A Conserved Helicase Processivity Factor Is Needed for Conjugation and Replication of an Integrative and Conjugative Element

Integrative and conjugative elements (ICEs) are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT) by the ICE–encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP), encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability.     

Selective Pressures to Maintain Attachment Site Specificity of Integrative and Conjugative Elements

Integrative and conjugative elements (ICEs) are widespread mobile genetic elements that are usually found integrated in bacterial chromosomes. They are important agents of evolution and contribute to the acquisition of new traits, including antibiotic resistances. ICEs can excise from the chromosome and transfer to recipients by conjugation. Many ICEs are site-specific in that they integrate preferentially into a primary attachment site in the bacterial genome. Site-specific ICEs can also integrate into secondary locations, particularly if the primary site is absent. However, little is known about the consequences of integration of ICEs into alternative attachment sites or what drives the apparent maintenance and prevalence of the many ICEs that use a single attachment site. Using ICEBs1, a site-specific ICE from Bacillus subtilis that integrates into a tRNA gene, we found that integration into secondary sites was detrimental to both ICEBs1 and the host cell. Excision of ICEBs1 from secondary sites was impaired either partially or completely, limiting the spread of ICEBs1. Furthermore, induction of ICEBs1 gene expression caused a substantial drop in proliferation and cell viability within three hours. This drop was dependent on rolling circle replication of ICEBs1 that was unable to excise from the chromosome. Together, these detrimental effects provide selective pressure against the survival and dissemination of ICEs that have integrated into alternative sites and may explain the maintenance of site-specific integration for many ICEs.     

Polar Positioning of a Conjugation Protein from the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

ICEBs1 is an integrative and conjugative element found in the chromosome of Bacillus subtilis. ICEBs1 encodes functions needed for its excision and transfer to recipient cells. We found that the ICEBs1 gene conE (formerly yddE) is required for conjugation and that conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE. ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4. We found that a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells. At least one ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles, as ConE-GFP was dispersed throughout the cytoplasm in a strain lacking ICEBs1. We also visualized the subcellular location of ICEBs1. When integrated in the chromosome, ICEBs1 was located near midcell along the length of the cell, a position characteristic of that chromosomal region. Following excision, ICEBs1 was more frequently found near a cell pole. Excision of ICEBs1 also caused altered positioning of at least one component of the replisome. Taken together, our findings indicate that ConE is a critical component of the ICEBs1 conjugation machinery, that conjugative transfer of ICEBs1 from B. subtilis likely initiates at a donor cell pole, and that ICEBs1 affects the subcellular position of the replisome.Integrative and conjugative elements (also known as conjugative transposons) and conjugative plasmids are key elements in horizontal gene transfer and are capable of mediating their own transfer from donor to recipient cells. ICEBs1 is an integrative and conjugative element found in some Bacillus subtilis strains. Where found, ICEBs1 is integrated into the leucine tRNA gene trnS-leu2 (Fig. ​(Fig.1)1) (7, 14, 21).Open in a separate windowFIG. 1.Genetic map of ICEBs1. conE (formerly yddE), regulatory genes (gray arrows), and genes required for integration, excision, and nicking (hatched arrows) are indicated. The number of transmembrane (TM) segments for each protein predicted by cPSORTdb (46) is indicated below each gene. Other topology programs yield similar but not identical predictions.ICEBs1 gene expression, excision, and potential mating are induced by activation of RecA during the SOS response following DNA damage (7). In addition, ICEBs1 is induced by increased production or activation of the ICEBs1-encoded regulatory protein RapI. Production and activity of RapI are indicative of the presence of potential mating partners that do not contain a copy of ICEBs1 (7). Under inducing conditions, the ICEBs1 repressor ImmR (6) is inactivated by proteolytic cleavage mediated by the antirepressor and protease ImmA (12). Most ICEBs1 genes then become highly expressed (7). One of these genes (xis) encodes an excisionase, which in combination with the element''s integrase causes efficient excision and formation of a double-stranded circle (7, 38). The circular form is nicked at the origin of transfer, oriT, by a DNA relaxase, the product of nicK (39). Under appropriate conditions, ICEBs1 can then be transferred by mating into B. subtilis and other species, including the pathogens Listeria monocytogenes and Bacillus anthracis (7). Once transferred to a recipient, ICEBs1 can be stably integrated into the genome at its attachment site in trnS-leu2 by the ICEBs1-encoded integrase (38).In contrast to what is known about ICEBs1 genes and proteins involved in excision, integration, and gene regulation, less is known about the components that make up gram-positive organisms'' mating machinery, defined as the conjugation proteins involved in DNA transfer (18, 24). The well-characterized mating machinery of gram-negative organisms can serve as a preliminary model (15, 16, 37, 48). Gram-negative organisms'' mating machinery is a type IV secretion system composed of at least eight conserved proteins that span the cell envelope. For example, the conjugation apparatus of the Agrobacterium tumefaciens Ti plasmid (pTi) is composed of 11 proteins (VirB1 through VirB11), including the ATPase VirB4 (16). VirB4 family members interact with several components of their cognate secretion systems and may energize machine assembly and/or substrate transfer (16, 48). The secretion substrate is targeted to the conjugation machinery by a coupling protein. Coupling proteins, such as VirD4 of pTi, interact with a protein attached to the end of the DNA substrate and couple the substrate to other components of the conjugation machinery. Coupling proteins might also energize the translocation of DNA through the machinery. Both VirB4 and VirD4 belong to the large HerA/FtsK superfamily of ATPases (29). Two other characterized members of this superfamily are the chromosome-partitioning proteins FtsK and SpoIIIE (29), which are ATP-dependent DNA pumps (reviewed in reference 2).Some of the proteins encoded by the conjugative elements of gram-positive organisms are homologous to components of the conjugation machinery from gram-negative organisms (1, 9, 14, 29), indicating that some aspects of conjugative DNA transfer may be similar in gram-positive and gram-negative organisms. For example, ConE (formerly YddE) of ICEBs1 has sequence similarities to VirB4 (29). YdcQ may be the ICEBs1-encoded coupling protein, as it is phylogenetically related to other coupling proteins (29, 44). Despite some similarities, the cell envelopes and many of the genes encoding the conjugation machinery are different between gram-positive and gram-negative organisms, indicating that there are likely to be significant structural and mechanistic differences as well.To begin to define the conjugation machinery of ICEBs1 and to understand spatial aspects of conjugation, we examined the function and subcellular location of ConE of ICEBs1. Our results indicate that ConE is likely a crucial ATPase component of the ICEBs1 conjugation machinery. We found that ConE and excised ICEBs1 DNA were located at or near the cell poles. We propose that the conjugation machinery is likely located at the cell poles and that mating might occur from a donor cell pole.     

Identification,characterization and benefits of an exclusion system in an integrative and conjugative element of Bacillus subtilis

Integrative and conjugative elements (ICEs) are mobile genetic elements that transfer from cell to cell by conjugation (like plasmids) and integrate into the chromosomes of bacterial hosts (like lysogenic phages or transposons). ICEs are prevalent in bacterial chromosomes and play a major role in bacterial evolution by promoting horizontal gene transfer. Exclusion prevents the redundant transfer of conjugative elements into host cells that already contain a copy of the element. Exclusion has been characterized mostly for conjugative elements of Gram‐negative bacteria. Here, we report the identification and characterization of an exclusion mechanism in ICEBs1 from the Gram‐positive bacterium Bacillus subtilis. We found that cells containing ICEBs1 inhibit the activity of the ICEBs1‐encoded conjugation machinery in other cells. This inhibition (exclusion) was specific to the cognate conjugation machinery and the ICEBs1 gene yddJ was both necessary and sufficient to mediate exclusion by recipient cells. Through a mutagenesis and enrichment screen, we identified exclusion‐resistant mutations in the ICEBs1 gene conG. Using genes from a heterologous but related ICE, we found that the exclusion specificity was determined by ConG and YddJ. Finally, we found that under conditions that support conjugation, exclusion provides a selective advantage to the element and its host cells.     

Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

We identified a functional single strand origin of replication (sso) in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA). In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA) by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso''s in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.     

Autonomous plasmid‐like replication of a conjugative transposon

Integrative and conjugative elements (ICEs), a.k.a. conjugative transposons, are mobile genetic elements involved in many biological processes, including pathogenesis, symbiosis and the spread of antibiotic resistance. Unlike conjugative plasmids that are extra‐chromosomal and replicate autonomously, ICEs are integrated in the chromosome and replicate passively during chromosomal replication. It is generally thought that ICEs do not replicate autonomously. We found that when induced, Bacillus subtilis ICEBs1 undergoes autonomous plasmid‐like replication. Replication was unidirectional, initiated from the ICEBs1 origin of transfer, oriT, and required the ICEBs1‐encoded relaxase NicK. Replication also required several host proteins needed for chromosomal replication, but did not require the replicative helicase DnaC or the helicase loader protein DnaB. Rather, replication of ICEBs1 required the helicase PcrA that is required for rolling circle replication of many plasmids. Transfer of ICEBs1 from the donor required PcrA, but did not require replication, indicating that PcrA, and not DNA replication, facilitates unwinding of ICEBs1 DNA for horizontal transfer. Although not needed for horizontal transfer, replication of ICEBs1 was needed for stability of the element. We propose that autonomous plasmid‐like replication is a common property of ICEs and contributes to the stability and maintenance of these mobile genetic elements in bacterial populations.     

Identification of host genes that affect acquisition of an integrative and conjugative element in Bacillus subtilis

Conjugation, a major type of horizontal gene transfer in bacteria, involves transfer of DNA from a donor to a recipient using donor‐encoded conjugation machinery. Using a high‐throughput screen (Tn‐seq), we identified genes in recipients that contribute to acquisition of the integrative and conjugative element ICEBs1 by Bacillus subtilis. We found that null mutations in some genes caused an increase, and others a decrease in conjugation efficiency. Some mutations affected conjugation only when present in recipients. Other mutations affected conjugation when present in donors or recipients. Most of the genes identified are known or predicted to affect the cell envelope. Several encode enzymes involved in phospholipid biosynthesis and one encodes a homologue of penicillin‐binding proteins. Two of the genes identified also affected conjugation of Tn916, indicating that their roles in conjugation may be general. We did not identify any genes in recipients that were essential for ICEBs1 conjugation, indicating that if there are such genes, then these are either essential for cell growth or redundant. Our results indicate that acquisition of ICEBs1, and perhaps other conjugative elements, is robust and not easily avoided by mutation and that several membrane‐related functions affect the efficiency of conjugation.     

Biology and engineering of integrative and conjugative elements: Construction and analyses of hybrid ICEs reveal element functions that affect species-specific efficiencies

Integrative and conjugative elements (ICEs) are mobile genetic elements that reside in a bacterial host chromosome and are prominent drivers of bacterial evolution. They are also powerful tools for genetic analyses and engineering. Transfer of an ICE to a new host involves many steps, including excision from the chromosome, DNA processing and replication, transfer across the envelope of the donor and recipient, processing of the DNA, and eventual integration into the chromosome of the new host (now a stable transconjugant). Interactions between an ICE and its host throughout the life cycle likely influence the efficiencies of acquisition by new hosts. Here, we investigated how different functional modules of two ICEs, Tn916 and ICEBs1, affect the transfer efficiencies into different host bacteria. We constructed hybrid elements that utilize the high-efficiency regulatory and excision modules of ICEBs1 and the conjugation genes of Tn916. These elements produced more transconjugants than Tn916, likely due to an increase in the number of cells expressing element genes and a corresponding increase in excision. We also found that several Tn916 and ICEBs1 components can substitute for one another. Using B. subtilis donors and three Enterococcus species as recipients, we found that different hybrid elements were more readily acquired by some species than others, demonstrating species-specific interactions in steps of the ICE life cycle. This work demonstrates that hybrid elements utilizing the efficient regulatory functions of ICEBs1 can be built to enable efficient transfer into and engineering of a variety of other species.     

Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage

Most bacterial genomes contain horizontally acquired and transmissible mobile genetic elements, including temperate bacteriophages and integrative and conjugative elements. Little is known about how these elements interact and co-evolved as parts of their host genomes. In many cases, it is not known what advantages, if any, these elements provide to their bacterial hosts. Most strains of Bacillus subtilis contain the temperate phage SPß and the integrative and conjugative element ICEBs1. Here we show that the presence of ICEBs1 in cells protects populations of B. subtilis from predation by SPß, likely providing selective pressure for the maintenance of ICEBs1 in B. subtilis. A single gene in ICEBs1 (yddK, now called spbK for SPß killing) was both necessary and sufficient for this protection. spbK inhibited production of SPß, during both activation of a lysogen and following de novo infection. We found that expression spbK, together with the SPß gene yonE constitutes an abortive infection system that leads to cell death. spbK encodes a TIR (Toll-interleukin-1 receptor)-domain protein with similarity to some plant antiviral proteins and animal innate immune signaling proteins. We postulate that many uncharacterized cargo genes in ICEs may confer selective advantage to cells by protecting against other mobile elements.     

Exploration of DNA processing features unravels novel properties of ICE conjugation in Gram-positive bacteria

Integrative and conjugative elements (ICEs) are important drivers of horizontal gene transfer in prokaryotes. They are responsible for antimicrobial resistance spread, a major current health concern. ICEs are initially processed by relaxases that recognize the binding site of oriT sequence and nick at a conserved nic site. The ICESt3/Tn916/ICEBs1 superfamily, which is widespread among Firmicutes, encodes uncanonical relaxases belonging to a recently identified family called MOB_T. This family is related to the rolling circle replication initiators of the Rep_trans family. The nic site of these MOB_T relaxases is conserved but their DNA binding site is still unknown. Here, we identified the bind site of RelSt3, the MOB_T relaxase from ICESt3. Unexpectedly, we found this bind site distantly located from the nic site. We revealed that the binding of the RelSt3 N-terminal HTH domain is required for efficient nicking activity. We also deciphered the role of RelSt3 in the initial and final stages of DNA processing during conjugation. Especially, we demonstrated a strand transfer activity, and the formation of covalent DNA-relaxase intermediate for a MOB_T relaxase.     

An ICEBs1-like element may be associated with the extreme radiation and desiccation resistance of Bacillus pumilus SAFR-032 spores

Comparisons of the genomes of Bacillus pumilus SAFR-032 and the closely related type strain, B. pumilus ATCC7061^T, exposed an extended region of non-homologous genes. A detailed examination of this region revealed the presence of an ICEBs1-like integrative conjugative element in SAFR-032. A similar element was subsequently located elsewhere in the ATCC7061^T genome. A detailed comparison of these elements and the ICEBs1 of B. subtilis revealed extremely rapid flux in gene content, genome organization and sequence similarity. It is not clear if the B. pumilus elements as they are currently structured are functional. However, it is clear that the past involvement of these elements has brought multiple genes of unknown function to the SAFR-032 genome and these genes may be responsible for the rapid evolution that led to the extreme radiation and desiccation resistance of this organism’s spores.     

LytR-CpsA-Psr Enzymes as Determinants of Bacillus anthracis Secondary Cell Wall Polysaccharide Assembly

Bacillus anthracis, the causative agent of anthrax, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and associated proteins (BSLs) function as chain length determinants and bind to the secondary cell wall polysaccharide (SCWP). In this study, we identified the B. anthracis lcpD mutant, which displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, the B. anthracis lcpB3 variant displayed reduced cell size and chain length, which could be attributed to increased deposition of BSLs. In other bacteria, LytR-CpsA-Psr (LCP) proteins attach wall teichoic acid (WTA) and polysaccharide capsule to peptidoglycan. B. anthracis does not synthesize these polymers, yet its genome encodes six LCP homologues, which, when expressed in S. aureus, promote WTA attachment. We propose a model whereby B. anthracis LCPs promote attachment of SCWP precursors to discrete locations in the peptidoglycan, enabling BSL assembly and regulated separation of septal peptidoglycan.     

Autonomous plasmid‐like replication of Bacillus ICEBs1: a general feature of integrative conjugative elements?

Integrative conjugative elements (ICEs) occur frequently in Gram‐positive and Gram‐negative bacteria. In contrast to plasmids, they are stably integrated in the bacterial genome, often inserted in a tRNA gene. They are excised from the host chromosome upon induction in order to be transferred to a recipient cell. When conjugative transfer is completed, they stably reintegrate in the chromosome. It is generally thought that ICEs are incapable of autonomous replication, instead relying on replication and segregation along with the host chromosome. In this issue of Molecular Microbiology Lee and co‐workers demonstrate that ICEBs1 from Bacillus subtilis is capable of autonomous plasmid‐like replication in its circular form after excision. The authors show that ICEBs1 replication is unidirectional; it initiates at oriT_ICEBs1 and requires the ICEBs1‐encoded conjugative relaxase NicK. Replication also requires the catalytic subunit of the host DNA polymerase PolC, the host processivity clamp DnaN and the host‐encoded alternative helicase PcrA. Autonomous replication of ICEBs1 appears to be important for its stable maintenance, but not for horizontal transfer of the element. Lee and co‐workers argue that plasmid‐like replication is likely a common property of ICEs, probably contributing to stability and maintenance of ICEs in bacterial populations. I discuss these findings in context with data on other ICEs from Gram‐positive and Gram‐negative bacteria and with respect to possible consequences of the findings for basic research on mobile genetic elements from Gram‐positive bacteria and their applications in biotechnology.     

Identification and characterization of novel cell wall hydrolase CwlT: a two-domain autolysin exhibiting n-acetylmuramidase and DL-endopeptidase activities

A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1), and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C was added to the culture. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to dl-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of d-gamma-glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a dl-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was determined to act as an N-acetylmuramidase and not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents "a new category of cell wall hydrolases."     

GneZ,a UDP-GlcNAc 2-Epimerase,Is Required for S-Layer Assembly and Vegetative Growth of Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax, forms an S-layer atop its peptidoglycan envelope and displays S-layer proteins and Bacillus S-layer-associated (BSL) proteins with specific functions to support cell separation of vegetative bacilli and growth in infected mammalian hosts. S-layer and BSL proteins bind via the S-layer homology (SLH) domain to the pyruvylated secondary cell wall polysaccharide (SCWP) with the repeat structure [→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→]_n, where α-GlcNAc and β-GlcNAc are substituted with two and one galactosyl residues, respectively. B. anthracis gneY (BAS5048) and gneZ (BAS5117) encode nearly identical UDP-GlcNAc 2-epimerase enzymes that catalyze the reversible conversion of UDP-GlcNAc and UDP-ManNAc. UDP-GlcNAc 2-epimerase enzymes have been shown to be required for the attachment of the phage lysin PlyG with the bacterial envelope and for bacterial growth. Here, we asked whether gneY and gneZ are required for the synthesis of the pyruvylated SCWP and for S-layer assembly. We show that gneZ, but not gneY, is required for B. anthracis vegetative growth, rod cell shape, S-layer assembly, and synthesis of pyruvylated SCWP. Nevertheless, inducible expression of gneY alleviated all the defects associated with the gneZ mutant. In contrast to vegetative growth, neither germination of B. anthracis spores nor the formation of spores in mother cells required UDP-GlcNAc 2-epimerase activity.     

Bacillusanthracis Surface-Layer Proteins Assemble by Binding to the Secondary Cell Wall Polysaccharide in a Manner that Requires csaB and tagO

Bacillus anthracis, the causative agent of anthrax, requires surface (S)-layer proteins for the pathogenesis of infection. Previous work characterized S-layer protein binding via the surface layer homology domain to a pyruvylated carbohydrate in the envelope of vegetative forms. The molecular identity of this carbohydrate and the mechanism of its display in the bacterial envelope are still unknown. Analyzing acid-solubilized, purified carbohydrates by mass spectrometry and NMR spectroscopy, we identify secondary cell wall polysaccharide (SCWP) as the ligand of S-layer proteins. In agreement with the model that surface layer homology domains bind to pyruvylated carbohydrate, SCWP was observed to be linked to pyruvate in a manner requiring csaB, the only structural gene known to be required for S-layer assembly. B. anthracis does not elaborate wall teichoic acids; however, its genome harbors tagO and tagA, genes responsible for the synthesis of the linkage unit that tethers teichoic acids to the peptidoglycan layer. The tagO gene appears essential for B. anthracis growth and complements the tagO mutant phenotypes of staphylococci. Tunicamycin-mediated inhibition of TagO resulted in deformed, S-layer-deficient bacilli. Together, these results suggest that tagO-mediated assembly of linkage units tethers pyruvylated SCWP to the B. anthracis envelope, thereby enabling S-layer assembly and providing for the pathogenesis of anthrax infections.     

Interrogation of Bacillus anthracis SrtA active site loop forming open/close lid conformations through extensive MD simulations for understanding binding selectivity of SrtA inhibitors

Bacillus anthracis is a gram positive, deadly spore forming bacteria causing anthrax and these bacteria having the complex mechanism in the cell wall envelope, which can adopt the changes in environmental conditions. In this, the membrane bound cell wall proteins are said to progressive drug target for the inhibition of Bacillus anthracis. Among the cell wall proteins, the SrtA is one of the important mechanistic protein, which mediate the ligation with LPXTG motif by forming the amide bonds. The SrtA plays the vital role in cell signalling, cell wall formation, and biofilm formations. Inhibition of SrtA leads to rupture of the cell wall and biofilm formation, and that leads to inhibition of Bacillus anthracis and thus, SrtA is core important enzyme to study the inhibition mechanism. In this study, we have examined 28 compounds, which have the inhibitory activity against the Bacillus anthracis SrtA for developing the 3D-QSAR and also, compounds binding selectivity with both open and closed SrtA conformations, obtained from 100 ns of MD simulations. The binding site loop deviate in forming the open and closed gate mechanism is investigated to understand the inhibitory profile of reported compounds, and results show the closed state active site conformations are required for ligand binding specificity. Overall, the present study may offer an opportunity for better understanding of the mechanism of action and can be aided to further designing of a novel and highly potent SrtA inhibitors.     

Evidence of a Bacterial Receptor for Lysozyme: Binding of Lysozyme to the Anti-σ Factor RsiV Controls Activation of the ECF σ Factor σV

σ factors endow RNA polymerase with promoter specificity in bacteria. Extra-Cytoplasmic Function (ECF) σ factors represent the largest and most diverse family of σ factors. Most ECF σ factors must be activated in response to an external signal. One mechanism of activation is the stepwise proteolytic destruction of an anti-σ factor via Regulated Intramembrane Proteolysis (RIP). In most cases, the site-1 protease required to initiate the RIP process directly senses the signal. Here we report a new mechanism in which the anti-σ factor rather than the site-1 protease is the sensor. We provide evidence suggesting that the anti-σ factor RsiV is the bacterial receptor for the innate immune defense enzyme, lysozyme. The site-1 cleavage site is similar to the recognition site of signal peptidase and cleavage at this site is required for σ^V activation in Bacillus subtilis. We reconstitute site-1 cleavage in vitro and demonstrate that it requires both signal peptidase and lysozyme. We demonstrate that the anti-σ factor RsiV directly binds to lysozyme and muramidase activity is not required for σ^V activation. We propose a model in which the binding of lysozyme to RsiV activates RsiV for signal peptidase cleavage at site-1, initiating proteolytic destruction of RsiV and activation of σ^V. This suggests a novel mechanism in which conformational change in a substrate controls the cleavage susceptibility for signal peptidase. Thus, unlike other ECF σ factors which require regulated intramembrane proteolysis for activation, the sensor for σ^V activation is not the site-1 protease but the anti-σ factor.     

Effect of pH on the Electrophoretic Mobility of Spores of Bacillus anthracis and Its Surrogates in Aqueous Solutions

The electrophoretic mobility (EPM) of endospores of Bacillus anthracis and surrogates was measured in aqueous solution across a broad pH range and several ionic strengths. EPM values trended around phylogenetic clustering based on the 16S rRNA gene. Measurements reported here provide new insight for Bacillus anthracis surrogate selection and for attachment/detachment and transport studies.     

Crystal Structure of the Catalytic Domain of the Bacillus cereus SleB Protein, Important in Cortex Peptidoglycan Degradation during Spore Germination

The SleB protein is one of two redundant cortex-lytic enzymes (CLEs) that initiate the degradation of cortex peptidoglycan (PG), a process essential for germination of spores of Bacillus species, including Bacillus anthracis. SleB has been characterized as a soluble lytic transglycosylase that specifically recognizes spore cortex PG and catalyzes the cleavage of glycosidic bonds between N-acetylmuramic acid (NAM) and N-acetylglucosamine residues with concomitant formation of a 1,6-anhydro bond in the NAM residue. We found that like the full-length Bacillus cereus SleB, the catalytic C-terminal domain (SleB^C) exhibited high degradative activity on cortex PG in vitro, although SleB''s N-terminal domain, thought to bind PG, was inactive. The 1.85-Å crystal structure of SleB^C reveals an ellipsoid molecule with two distinct domains dominated by either α helices or β strands. The overall fold of SleB closely resembles that of the catalytic domain of the family 1 lytic transglycosylases but with a completely different topological arrangement. Structural analysis shows that an invariant Glu157 of SleB is in a position equivalent to that of the catalytic glutamate in other lytic transglycosylases. Indeed, SleB bearing a Glu157-to-Gln mutation lost its cortex degradative activity completely. In addition, the other redundant CLE (called CwlJ) in Bacillus species likely has a three-dimensional structure similar to that of SleB, including the invariant putative catalytic Glu residue. SleB and CwlJ may offer novel targets for the development of anti-spore agents.