoters generated by site-directed mutagenesis and sigG promoters from other species suggests the presence of a binding site for a transcriptional repressor within the sigG promoter region. Replacement of the wild-type promoter with sigma(E)-independent promoters resulted in impairment of sporulation. These data support the idea that sigma(E) activity is required for the transcription of sigG.

Activity of phosphoforms and truncated versions of Ndt80, a checkpoint-regulated sporulation-specific transcription factor of Saccharomyces cerevisiae.

Ndt80 contributes to the highly regulated cascade of sequential gene expression that directs spore formation in Saccharomyces cerevisiae. This DNA-binding transcriptional activator, which is responsible for the expression of a set of middle sporulation-specific genes, is a target of the meiotic recombination checkpoint. Triggering of this checkpoint prevents phosphorylation and accumulation of active Ndt80. In this study we have investigated the requirements for the activation function of Ndt80 by exploring the role of phosphorylation in the regulation of its activity and by examining the effect of C-terminal truncations. Of three phosphoforms of Ndt80 that we resolved, which we refer to as P approximately Ndt80", P approximately Ndt80', and P approximately Ndt80 in order of increasing electrophoretic mobility, the P approximately Ndt80" and P approximately Ndt80' isoforms correlated with active Ndt80. In particular, P approximately Ndt80" was present in lysates from wild-type sporulating cells and in cells that bypassed checkpoint-mediated arrest as a result of mutations in RAD17, SUM1, or SWE1, or overexpression of NDT80. P approximately Ndt80' was the slowest-migrating isoform that accumulated in Delta ime2/Delta ime2 Delta sum1/Delta sum1 cells in sporulation medium and in mitotic cells that ectopically expressed NDT80. Nonphosphorylated Ndt80 and P approximately Ndt80, which had a slightly lower mobility than nonphosphorylated Ndt80 and was the predominant phosphoform present in checkpoint-arrested cells, correlated with inactive Ndt80. These data are consistent with the notion that extensive phosphorylation, but not Ime2-dependent phosphorylation, of Ndt80 is required for its activity. Examination of the effect of increasingly extensive truncation of the C terminal region of Ndt80 revealed that some functions of Ndt80 were more sensitive to a reduction in its activity than others. In particular, we found that a truncated version of Ndt80 that lacked the last 110 residues was able to promote expression of some middle sporulation-specific genes, but could not direct spore formation. Full activity, however, could be restored to this version of Ndt80 by increasing its level of expression.

Identification of sporulation genes by genome-wide analysis of the sigmaE regulon of Bacillus subtilis.

Differentiation in the spore-forming bacterium Bacillus subtilis is governed by the sequential activation of five sporulation-specific transcription factors. The early mother-cell-specific transcription factor, sigma(E), directs the transcription of many genes that contribute to the formation of mature, dormant spores. In this study, DNA microarrays were used to identify genes belonging to the sigma(E) regulon. In total, 171 genes were found to be under the control of sigma(E). Of these, 101 genes had not previously been described as being sigma(E) dependent. Disruption of some of the previously unknown genes (ydcC, yhaL, yhbH, yjaV and yqfD) resulted in a defect in sporulation.

Tethering of the Bacillus subtilis sigma E proprotein to the cell membrane is necessary for its processing but insufficient for its stabilization.

sigma(E), a sporulation-specific transcription factor of Bacillus subtilis, is synthesized as an inactive proprotein with a 27-amino acid extension at its amino terminus. This "pro" sequence is removed by a developmentally regulated protease, but when present, it blocks sigma(E) activity, tethers sigma(E) to the bacterium's cytoplasmic membrane, and promotes sigma(E) stability. To investigate whether pro-sigma(E) processing and/or stabilization are tied to membrane sequestration, we used fluorescent protein fusions to examine the membrane binding of SigE variants. The results are consistent with membrane association as a prerequisite for pro-sigma(E) processing but not as a sufficient cause for the proprotein's stability.

Structure of the sporulation-specific transcription factor Ndt80 bound to DNA.

Progression through the middle phase of sporulation in Saccharomyces cerevisiae is promoted by the successful completion of recombination at the end of prophase I. Completion of meiotic recombination allows the activation of the sporulation-specific transcription factor Ndt80, which binds to a specific DNA sequence, the middle sporulation element (MSE), and activates approximately 150 genes to enable progression through meiosis. Here, we isolate the DNA-binding domain of Ndt80 and determine its crystal structure both free and in complex with an MSE-containing DNA. The structure reveals that Ndt80 is a member of the Ig-fold family of transcription factors. The structure of the DNA-bound form, refined at 1.4 A, reveals an unexpected mode of recognition of 5'-pyrimidine- guanine-3' dinucleotide steps by arginine residues that simultaneously recognize the 3'-guanine base through hydrogen bond interactions and the 5'-pyrimidine through stacking/van der Waals interactions. Analysis of the DNA-binding affinities of MSE mutants demonstrates the central importance of these interactions, and of the AT-rich portion of the MSE. Functional similarities between Ndt80 and the Caenorhabditis elegans p53 homolog suggest an evolutionary link between Ndt80 and the p53 family.

NMR studies of the interactions of SpoIIAA with its partner proteins that regulate sporulation in Bacillus subtilis

SpoIIAA is a key component in the network of interactions that regulate the first sporulation-specific transcription factor, σF, in Bacillus subtilis. In its unphosphorylated form SpoIIAA is either phosphorylated by or forms a non-covalent complex with SpoIIAB, whereas in its phosphorylated form it is dephosphorylated by SpoIIE. In this work we present NMR studies of the SpoIIAA2.SpoIIAB complex and of mutant proteins that are deficient in their ability to interact with SpoIIAB or SpoIIE. The NMR studies of the SpoIIAA2.SpoIIAB complex allowed us to define a contiguous patch that is perturbed upon complex formation. By examining the chemical shift perturbations in the mutant proteins we have identified more specific areas that contain residues critical for the SpoIIAB and SpoIIE interactions.

Bacillus subtilis mutations that alter the pathway of phosphorylation of the anti-anti-sigmaF factor SpoIIAA lead to a Spo- phenotype.

Sigma-F, the first sporulation-specific transcription factor of Bacillus subtilis, is regulated by an anti-sigma factor SpoIIAB, which can also act as a protein kinase that phosphorylates the anti-anti-sigma factor SpoIIAA. The time course of phosphorylation reaction is biphasic, a fact that has been interpreted in terms of a mechanism for sequestering SpoIIAB away from sigmaF and thus allowing activation of sigmaF when needed. Site-directed mutagenesis of SpoIIAA has allowed us to isolate two mutants that cannot activate sigmaF and which are therefore Spo-. The two mutant SpoIIAA proteins, SpoIIAAL61A and SpoIIAAL90A, are phosphorylated with linear kinetics; in addition they are less able to form the stable non-covalent complex that wild-type SpoIIAA makes with SpoIIAB in the presence of ADP. The phosphorylated form of SpoIIAAL90A was hydrolysed by the specific phosphatase SpoIIE at the same rate as wild-type SpoIIAA-P, but the rate of hydrolysis of SpoIIAAL61A-P was much slower. The secondary structure and the global fold of the mutant proteins were unchanged from the wild type. The results are interpreted in terms of a model for the wild type in which SpoIIAB, after phosphorylating SpoIIAA, is released in a form that is tightly bound to ADP and which then makes a ternary complex with an unreacted SpoIIAA. We propose that it is the inability to make this ternary complex that deprives the mutant cells of a means of keeping SpoIIAB from inhibiting sigmaF.

Membrane topology of the Bacillus subtilis pro-sigma(K) processing complex.

Activation of the final sporulation-specific transcription factor, sigma(K), is regulated by a signal emanating from the forespore which interacts with the pro-sigma(K) processing complex, comprising SpoIVFA, BofA, and the pro-sigma(K) processing protease, SpoIVFB. Mature sigma(K) then directs late gene expression in the parental compartment of the developing sporangial cell. The nature of this complex and how it is activated to process pro-sigma(K) are not understood. All three proteins are predicted to be integral membrane proteins. Here, we have analyzed the membrane topology of SpoIVFA and SpoIVFB by constructing chimeric forms of spoIVFA and spoIVFB with the complementary reporters phoA and lacZ and analyzing activity in Escherichia coli. SpoIVFA was found to have a single transmembrane-spanning domain, while SpoIVFB was shown to have six transmembrane-spanning domains (6-transmembrane configuration). Further, SpoIVFA is required to stabilize SpoIVFB in the membrane. SpoIVFB was shown to have a 4-transmembrane configuration when expressed on its own but was found to have a 6-transmembrane configuration when coexpressed with SpoIVFA, while BofA had a positive effect on the assembly of both SpoIVFA and SpoIVFB. The single transmembrane domain of SpoIVFA (approximately residues 73 to 90) was shown to be the principle determinant in stabilizing the 6-transmembrane configuration of SpoIVFB. Although the bofB8 allele, which uncouples the sigma(K) checkpoint, did not appear to promote a conformational change from a 6- to 4-transmembrane configuration of SpoIVFB (apparently ruling out a profound conformational change as the mechanism of activating SpoIVFB proteolytic activity), instability of SpoIVFB may be an important factor in SpoIVFB-mediated processing of pro-sigma(K).