Biomedical subjects

Alan D Grossman

Publications and source records attributed to Alan D Grossman.

At least 19 recordsLinked to original sources

PubMed2025

Activation and modulation of the host response to DNA damage by an integrative and conjugative element.

Mobile genetic elements help drive horizontal gene transfer and bacterial evolution. Conjugative elements and temperate bacteriophages can be stably maintained in host cells. They can alter host physiology and regulatory responses and typically carry genes that are beneficial to their hosts. We found that ICEBs1, an integrative and conjugative element (ICE) of Bacillus subtilis, inhibits the host response to DNA damage (the SOS response). Activation of ICEBs1 before DNA damage reduced host cell lysis that was caused by SOS-mediated activation of two resident prophages. Further, activation of ICEBs1 itself activated the SOS response in a subpopulation of cells, and this activation was attenuated by the functions of the ICEBs1 genes ydcT and yddA (now ramT and ramA; ram for RecA modulator). Double-mutant analyses indicated that RamA functions to inhibit and RamT functions to both inhibit and activate the SOS response. Both RamT and RamA caused a reduction in RecA filaments, one of the early steps in activation of the SOS response. We suspect that there are several different mechanisms by which mobile genetic elements that generate single-stranded DNA (ssDNA) during their life cycle inhibit the host SOS response and RecA function, as RamT and RamA differ from the known SOS inhibitors encoded by conjugative elements.IMPORTANCEBacterial genomes typically contain mobile genetic elements, including bacteriophages (viruses) and integrative and conjugative elements, that affect host physiology. ICEs can excise from the chromosome and undergo rolling-circle replication, producing ssDNA, a signal that indicates DNA damage and activates the host SOS response. We found that following excision and replication, ICEBs1 of B. subtilis stimulates the host SOS response and that ICEBs1 encodes two proteins that limit the extent of this response. These proteins also reduce the amount of cell killing caused by resident prophages following their activation by DNA damage. These proteins are different from those previously characterized that inhibit the host SOS response and represent a new way in which ICEs can affect their host cells.

Bacillus subtilis↗

PubMed2024

It's complicated: relationships between integrative and conjugative elements and their bacterial hosts.

Integrative and conjugative elements (ICEs) are typically found integrated in a bacterial host chromosome. They can excise, replicate, and transfer from cell to cell. Many contain genes that confer phenotypes to host cells, including antibiotic resistances, specialized metabolisms, phage defense, and symbiosis or pathogenesis determinants. Recent studies revealed that at least three ICEs (ICEclc, Tn916, and TnSmu1) cause growth arrest or death of host cells upon element activation. This review highlights the complex interactions between ICEs and their hosts, including the recent examples of the significant costs to host cells. We contrast two examples of killing, ICEclc and Tn916, in which killing, respectively, benefits or impairs conjugation and emphasize the importance of understanding the impacts of ICE-host relationships on conjugation. ICEs are typically only active in a small fraction of cells in a population, and we discuss how phenotypes normally occurring in a small subset of host cells can be uncovered.

Conjugation, Genetic↗

PubMed2007

Replication is required for the RecA localization response to DNA damage in Bacillus subtilis.

In both prokaryotes and eukaryotes, proteins involved in DNA repair often organize into multicomponent complexes that can be visualized as foci in living cells. We used a RecA-GFP fusion to examine the subcellular cues that direct RecA-GFP to assemble as foci in response to DNA damage. We used two different methods to inhibit initiation of DNA replication and determined that DNA replication is required for the cell to establish RecA-GFP foci after exposure to DNA-damaging agents. Furthermore, use of endonuclease cleavage to generate a site-specific double-strand break demonstrated that the replication machinery (replisome) and DNA synthesis are required for assembly of RecA-GFP foci during repair of a double-strand break. We monitored the cellular levels of RecA and found that focus formation does not require further induction of protein levels, suggesting that foci result from a redistribution of existing protein to sites of damage encountered by the replisome. Taken together, our results support the model that existing RecA protein is recruited to ssDNA generated by the replisome at sites of DNA damage. These results provide insight into the mechanisms that the cell uses to recruit repair proteins to damaged DNA in living cells.

Bacillus subtilis↗

PubMed2006

Subcellular positioning of the origin region of the Bacillus subtilis chromosome is independent of sequences within oriC, the site of replication initiation, and the replication initiator DnaA.

Regions of bacterial chromosomes occupy characteristic locations within the cell. In Bacillus subtilis, the origin of replication, oriC, is located at 0 degrees /360 degrees on the circular chromosome. After duplication, sister 0 degrees regions rapidly move to and then reside near the cell quarters. It has been hypothesized that origin function or oriC sequences contribute to positioning and movement of the 0 degrees region. We found that the position of a given chromosomal region does not depend on initiation of replication from the 0 degrees region. In an oriC mutant strain that replicates from a heterologous origin (oriN) at 257 degrees , the position of both the 0 degrees and 257 degrees regions was similar to that in wild-type cells. Thus, positioning of chromosomal regions appears to be independent of which region is replicated first. Furthermore, we found that neither oriC sequences nor the replication initiator DnaA is required or sufficient for positioning a region near the cell quarters. A sequence within oriC previously proposed to play a critical role in chromosome positioning and partitioning was found to make little, if any, contribution. We propose that uncharacterized sites outside of oriC are involved in moving and/or maintaining the 0 degrees region near the cell quarters.

Bacillus subtilis↗

PubMed2006

Spatial and temporal organization of the Bacillus subtilis replication cycle.

DNA replication occurs at discrete sites in the cell. To gain insight into the spatial and temporal organization of the Bacillus subtilis replication cycle, we simultaneously visualized replication origins and the replication machinery (replisomes) inside live cells. We found that the origin of replication is positioned near midcell prior to replication. After initiation, the replisome colocalizes with the origin, confirming that replication initiates near midcell. The replisome remains near midcell after duplicated origins separate. Artificially mispositioning the origin region leads to mislocalization of the replisome indicating that the location of the origin at the time of initiation establishes the position of the replisome. Time-lapse microscopy revealed that a single replisome focus reversibly splits into two closely spaced foci every few seconds in many cells, including cells that recently initiated replication. Thus, sister replication forks are likely not intimately associated with each other throughout the replication cycle. Fork dynamics persisted when replication elongation was halted, and is thus independent of the relative movement of DNA through the replisome. Our results provide new insights into how the replisome is positioned in the cell and refine our current understanding of the spatial and temporal events of the B. subtilis replication cycle.

Bacillus subtilis↗

PubMed2006

The chromosome partitioning proteins Soj (ParA) and Spo0J (ParB) contribute to accurate chromosome partitioning, separation of replicated sister origins, and regulation of replication initiation in Bacillus subtilis.

Soj (ParA) and Spo0J (ParB) of Bacillus subtilis belong to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. Unlike most Par systems, for which intact copies of both parA and parB are required for the Par system to function, inactivating soj does not cause a detectable chromosome partitioning phenotype whereas inactivating spo0J leads to a 100-fold increase in the production of anucleate cells. This suggested either that Soj does not function like other ParA homologues, or that a cellular factor might compensate for the absence of soj. We found that inactivating smc, the gene encoding the structural maintenance of chromosomes (SMC) protein, unmasked a role for Soj in chromosome partitioning. A soj null mutation dramatically enhanced production of anucleate cells in an smc null mutant. To look for effects of a soj null on other phenotypes perturbed in a spo0J null mutant, we analysed replication initiation and origin positioning in (soj-spo0J)+, Deltasoj, Deltaspo0J and Delta(soj-spo0J) cells. All of the mutations caused increased initiation of replication and, to varying extents, affected origin positioning. Using a new assay to measure separation of the chromosomal origins, we found that inactivating soj, spo0J or both led to a significant defect in separating replicated sister origins, such that the origins remain too close to be spatially resolved. Separation of a region outside the origin was not affected. These results indicate that there are probably factors helping to pair sister origin regions for part of the replication cycle, and that Soj and Spo0J may antagonize this pairing to contribute to timely separation of replicated origins. The effects of Deltasoj, Deltaspo0J and Delta(soj-spo0J) mutations on origin positioning, chromosome partitioning and replication initiation may be a secondary consequence of a defect in separating replicated origins.

Bacillus subtilis↗

PubMed2006

Modulation of the ComA-dependent quorum response in Bacillus subtilis by multiple Rap proteins and Phr peptides.

In Bacillus subtilis, extracellular peptide signaling regulates several biological processes. Secreted Phr signaling peptides are imported into the cell and act intracellularly to antagonize the activity of regulators known as Rap proteins. B. subtilis encodes several Rap proteins and Phr peptides, and the processes regulated by many of these Rap proteins and Phr peptides are unknown. We used DNA microarrays to characterize the roles that several rap-phr signaling modules play in regulating gene expression. We found that rapK-phrK regulates the expression of a number of genes activated by the response regulator ComA. ComA activates expression of genes involved in competence development and the production of several secreted products. Two Phr peptides, PhrC and PhrF, were previously known to stimulate the activity of ComA. We assayed the roles that PhrC, PhrF, and PhrK play in regulating gene expression and found that these three peptides stimulate ComA-dependent gene expression to different levels and are all required for full expression of genes activated by ComA. The involvement of multiple Rap proteins and Phr peptides allows multiple physiological cues to be integrated into a regulatory network that modulates the timing and magnitude of the ComA response.

Bacillus subtilis↗

PubMed2006

Characterization of the global transcriptional responses to different types of DNA damage and disruption of replication in Bacillus subtilis.

DNA damage and perturbations in DNA replication can induce global transcriptional responses that can help organisms repair the damage and survive. RecA is known to mediate transcriptional responses to DNA damage in several bacterial species by inactivating the repressor LexA and phage repressors. To gain insight into how Bacillus subtilis responds to various types of DNA damage, we measured the effects of DNA damage and perturbations in replication on mRNA levels by using DNA microarrays. We perturbed replication either directly with p-hydroxyphenylazo-uracil (HPUra), an inhibitor of DNA polymerase, or indirectly with the DNA-damaging reagents mitomycin C (MMC) and UV irradiation. Our results indicate that the transcriptional responses to HPUra, MMC, and UV are only partially overlapping. recA is the major transcriptional regulator under all of the tested conditions, and LexA appears to directly repress the expression of 63 genes in 26 operons, including the 18 operons previously identified as LexA targets. MMC and HPUra treatments caused induction of an integrative and conjugative element (ICEBs1) and resident prophages (PBSX and SPbeta), which affected the expression of many host genes. Consistent with previous results, the induction of these mobile elements required recA. Induction of the phage appeared to require inactivation of LexA. Unrepaired UV damage and treatment with MMC also affected the expression of some of the genes that are controlled by DnaA. Furthermore, MMC treatment caused an increase in origin-proximal gene dosage. Our results indicate that different types of DNA damage have different effects on replication and on the global transcriptional profile.

Bacillus Phages↗

PubMed2005

A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA.

Organisms respond to perturbations in DNA replication. We characterized the global transcriptional response to inhibition of DNA replication in Bacillus subtilis. We focused on changes that were independent of the known recA-dependent global DNA damage (SOS) response. We found that overlapping sets of genes are affected by perturbations in replication elongation or initiation and that this transcriptional response serves to inhibit cell division and maintain cell viability. Approximately 20 of the operons (>50 genes) affected have potential DnaA-binding sites and are probably regulated directly by DnaA, the highly conserved replication initiation protein and transcription factor. Many of these genes have homologues and recognizable DnaA-binding sites in other bacteria, indicating that a DnaA-mediated response, elicited by changes in DNA replication status, may be conserved.

Bacillus subtilis↗

PubMed2005

Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response.

Horizontal gene transfer contributes to the evolution of bacterial species. Mobile genetic elements play an important role in horizontal gene transfer, and characterization of the regulation of these elements should provide insight into conditions that influence bacterial evolution. We characterized a mobile genetic element, ICEBs1, in the Gram-positive bacterium Bacillus subtilis and found that it is a functional integrative and conjugative element (ICE) capable of transferring to Bacillus and Listeria species. We identified two conditions that promote ICEBs1 transfer: conditions that induce the global DNA damage response and crowding by potential recipients that lack ICEBs1. Transfer of ICEBs1 into cells that already contain the element is inhibited by an intercellular signaling peptide encoded by ICEBs1. The dual regulation of ICEBs1 allows for passive propagation in the host cell until either the potential mating partners lacking ICEBs1 are present or the host cell is in distress.

Bacillus↗

PubMed2005

Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis.

Quorum or diffusion responses in bacteria are mediated by secreted signalling molecules that accumulate extracellularly as cultures grow to high density. The regulatory response to these signalling molecules can result in dramatic changes in gene expression. In Bacillus subtilis, a quorum response is mediated by a secreted 10-amino-acid modified peptide (ComX pheromone) that activates a receptor histidine kinase (ComP) that activates a response regulator transcription factor (ComA). We have used DNA microarrays to identify genes controlled by the ComX-ComP-ComA quorum-sensing pathway. We found that ComX, ComP and ComA affect the same set of genes, indicating that the kinase ComP is the only receptor for the signalling molecule ComX, and that ComA is the only transcription factor activated directly by ComP, under the conditions tested. Expression of over 20 genes appears to be controlled directly by this signalling pathway, and expression of over 150 additional genes, including those involved in competence development, appears to be controlled indirectly. The genes affected appear to have three general functions: (i) to co-ordinate physiological changes involved in developmental pathways, (ii) to produce extracellular products under conditions in which high concentrations of product are needed to be effective and (iii) to enhance survival, growth and colonization under conditions of crowding or limited diffusion. Many of the genes and processes controlled by the quorum response in B. subtilis are also regulated by quorum sensing in Gram-positive and Gram-negative bacteria. The quorum-sensing signalling molecules and regulatory proteins are quite different between Gram-positives and Gram-negatives and the convergent physiological regulation of similar genes and processes indicate the important and conserved nature of the quorum response.

Arginine↗

PubMed2005

Cytoplasmic degradation of ssrA-tagged proteins.

Degradation of ssrA-tagged proteins is a central feature of protein-quality control in all bacteria. In Escherichia coli, the ATP-dependent ClpXP and ClpAP proteases are thought to participate in this process, but their relative contributions to degradation of ssrA-tagged proteins in vivo have been uncertain because two adaptor proteins, ClpS and SspB, can modulate proteolysis of these substrates. Here, intracellular levels of these protease components and adaptors were determined during exponential growth and as cells entered early stationary phase. Levels of ClpA and ClpP increased about threefold during this transition, whereas ClpX, ClpS and SspB levels remained nearly constant. Using GFP-ssrA expressed from the chromosome as a degradation reporter, the effects of altered concentrations of different protease components or adaptor proteins were explored. Both ClpXP and ClpAP degraded GFP-ssrA in the cell, demonstrating that wild-type levels of SspB and ClpS do not inhibit ClpAP completely. Upon entry into stationary phase, increased levels of ClpAP resulted in increased degradation of ssrA-tagged substrates. As measured by maximum turnover rates, ClpXP degradation of GFP-ssrA in vivo was significantly more efficient than in vitro. Surprisingly, ClpX-dependent ClpP-independent degradation of GFP-ssrA was also observed. Thus, unfolding of this substrate by ClpX appears to enhance intracellular degradation by other proteases.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2005

Genetic composition of the Bacillus subtilis SOS system.

The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA, which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage. To identify potential RecA-LexA-regulated genes in Bacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for the B. subtilis operator: 5'-CGAACATATGTTCG-3'. Although the number of genes controlled by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E. coli.

5' Flanking Region↗

PubMed2004

Structure and mechanism of action of Sda, an inhibitor of the histidine kinases that regulate initiation of sporulation in Bacillus subtilis.

Histidine kinases are used extensively in prokaryotes to monitor and respond to changes in cellular and environmental conditions. In Bacillus subtilis, sporulation-specific gene expression is controlled by a histidine kinase phosphorelay that culminates in phosphorylation of the Spo0A transcription factor. Sda provides a developmental checkpoint by inhibiting this phosphorelay in response to DNA damage and replication defects. We show that Sda acts at the first step in the relay by inhibiting autophosphorylation of the histidine kinase KinA. The structure of Sda, which we determined using NMR, comprises a helical hairpin. A cluster of conserved residues on one face of the hairpin mediates an interaction between Sda and the KinA dimerization/phosphotransfer domain. This interaction stabilizes the KinA dimer, and the two proteins form a stable heterotetramer. The data indicate that Sda forms a molecular barricade that inhibits productive interaction between the catalytic and phosphotransfer domains of KinA.

Amino Acid Sequence↗

PubMed2004

Identification of AbrB-regulated genes involved in biofilm formation by Bacillus subtilis.

Bacillus subtilis is a ubiquitous soil bacterium that forms biofilms in a process that is negatively controlled by the transcription factor AbrB. To identify the AbrB-regulated genes required for biofilm formation by B. subtilis, genome-wide expression profiling studies of biofilms formed by spo0A abrB and sigH abrB mutant strains were performed. These data, in concert with previously published DNA microarray analysis of spo0A and sigH mutant strains, led to the identification of 39 operons that appear to be repressed by AbrB. Eight of these operons had previously been shown to be repressed by AbrB, and we confirmed AbrB repression for a further six operons by reverse transcription-PCR. The AbrB-repressed genes identified in this study are involved in processes known to be regulated by AbrB, such as extracellular degradative enzyme production and amino acid metabolism, and processes not previously known to be regulated by AbrB, such as membrane bioenergetics and cell wall functions. To determine whether any of these AbrB-regulated genes had a role in biofilm formation, we tested 23 mutants, each with a disruption in a different AbrB-regulated operon, for the ability to form biofilms. Two mutants had a greater than twofold defect in biofilm formation. A yoaW mutant exhibited a biofilm structure with reduced depth, and a sipW mutant exhibited only surface-attached cells and did not form a mature biofilm. YoaW is a putative secreted protein, and SipW is a signal peptidase. This is the first evidence that secreted proteins have a role in biofilm formation by Bacillus subtilis.

Bacillus subtilis↗

PubMed2004

Control of DNA replication initiation by recruitment of an essential initiation protein to the membrane of Bacillus subtilis.

The Bacillus subtilis proteins DnaD and DnaB are essential for replication initiation and are conserved in low G+C content Gram-positive bacteria. Previous work indicated that DnaD and DnaB are involved in helicase loading during the process of restarting stalled replication forks. We have investigated the roles of DnaD and DnaB in replication initiation at oriC in vivo. Using chromatin immunoprecipitation (ChIP), we found that DnaD and DnaB functions are needed to load the replicative helicase at oriC. To investigate further the functions of DnaD and DnaB in replication initiation, we isolated and characterized suppressors of the temperature sensitivity of dnaD and dnaB mutant cells. In both cases, we isolated the identical missense mutation in dnaB, dnaBS371P. Using yeast two-hybrid analysis, we found that dnaBS371P uncovers a previously undetected physical interaction between DnaD and DnaB. We also found that DnaBS371P constitutively recruits DnaD to the membrane fraction of cells, where DnaB and oriC are enriched. Phenotypes of cells expressing DnaBS371P are consistent with aberrant replication control. We hypothesize that B. subtilis regulates replication initiation by regulating a physical interaction between two proteins essential for helicase loading at chromosomal origins.

Bacillus subtilis↗

PubMed2004

Multicopy plasmids affect replisome positioning in Bacillus subtilis.

The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that in B. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.

Bacillus subtilis↗

PubMed2003

Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis.

The Spx protein of Bacillus subtilis represses activator-stimulated transcription by interacting with the C-terminal domain of RNA polymerase (RNAP) alpha subunit. Its concentration increases in cells lacking the ATP-dependent protease, ClpXP, resulting in severe effects on growth and developmental processes. Microarray analysis was undertaken to identify genes that are induced or repressed when Spx interacts with RNAP. The induced genes included those encoding products known to function in maintaining thiol homeostasis. Two genes, thioredoxin (trxA) and thioredoxin reductase (trxB), are transcriptionally induced under conditions of thiol-specific oxidative (disulfide) stress by a mechanism involving Spx-RNAP interaction. Disulfide stress also results in an increase in Spx-dependent transcriptional repression. The increase in Spx activity in cells encountering disulfide stress is due in part to a posttranscriptional mechanism of spx control resulting in an increase in Spx concentration. An spx null mutant and a strain bearing an allele of rpoA that prevents Spx-RNAP interaction show hypersensitivity to disulfide stress. From these results, it is proposed that Spx is an activator that mobilizes the operations necessary to reverse the effects of oxidative damage, but it also serves as a negative regulator that causes the postponement of developmental programs and energy-consuming growth-related functions while the cell copes with the period of stress.

Alleles↗