Bacterial Two-hybrid Assays Research Articles

602. Mechanism of LiaY-Mediated Daptomycin Resistance in Enterococcus faecalis

BackgroundDaptomycin (DAP) is a lipopeptide antibiotic that targets the cell membrane (CM) at the division septum. DAP resistance (DAP-R) in E. faecalis (Efs) has been linked to mutations in genes encoding the LiaFSR stress response system and lipid biosynthetic enzymes, including cardiolipin synthase (Cls). The signature phenotype of DAP-R is redistribution of CM anionic phospholipid (APL) microdomains. Using a genetic approach, we have identified a transmembrane protein (LiaY) as a major mediator of cell membrane APL redistribution associated with DAP-R. Here, we explore the mechanism of LiaY-mediated changes in the CM under the hypothesis that CM remodeling occurs through interactions with Cls.Methods Efs encodes two cls genes (cls1 and cls2). Deletion mutants of both cls genes were generated using the Crispr/cas9 system in the daptomycin-sensitive strain Efs OG117 and Efs OG117∆liaX (a DAP-R derivative of OG117). DAP minimum inhibitory concentration (MIC) was determined using E-test on Mueller–Hinton II agar. Visualization of APL microdomains was performed by staining mid-logarithmic phase cells with 1 µM of 10-N-nonyl-acridine orange (NAO) and fluorescence microscopy. Bacterial two-hybrid system was used to study interactions between LiaY with Cls1 or Cls2.ResultsSingle or double deletion of cls1 or cls2 in Efs OG117 did not affect DAP MIC, and no changes in CM architecture were seen by NAO staining. In contrast,deletion of cls1 (alone or in conjunction with a deletion of cls2) in a DAP-R derivative of OG117 OG117∆liaX, resulted in a marked decrease in DAP MIC, and NAO staining of Efs OG117∆liaX∆cls1∆cls2 shows a restoration of septal APL microdomain localization.In the same DAP-R background, deletion of cls2 alone did not have any effect on DAP MIC or APL microdomain distribution. Additionally, bacterial two-hybrid assays showed a positive interaction of LiaY with Cls1 but not with Cls2.ConclusionWe have identified the biochemical basis for DAP-R associated CM remodeling. In a proposed model, the LiaR-mediated activation of the LiaY triggers specific interactions with Cls1 displacing the protein away from the septum, resulting in local generation of APL microdomains that prevents DAP-mediated damage to the CM.Disclosures All authors: No reported disclosures.

The Group B Streptococcal surface antigen I/II protein, BspC, interacts with host vimentin to promote adherence to brain endothelium and inflammation during the pathogenesis of meningitis.

Streptococcus agalactiae (Group B Streptococcus, GBS) normally colonizes healthy adults but can cause invasive disease, such as meningitis, in the newborn. To gain access to the central nervous system, GBS must interact with and penetrate brain or meningeal blood vessels; however, the exact mechanisms are still being elucidated. Here, we investigate the contribution of BspC, an antigen I/II family adhesin, to the pathogenesis of GBS meningitis. Disruption of the bspC gene reduced GBS adherence to human cerebral microvascular endothelial cells (hCMEC), while heterologous expression of BspC in non-adherent Lactococcus lactis conferred bacterial attachment. In a murine model of hematogenous meningitis, mice infected with ΔbspC mutants exhibited lower mortality as well as decreased brain bacterial counts and inflammatory infiltrate compared to mice infected with WT GBS strains. Further, BspC was both necessary and sufficient to induce neutrophil chemokine expression. We determined that BspC interacts with the host cytoskeleton component vimentin and confirmed this interaction using a bacterial two-hybrid assay, microscale thermophoresis, immunofluorescent staining, and imaging flow cytometry. Vimentin null mice were protected from WT GBS infection and also exhibited less inflammatory cytokine production in brain tissue. These results suggest that BspC and the vimentin interaction is critical for the pathogenesis of GBS meningitis.

Structure of the type VI secretion system TssK\u2013TssF\u2013TssG baseplate subcomplex revealed by cryo-electron microscopy

Type VI secretion systems (T6SSs) translocate effectors into target cells and are made of a contractile sheath and a tube docked onto a multi-protein transmembrane complex via a baseplate. Although some information is available about the mechanisms of tail contraction leading to effector delivery, the detailed architecture and function of the baseplate remain unknown. Here, we report the 3.7 Å resolution cryo-electron microscopy reconstruction of an enteroaggregative Escherichia coli baseplate subcomplex assembled from TssK, TssF and TssG. The structure reveals two TssK trimers interact with a locally pseudo-3-fold symmetrical complex comprising two copies of TssF and one copy of TssG. TssF and TssG are structurally related to each other and to components of the phage T4 baseplate and of the type IV secretion system, strengthening the evolutionary relationships among these macromolecular machines. These results, together with bacterial two-hybrid assays, provide a structural framework to understand the T6SS baseplate architecture.

Cryptic protein interactions regulate DNA replication initiation.

DNA replication is a fundamental biological process that is tightly regulated in all cells. In bacteria, DnaA controls when and where replication begins by building a step-wise complex that loads the replicative helicase onto chromosomal DNA. In many low-GC Gram-positive species, DnaA recruits the DnaD and DnaB proteins to function as adaptors to assist in helicase loading. How DnaA, its adaptors and the helicase form a complex at the origin is unclear. We addressed this question using the bacterial two-hybrid assay to determine how the initiation proteins from Bacillus subtilis interact with each other. We show that cryptic interaction sites play a key role in this process and we map these regions for the entire pathway. In addition, we found that the SirA regulator that blocks initiation in sporulating cells binds to a surface on DnaA that overlaps with DnaD. The interaction between DnaA and DnaD was also mapped to the same DnaA surface in the human pathogen Staphylococcus aureus, demonstrating the broad conservation of this surface. Therefore, our study has unveiled key protein interactions essential for initiation and our approach is widely applicable for mapping interactions in other signaling pathways that are governed by cryptic binding surfaces.

An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases in Myxococcus xanthus DK1622.

One mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 from Myxococcus xanthus DK1622 for characterization. The M. xanthus DK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination of in vivo and in vitro assays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping of M. xanthus DK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a "universal" MLP for generating functional hybrid NRPSs.IMPORTANCE MbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP from Myxococcus xanthus DK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a "universal" MLP during the construction of functional hybrid NRPSs.

Identification of drug target candidates of the swine pathogen Actinobacillus pleuropneumoniae by construction of protein-protein interaction network.

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae has led to severe economic losses in the pig industry worldwide. A. pleuropneumoniae displays various levels of antimicrobial resistance, leading to the dire need to identify new drug targets. Protein-protein interaction (PPI) network can aid the identification of drug targets by discovering essential proteins during the life of bacteria. The aim of this study is to identify drug target candidates of A. pleuropneumoniae from essential proteins in PPI network. The homologous protein mapping method (HPM) was utilized to construct A. pleuropneumoniae PPI network. Afterwards, the subnetwork centered with H-NS was selected to verify the PPI network using bacterial two-hybrid assays. Drug target candidates were identified from the hub proteins by analyzing the topology of the network using interaction degree and homologous comparison with the pig proteome. An A. pleuropneumoniae PPI network containing 2737 non-redundant interaction pairs among 533 proteins was constructed. These proteins were distributed in 21 COG functional categories and 28 KEGG metabolic pathways. The A. pleuropneumoniae PPI network was scale free and the similar topological tendencies were found when compared with other bacteria PPI network. Furthermore, 56.3% of the H-NS subnetwork interactions were validated. 57 highly connected proteins (hub proteins) were identified from the A. pleuropneumoniae PPI network. Finally, 9 potential drug targets were identified from the hub proteins, with no homologs in swine. This study provides drug target candidates, which are promising for further investigations to explore lead compounds against A. pleuropneumoniae.

Leptospiral flagellar sheath protein FcpA interacts with FlaA2 and FlaB1 in Leptospira biflexa.

Leptospira spp. are spirochete bacteria that possess periplasmic flagella (PFs) underneath the outer membrane; each flagellum is attached to each end of the protoplasmic cylinder. PFs of Leptospira have a coiled shape that bends the end of the cell body. However, the molecular mechanism by which multiple flagellar proteins organize to form the distinctively curled PF of Leptospira remains unclear. Here we obtained a slow-motility mutant of L. biflexa MD4-3 by random insertion mutagenesis using a Himar1 transposon. In MD4-3, the gene encoding the flagellar sheath protein, flagellar-coiling protein A (FcpA), which was recently identified in L. interrogans, was inactivated. As with L. interrogans ΔfcpA strains, the L. biflexa ΔfcpA strain lacked a distinct curvature at both ends of the cell body, and its motility was significantly reduced as compared with that of the wild-type strain. PFs isolated from the ΔfcpA strain were straight and were thinner than those isolated from the wild-type strain. Western blot analysis revealed that flagellar proteins FlaA1, FlaA2, FlaB1, and FlaB2 were expressed in the ΔfcpA strain but the flagellar proteins, except for FlaB2 were not incorporated in its PFs. Immunoprecipitation assay using anti-FcpA antiserum demonstrated that FcpA associates with FlaA2 and FlaB1. The association between FcpA and FlaA2 was also verified using pull-down assay. The regions of FlaA2 and FlaB1 interacting with FcpA were determined using a bacterial two-hybrid assay. These results suggest that FcpA together with FlaA2, produces coiling of PF of the Leptospira, and the interaction between the sheath and core filament may be mediated by FcpA and FlaB1.

A Multimodal Strategy Used by a Large c-di-GMP Network.

The Pseudomonas fluorescens genome encodes more than 50 proteins predicted to be involved in c-di-GMP signaling. Here, we demonstrated that, tested across 188 nutrients, these enzymes and effectors appeared capable of impacting biofilm formation. Transcriptional analysis of network members across ∼50 nutrient conditions indicates that altered gene expression can explain a subset of but not all biofilm formation responses to the nutrients. Additional organization of the network is likely achieved through physical interaction, as determined via probing ∼2,000 interactions by bacterial two-hybrid assays. Our analysis revealed a multimodal regulatory strategy using combinations of ligand-mediated signals, protein-protein interaction, and/or transcriptional regulation to fine-tune c-di-GMP-mediated responses. These results create a profile of a large c-di-GMP network that is used to make important cellular decisions, opening the door to future model building and the ability to engineer this complex circuitry in other bacteria.IMPORTANCE Cyclic diguanylate (c-di-GMP) is a key signaling molecule regulating bacterial biofilm formation, and many microbes have up to dozens of proteins that make, break, or bind this dinucleotide. A major open issue in the field is how signaling specificity is conferred in the unpartitioned space of a bacterial cell. Here, we took a systems approach, using mutational analysis, transcriptional studies, and bacterial two-hybrid analysis to interrogate this network. We found that a majority of enzymes are capable of impacting biofilm formation in a context-dependent manner, and we revealed examples of two or more modes of regulation (i.e., transcriptional control with protein-protein interaction) being utilized to generate an observable impact on biofilm formation.

IcmF and DotU are required for the virulence of Acidovorax oryzae strain RS-1.

Acidovorax oryzae (Ao) cause bacterial brown stripe in rice that result in great economic loss. However, the pathogenic mechanism of this rice bacterial pathogen still remains unclear. Interestingly, transcriptomic and proteomic analysis of in vivo infection indicate that the pathogenicity of Ao strain RS-1 may be associated with the type six secretion system (T6SS), which was identified by in silico comparative genomic analysis in our previous studies. This makes it necessary to further examine the role of each core component of T6SS in the pathogenicity of Ao strain RS-1 to rice plants. Results from this study highlight the mutual interaction between IcmF and DotU, which was determined by bacterial two-hybrid and bimolecular fluorescence complementation assays. Furthermore, a difference was observed in bacterial pathogenicity, biofilm formation, secreted proteins identified by LC-MS/MS analysis and the expression of T6SS other genes examined by quantitative real-time PCR between the wild-type and both single-gene knockout mutants, which were respectively constructed based on the insertional mutagenesis of Ao strain RS-1 in this study. Overall, our results clearly revealed the importance of T6SS icmF and dotU in pathogenicity of Ao strain RS-1 to rice plants.

A bacterial three-hybrid assay detects Escherichia coli Hfq-sRNA interactions in vivo.

The interaction of RNA molecules with proteins is a critical aspect of gene regulation across all domains of life. Here, we report the development of a bacterial three-hybrid (B3H) assay to genetically detect RNA–protein interactions. The basis for this three-hybrid assay is a transcription-based bacterial two-hybrid assay that has been used widely to detect and dissect protein–protein interactions. In the three-hybrid assay, a DNA-bound protein with a fused RNA-binding moiety (the coat protein of bacteriophage MS2 (MS2CP)) is used to recruit a hybrid RNA upstream of a test promoter. The hybrid RNA consists of a constant region that binds the tethered MS2CP and a variable region. Interaction between the variable region of the hybrid RNA and a target RNA-binding protein that is fused to a subunit of Escherichia coli RNA polymerase (RNAP) stabilizes the binding of RNAP to the test promoter, thereby activating transcription of a reporter gene. We demonstrate that this three-hybrid assay detects interaction between non-coding small RNAs (sRNAs) and the hexameric RNA chaperone Hfq from E. coli and enables the identification of Hfq mutants with sRNA-binding defects. Our findings suggest that this B3H assay will be broadly applicable for the study of RNA–protein interactions.

An Escherichia coli expression model reveals the species-specific function of FtsA from Neisseria gonorrhoeae in cell division.

Escherichia coli (Ec) has been used to study the function of cell division proteins from different microorganisms, especially when genetic tools are limited for studying these proteins in their native hosts. The expression of ftsA from Neisseria gonorrhoeae (Ng) disrupted cell division in E. coli resulting in a significant increase in cell length. In some cells, FtsANg localised to the division site and the poles of E. coli cells, but the majority of cells showed no specifical localisation. FtsANg did not complement an E. coli ftsA mutant strain. Bacterial two-hybrid and GST pull-down assays indicated that FtsANg interacted with FtsNEc, but no other cell division proteins from E. coli. This interaction was mediated through the 2A and 2B subdomains of FtsANg. This evidence suggests that the function of FtsANg is species specific.

Specific Glucoside Transporters Influence Septal Structure and Function in the Filamentous, Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

When deprived of combined nitrogen, some filamentous cyanobacteria contain two cell types: vegetative cells that fix CO2 through oxygenic photosynthesis and heterocysts that are specialized in N2 fixation. In the diazotrophic filament, the vegetative cells provide the heterocysts with reduced carbon (mainly in the form of sucrose) and heterocysts provide the vegetative cells with combined nitrogen. Septal junctions traverse peptidoglycan through structures known as nanopores and appear to mediate intercellular molecular transfer that can be traced with fluorescent markers, including the sucrose analog esculin (a coumarin glucoside) that is incorporated into the cells. Uptake of esculin by the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was inhibited by the α-glucosides sucrose and maltose. Analysis of Anabaena mutants identified components of three glucoside transporters that move esculin into the cells: GlsC (Alr4781) and GlsP (All0261) are an ATP-binding subunit and a permease subunit of two different ABC transporters, respectively, and HepP (All1711) is a major facilitator superfamily (MFS) protein that was shown previously to be involved in formation of the heterocyst envelope. Transfer of fluorescent markers (especially calcein) between vegetative cells of Anabaena was impaired by mutation of glucoside transporter genes. GlsP and HepP interact in bacterial two-hybrid assays with the septal junction-related protein SepJ, and GlsC was found to be necessary for the formation of a normal number of septal peptidoglycan nanopores and for normal subcellular localization of SepJ. Therefore, beyond their possible role in nutrient uptake in Anabaena, glucoside transporters influence the structure and function of septal junctions.IMPORTANCE Heterocyst-forming cyanobacteria have the ability to perform oxygenic photosynthesis and to assimilate atmospheric CO2 and N2 These organisms grow as filaments that fix these gases specifically in vegetative cells and heterocysts, respectively. For the filaments to grow, these types of cells exchange nutrients, including sucrose, which serves as a source of reducing power and of carbon skeletons for the heterocysts. Movement of sucrose between cells in the filament takes place through septal junctions and has been traced with a fluorescent sucrose analog, esculin, that can be taken up by the cells. Here, we identified α-glucoside transporters of Anabaena that mediate uptake of esculin and, notably, influence septal structure and the function of septal junctions.

Understanding the Role of the N-Terminal CrgA Interactions with FtsZ in the Mycobacterium Tuberculosis Divisome

CrgA is a 93-residue helical membrane protein found in Mycobacterium Tuberculosis (Mtb) divisome, the cell division apparatus, where it has been argued that it recruits other Mtb proteins to the divisome. Bacterial two-hybrid assays and pull down assays have documented 5 binding partners including FtsZ, FtsQ, FtsI (PBPB), PBPA, and CwsA and have also shown that CrgA is a dimer. A structure of the transmembrane domain of the monomer has been published (Das et al., 2015, PNAS 112:E119-E126) showing a pair of TM helices with a 30 residue N-terminus and an insignificant 2 residue C-terminus, both on the cytoplasmic side of the membrane while an interhelical loop of 20 residues is on the periplasmic side. A sampling of the first 18 residues show little anisotropy in the solid state NMR spectra indicating that it is intrinsically disordered, while a sampling of residues 19-30 show considerable anisotropy and are therefore thought to be structured. Since FtsZ is cytoplasmic it is thought to bind to this N-terminus. A combination of mutagenesis and solid-state NMR of CrgA samples in liquid crystalline bilayer preparations of POPC/POPG bilayers is underway to characterize the structured domain of the N-terminus. The Cα isotropic chemical shifts for this region suggest that it is a β-strand conformation, and since there is little evidence for a loop in this 12 residue structured domain the presumably this strand is stabilized by binding to the adjacent monomer, i.e. an interprotein β-sheet. Isotopic labeling strategies for solid state NMR are being used to document that our hypothesis is correct and to characterize the hydrogen bonding pattern. Initial results support our claim and raise an even more interesting question as to how FtsZ binds: does it bind using the intrinsically disordered domain of the N-terminus or does it bind to the β-sheet?

Interaction of E. coli Hsp90 with DnaK Involves the DnaJ Binding Region of DnaK

The 90-kDa heat shock protein (Hsp90) is a widely conserved and ubiquitous molecular chaperone that participates in ATP-dependent protein remodeling in both eukaryotes and prokaryotes. It functions in conjunction with Hsp70 and the Hsp70 cochaperones, an Hsp40 (J-protein) and a nucleotide exchange factor. In Escherichia coli, the functional collaboration between Hsp90Ec and Hsp70, DnaK, requires that the two chaperones directly interact. We used molecular docking to model the interaction of Hsp90Ec and DnaK. The top-ranked docked model predicted that a region in the nucleotide-binding domain (NBD) of DnaK interacted with a region in the middle domain of Hsp90Ec. We then made substitution mutants in DnaK residues suggested by the model to interact with Hsp90Ec. Of the 12 mutants tested, 11 were defective or partially defective in their ability to interact with Hsp90Ecin vivo in a bacterial two-hybrid assay and in vitro in a bio-layer interferometry assay. These DnaK mutants were also defective in their ability to function collaboratively in protein remodeling with Hsp90Ec but retained the ability to act with DnaK cochaperones. Taken together, these results suggest that a specific region in the NBD of DnaK is involved in the interaction with Hsp90Ec, and this interaction is functionally important. Moreover, the region of DnaK that we found to be necessary for Hsp90Ec binding includes residues that are also involved in J-protein binding, suggesting a functional interplay among DnaK, DnaK cochaperones, and Hsp90Ec.

Characterization of the Direct Interaction between Hybrid Sensor Kinases PA1611 and RetS That Controls Biofilm Formation and the Type III Secretion System in Pseudomonas aeruginosa.

One of the leading causes of morbidity and mortality in cystic fibrosis (CF) patients is pulmonary infection with Pseudomonas aeruginosa, and the pathophysiology of pulmonary infection in CF is affected by the lifestyle of this micro-organism. RetS-GacS/A-RsmA is a key regulatory pathway in P.aeruginosa that determines the bacterium's lifestyle choice. Previously, we identified PA1611, a hybrid sensor kinase, as a new player in this pathway that interacts with RetS and influences biofilm formation and type III secretion system. In this study, we explored the structural and mechanistic basis of the interaction between PA1611 and RetS. We identified the amino acid residues critical for PA1611-RetS interactions by molecular modeling. These residues were then targeted for site-directed mutagenesis. Amino acid substitutions were carried out at seven key positions in PA1611 and at six corresponding key positions in RetS. The influence of such substitutions in PA1611 on the interaction was analyzed by bacterial two-hybrid assays. We carried out functional analysis of these mutants in P.aeruginosa for their effect on specific phenotypes. Two residues, F269 and E276, located within the histidine kinase A and histidine kinase-like ATPase domains of PA1611 were found to play crucial roles in the PA1611-RetS interaction and had profound effects on phenotypes. Corresponding mutations in RetS demonstrated similar results. We further confirmed that these mutations in PA1611 function through the GacS/GacA-RsmY/Z signaling pathway. Collectively, our findings provide a noncognate sensor kinase direct interaction model for a signaling pathway, key for lifestyle selection in P.aeruginosa, and targeting such interaction may serve as a novel way of controlling infections with P.aeruginosa.

Structural Analysis and Inhibition of TraE from the pKM101 Type IV Secretion System

Gram-negative bacteria use type IV secretion systems (T4SSs) for a variety of macromolecular transport processes that include the exchange of genetic material. The pKM101 plasmid encodes a T4SS similar to the well-studied model systems from Agrobacterium tumefaciens and Brucella suis Here, we studied the structure and function of TraE, a homolog of VirB8 that is an essential component of all T4SSs. Analysis by X-ray crystallography revealed a structure that is similar to other VirB8 homologs but displayed an altered dimerization interface. The dimerization interface observed in the X-ray structure was corroborated using the bacterial two-hybrid assay, biochemical characterization of the purified protein, and in vivo complementation, demonstrating that there are different modes of dimerization among VirB8 homologs. Analysis of interactions using the bacterial two-hybrid and cross-linking assays showed that TraE and its homologs from Agrobacterium, Brucella, and Helicobacter pylori form heterodimers. They also interact with heterologous VirB10 proteins, indicating a significant degree of plasticity in the protein-protein interactions of VirB8-like proteins. To further assess common features of VirB8-like proteins, we tested a series of small molecules derived from inhibitors of Brucella VirB8 dimerization. These molecules bound to TraE in vitro, docking predicted that they bind to a structurally conserved surface groove of the protein, and some of them inhibited pKM101 plasmid transfer. VirB8-like proteins thus share functionally important sites, and these can be exploited for the design of specific inhibitors of T4SS function.

Vancomycin susceptibility in methicillin-resistant Staphylococcus aureus is mediated by YycHI activation of the WalRK essential two-component regulatory system.

The treatment of infections caused by methicillin-resistant Staphylococcus aureus is complicated by the emergence of strains with intermediate-level resistance to vancomycin (termed VISA). We have characterised a molecular pathway involved in the in vivo evolution of VISA mediated by the regulatory proteins YycH and YycI. In contrast to their function in other bacterial species, we report a positive role for these auxiliary proteins in regulation of the two-component regulator WalRK. Transcriptional profiling of yycH and yycI deletion mutants revealed downregulation of the ‘WalRK regulon’ including cell wall hydrolase genes atlA and sle1, with functional autolysis assays supporting these data by showing an impaired autolytic phenotype for each deletion strain. Using bacterial-two hybrid assays, we showed that YycH and YycI interact, and that YycHI also interacts with the sensor kinase WalK, forming a ternary protein complex. Mutation to YycH or YycI associated with clinical VISA strains had a deleterious impact on the YycHI/WalK complex, suggesting that the interaction is important for the regulation of WalRK. Taken together, we have described a novel antibiotic resistance strategy for the human pathogen S. aureus, whereby YycHI mutations are selected for in vivo leading to reduced WalRK activation, impaired cell wall turnover and ultimately reduced vancomycin efficacy.

The Last r Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin.

The latent period of phage T4, normally ∼25 min, can be extended indefinitely if the infected cell is superinfected after 5 min. This phenomenon, designated lysis inhibition (LIN), was first described in the 1940s and is genetically defined by mutations in diverse T4 r genes. RI, the main effector of LIN, has been shown to be secreted to the periplasm, where, upon activation by superinfection with a T-even virion, it binds to the C-terminal periplasmic domain of the T4 holin T and blocks its lethal permeabilization of the cytoplasmic membrane. Another r locus, rIII, has been the subject of conflicting reports. In this study, we show that RIII, an 82-amino-acid protein, is also required for LIN in both Escherichia coli B strains and E. coli K-12 strains. In T4ΔrIII infections, LIN was briefly established but was unstable. The overexpression of a cloned rIII gene alone impeded T-mediated lysis temporarily. However, coexpression of rIII and rI resulted in a stable LIN state. Bacterial two-hybrid assays and pulldown assays showed that RIII interacts with the cytoplasmic N terminus of T, which is a critical domain for holin function. We conclude that RIII is a T4 antiholin that blocks membrane hole formation by interacting directly with the holin. Accordingly, we propose an augmented model for T4 LIN that involves the stabilization of a complex of three proteins in two compartments of the cell: RI interacting with the C terminus of T in the periplasm and RIII interacting with the N terminus of T in the cytoplasm. Lysis inhibition is a unique feature of phage T4 in response to environmental conditions, effected by the antiholin RI, which binds to the periplasmic domain of the T holin and blocks its hole-forming function. Here we report that the T4 gene rIII encodes a cytoplasmic antiholin that, together with the main antiholin, RI, inhibits holin T by forming a complex of three proteins spanning two cell compartments.

Subunit orientation in the Escherichia coli enterobactin biosynthetic EntA-EntE complex revealed by a two-hybrid approach.

The siderophore enterobactin is synthesized by the enzymes EntA-F and EntH in the Escherichia coli cytoplasm. We previously reported invitro evidence of an interaction between tetrameric EntA and monomeric EntE. Here we used bacterial adenylate cyclase two-hybrid (BACTH) assays to demonstrate that the E.coli EntA-EntE interaction occurs intracellularly. Furthermore, to obtain information on subunit orientation in the EntA-EntE complex, we fused BACTH reporter fragments T18 and T25 to EntA and EntE in both N-terminal and C-terminal orientations. To validate functionality of our fusion proteins, we performed Chrome Azurol S (CAS) assays using E.coli entE(-) and entA(-) knockout strains transformed with our BACTH constructs. We found that transformants expressing N-terminal and C-terminal T18/T25 fusions to EntE exhibited CAS signals, indicating that these constructs could rescue the entE(-) phenotype. While expression of EntA with N-terminal T18/T25 fusions exhibited CAS signals, C-terminal fusions did not, presumably due to disruption of the EntA tetramer invivo. Bacterial growth assays supported our CAS findings. Co-transformation of functional T18/T25 fusions into cya(-)E.coli BTH101cells resulted in positive BACTH signals only when T18/T25 fragments were fused to the N-termini of both EntA and EntE. Co-expression of N-terminally fused EntA with C-terminally fused EntE resulted in no detectable BACTH signal. Analysis of protein expression by Western blotting confirmed that the loss of BACTH signal was not due to impaired expression of fusion proteins. Based on our results, we propose that the N-termini of EntA and EntE are proximal in the intracellular complex, while the EntA N-terminus and EntE C-terminus are distal. A protein-protein docking simulation using SwarmDock was in agreement with our experimental observations.

THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria.

Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP-binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors.