RND pumps across the genus Acinetobacter: AdeIJK is the universal efflux pump.

Acinetobacter are generally soil-dwelling organisms that can also cause serious human infections. A. baumannii is one of the most common causative agents of Acinetobacter infections and is often multidrug resistant. However, an additional 25 species within the genus have also been associated with infection. A. baumannii encodes six resistance nodulation division (RND) efflux pumps, the most clinically relevant class of efflux pumps for antibiotic export, but the distribution and types of RND efflux pumps across the genus is currently unknown. Sixty-four species making up the genus Acinetobacter were searched for RND systems within their genomes. We also developed a novel method using conserved RND residues to predict the total number of RND proteins including currently undescribed RND pump proteins. The total number of RND proteins differed both within a species and across the genus. Species associated with infection tended to encode more pumps. AdeIJK/AdeXYZ was found in all searched species of Acinetobacter, and through genomic, structural and phenotypic work we show that these genes are actually homologues of the same system. This interpretation is further supported by structural analysis of the potential drug-binding determinants of the associated RND-transporters, which reveal their close similarity to each other, and distinctiveness from other RND-pumps in Acinetobacter, such as AdeB. Therefore, we conclude that AdeIJK is the fundamental RND system for species in the genus Acinetobacter. AdeIJK can export a broad range of antibiotics and provides crucial functions within the cell, for example lipid modulation of the cell membrane, and therefore it is likely that all Acinetobacter require AdeIJK for survival and homeostasis. In contrast, additional RND systems, such as AdeABC and AdeFGH, were only found in a subset of Acinetobacter that are associated with infection. By understanding the roles and mechanisms of RND efflux systems in Acinetobacter, treatments for infections can avoid efflux-mediated resistance and improve patient outcomes.

Functionally distinct mutations within AcrB underpin antibiotic resistance in different lifestyles.

Antibiotic resistance is a pressing healthcare challenge and is mediated by various mechanisms, including the active export of drugs via multidrug efflux systems, which prevent drug accumulation within the cell. Here, we studied how Salmonella evolved resistance to two key antibiotics, cefotaxime and azithromycin, when grown planktonically or as a biofilm. Resistance to both drugs emerged in both conditions and was associated with different substitutions within the efflux-associated transporter, AcrB. Azithromycin exposure selected for an R717L substitution, while cefotaxime for Q176K. Additional mutations in ramR or envZ accumulated concurrently with the R717L or Q176K substitutions respectively, resulting in clinical resistance to the selective antibiotics and cross-resistance to other drugs. Structural, genetic, and phenotypic analysis showed the two AcrB substitutions confer their benefits in profoundly different ways. R717L reduces steric barriers associated with transit through the substrate channel 2 of AcrB. Q176K increases binding energy for cefotaxime, improving recognition in the distal binding pocket, resulting in increased efflux efficiency. Finally, we show the R717 substitution is present in isolates recovered around the world.

The role of bacterial transport systems in the removal of host antimicrobial peptides in Gram-negative bacteria.

Antibiotic resistance is a global issue that threatens our progress in healthcare and life expectancy. In recent years, antimicrobial peptides (AMPs) have been considered as promising alternatives to the classic antibiotics. AMPs are potentially superior due to their lower rate of resistance development, since they primarily target the bacterial membrane ('Achilles' heel' of the bacteria). However, bacteria have developed mechanisms of AMP resistance, including the removal of AMPs to the extracellular space by efflux pumps such as the MtrCDE or AcrAB-TolC systems, and the internalization of AMPs to the cytoplasm by the Sap transporter, followed by proteolytic digestion. In this review, we focus on AMP transport as a resistance mechanism compiling all the experimental evidence for the involvement of efflux in AMP resistance in Gram-negative bacteria and combine this information with the analysis of the structures of the efflux systems involved. Finally, we expose some open questions with the aim of arousing the interest of the scientific community towards the AMPs-efflux pumps interactions. All the collected information broadens our understanding of AMP removal by efflux pumps and gives some clues to assist the rational design of AMP-derivatives as inhibitors of the efflux pumps.

Cefotaxime Exposure Selects Mutations within the CA-Domain of envZ Which Promote Antibiotic Resistance but Repress Biofilm Formation in Salmonella.

Cephalosporins are important beta lactam antibiotics, but resistance can be mediated by various mechanisms including production of beta lactamase enzymes, changes in membrane permeability or active efflux. We used an evolution model to study how Salmonella adapts to subinhibitory concentrations of cefotaxime in planktonic and biofilm conditions and characterized the mechanisms underpinning this adaptation. We found that Salmonella rapidly adapts to subinhibitory concentrations of cefotaxime via selection of multiple mutations within the CA-domain region of EnvZ. We showed that changes in this domain affect the ATPase activity of the enzyme and in turn impact OmpC, OmpF porin expression and hence membrane permeability leading to increased tolerance to cefotaxime and low-level resistance to different classes of antibiotics. Adaptation to cefotaxime through EnvZ also resulted in a significant cost to biofilm formation due to downregulation of curli. We assessed the role of the mutations identified on the activity of EnvZ by genetic characterization, biochemistry and in silico analysis and confirmed that they are responsible for the observed phenotypes. We observed that sublethal cefotaxime exposure selected for heterogeneity in populations with only a subpopulation carrying mutations within EnvZ and being resistant to cefotaxime. Population structure and composition dynamically changed depending on the presence of the selection pressure, once selected, resistant subpopulations were maintained even in extended passage without drug. IMPORTANCE Understanding mechanisms of antibiotic resistance is crucial to guide how best to use antibiotics to minimize emergence of resistance. We used a laboratory evolution system to study how Salmonella responds to cefotaxime in both planktonic and biofilm conditions. In both contexts, we observed rapid selection of mutants within a single hot spot within envZ. The mutations selected altered EnvZ which in turn triggers changes in porin production at the outer membrane. Emergence of mutations within this region was repeatedly observed in parallel lineages in different conditions. We used a combination of genetics, biochemistry, phenotyping and structural analysis to understand the mechanisms. This data show that the changes we observe provide resistance to cefotaxime but come at a cost to biofilm formation and the fitness of mutants changes greatly depending on the presence or absence of a selective drug. Studying how resistance emerges can inform selective outcomes in the real world.

A role for the periplasmic adaptor protein AcrA in vetting substrate access to the RND efflux transporter AcrB.

Tripartite resistance-nodulation-division (RND) efflux pumps, such as AcrAB-TolC of Salmonella Typhimurium, contribute to antibiotic resistance and comprise an inner membrane RND-transporter, an outer membrane factor, and a periplasmic adaptor protein (PAP). The role of the PAP in the assembly and active transport process remains poorly understood. Here, we identify the functionally critical residues involved in PAP-RND-transporter binding between AcrA and AcrB and show that the corresponding RND-binding residues in the closely related PAP AcrE, are also important for its interaction with AcrB. We also report a residue in the membrane-proximal domain of AcrA, that when mutated, differentially affects the transport of substrates utilising different AcrB efflux channels, namely channels 1 and 2. This supports a potential role for the PAP in sensing the substrate-occupied state of the proximal binding pocket of the transporter and substrate vetting. Understanding the PAP's role in the assembly and function of tripartite RND pumps can guide novel ways to inhibit their function to combat antibiotic resistance.

Structure-function analysis of MmpL7-mediated lipid transport in mycobacteria.

Mycobacterial membrane protein Large (MmpL7) is a Resistance-Nodulation-Division (RND) family transporter required for the export of the virulence lipid, phthiocerol dimycocerosate (PDIM), in Mycobacterium tuberculosis. Using a null mutant of the related, vaccine strain Mycobacterium bovis BCG, we show that MmpL7 is also involved in the transport of the structurally related phenolic glycolipid (PGL), which is also produced by the hypervirulent M. tuberculosis strain HN878, but absent in M. tuberculosis H37Rv. Furthermore, we generated an in silico model of M. tuberculosis MmpL7 that revealed MmpL7 as a functional outlier within the MmpL-family, missing a canonical proton-relay signature sequence, suggesting that it employs a yet-unidentified mechanism for energy coupling for transport. In addition, our analysis demonstrates that the periplasmic porter domain 2 insert (PD2-insert), which doesn't share any recognisable homology, is highly alpha-helical in nature, suggesting an organisation similar to that seen in the hopanoid PD3/4 domains. Using the M. bovis BCG mmpL7 mutant for functional complementation with mutated alleles of mmpL7, we were able to identify residues present in the transmembrane domains TM4 and TM10, and the PD2 domain insert that play a crucial role in PDIM transport, and in certain cases, biosynthesis of PDIM.

Interchangeability of periplasmic adaptor proteins AcrA and AcrE in forming functional efflux pumps with AcrD in Salmonella enterica serovar Typhimurium.

BACKGROUND: Resistance-nodulation-division (RND) efflux pumps are important mediators of antibiotic resistance. RND pumps, including the principal multidrug efflux pump AcrAB-TolC in Salmonella, are tripartite systems with an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor (OMF). We previously identified the residues required for binding between the PAP AcrA and the RND transporter AcrB and have demonstrated that PAPs can function with non-cognate transporters. AcrE and AcrD/AcrF are homologues of AcrA and AcrB, respectively. Here, we show that AcrE can interact with AcrD, which does not possess its own PAP, and establish that the residues previously identified in AcrB binding are also involved in AcrD binding. METHODS: The acrD and acrE genes were expressed in a strain lacking acrABDEF (Δ3RND). PAP residues involved in promiscuous interactions were predicted based on previously defined PAP-RND interactions and corresponding mutations generated in acrA and acrE. Antimicrobial susceptibility of the mutant strains was determined. RESULTS: Co-expression of acrD and acrE significantly decreased susceptibility of the Δ3RND strain to AcrD substrates, showing that AcrE can form a functional complex with AcrD. The substrate profile of Salmonella AcrD differed from that of Escherichia coli AcrD. Mutations targeting the previously defined PAP-RND interaction sites in AcrA/AcrE impaired efflux of AcrD-dependent substrates. CONCLUSIONS: These data indicate that AcrE forms an efflux-competent pump with AcrD and thus presents an alternative PAP for this pump. Mutagenesis of the conserved RND binding sites validates the interchangeability of AcrA and AcrE, highlighting them as potential drug targets for efflux inhibition.

Quantitative real-time analysis of the efflux by the MacAB-TolC tripartite efflux pump clarifies the role of ATP hydrolysis within mechanotransmission mechanism.

Tripartite efflux pumps built around ATP-binding cassette (ABC) transporters are membrane protein machineries that perform vectorial export of a large variety of drugs and virulence factors from Gram negative bacteria, using ATP-hydrolysis as energy source. Determining the number of ATP molecules consumed per transport cycle is essential to understanding the efficiency of substrate transport. Using a reconstituted pump in a membrane mimic environment, we show that MacAB-TolC from Escherichia coli couples substrate transport to ATP-hydrolysis with high efficiency. Contrary to the predictions of the currently prevailing "molecular bellows" model of MacB-operation, which assigns the power stroke to the ATP-binding by the nucleotide binding domains of the transporter, by utilizing a novel assay, we report clear synchronization of the substrate transfer with ATP-hydrolysis, suggesting that at least some of the power stroke for the substrate efflux is provided by ATP-hydrolysis. Our findings narrow down the window for energy consumption step that results in substrate transition into the TolC-channel, expanding the current understanding of the efflux cycle of the MacB-based tripartite assemblies. Based on that we propose a modified model of the MacB cycle within the context of tripartite complex assembly.

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria.

Tripartite efflux pumps and the related type 1 secretion systems (T1SSs) in Gram-negative organisms are diverse in function, energization, and structural organization. They form continuous conduits spanning both the inner and the outer membrane and are composed of three principal components-the energized inner membrane transporters (belonging to ABC, RND, and MFS families), the outer membrane factor channel-like proteins, and linking the two, the periplasmic adaptor proteins (PAPs), also known as the membrane fusion proteins (MFPs). In this review we summarize the recent advances in understanding of structural biology, function, and regulation of these systems, highlighting the previously undescribed role of PAPs in providing a common architectural scaffold across diverse families of transporters. Despite being built from a limited number of basic structural domains, these complexes present a staggering variety of architectures. While key insights have been derived from the RND transporter systems, a closer inspection of the operation and structural organization of different tripartite systems reveals unexpected analogies between them, including those formed around MFS- and ATP-driven transporters, suggesting that they operate around basic common principles. Based on that we are proposing a new integrated model of PAP-mediated communication within the conformational cycling of tripartite systems, which could be expanded to other types of assemblies.

Structure of dual BON-domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation.

The Gram-negative outer-membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents, and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here, we reveal DolP is a lipoprotein functionally conserved amongst Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for Escherichia coli DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localisation of the protein to the cell division site, providing evidence of subcellular localisation of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

Mutations in the TolC Periplasmic Domain Affect Substrate Specificity of the AcrAB-TolC Pump.

TolC and the other members of the outer membrane factor (OMF) family are outer membrane proteins forming trimeric channels that serve as a conduit for most actively effluxed substrates in Gram-negative bacteria by providing a key component in a multitude of tripartite efflux-pumps. Current models of tripartite pump assembly ascribe substrate selection to the inner-membrane transporter and periplasmic-adapter protein (PAP) assembly, suggesting that TolC is a passive, non-selective channel. While the membrane-embedded portion of the protein adopts a porin-like fold, the periplasmic domain of TolC presents a unique "alpha-barrel" architecture. This alpha-barrel consists of pseudo-continuous α-helices forming curved coiled-coils, whose tips form α-helical hairpins, relaxation of which results in a transition of TolC from a closed to an open-aperture state allowing effective efflux of substrates through its channel. Here, we analyzed the effects of site-directed mutations targeting the alpha-barrel of TolC, of the principal tripartite efflux-pump Escherichia coli AcrAB-TolC, on the activity and specificity of efflux. Live-cell functional assays with these TolC mutants revealed that positions both at the periplasmic tip of, and partway up the TolC coiled-coil alpha-barrel domain are involved in determining the functionality of the complex. We report that mutations affecting the electrostatic properties of the channel, particularly the D371V mutation, significantly impact growth even in the absence of antibiotics, causing hyper-susceptibility to all tested efflux-substrates. These results suggest that inhibition of TolC functionality is less well-tolerated than deletion of tolC, and such inhibition may have an antibacterial effect. Significantly and unexpectedly, we identified antibiotic-specific phenotypes associated with novel TolC mutations, suggesting that substrate specificity may not be determined solely by the transporter protein or the PAP, but may reside at least partially with the TolC-channel. Furthermore, some of the effects of mutations are difficult to reconcile with the currently prevalent tip-to-tip model of PAP-TolC interaction due to their location higher-up on the TolC alpha-barrel relative to the proposed PAP-docking sites. Taken together our results suggest a possible new role for TolC in vetting of efflux substrates, alongside its established role in tripartite complex assembly.

Cryo-EM Structure and Molecular Dynamics Analysis of the Fluoroquinolone Resistant Mutant of the AcrB Transporter from Salmonella.

Salmonella is an important genus of Gram-negative pathogens, treatment of which has become problematic due to increases in antimicrobial resistance. This is partly attributable to the overexpression of tripartite efflux pumps, particularly the constitutively expressed AcrAB-TolC. Despite its clinical importance, the structure of the Salmonella AcrB transporter remained unknown to-date, with much of our structural understanding coming from the Escherichia coli orthologue. Here, by taking advantage of the styrene maleic acid (SMA) technology to isolate membrane proteins with closely associated lipids, we report the very first experimental structure of Salmonella AcrB transporter. Furthermore, this novel structure provides additional insight into mechanisms of drug efflux as it bears the mutation (G288D), originating from a clinical isolate of Salmonella Typhimurium presenting an increased resistance to fluoroquinolones. Experimental data are complemented by state-of-the-art molecular dynamics (MD) simulations on both the wild type and G288D variant of Salmonella AcrB. Together, these reveal several important differences with respect to the E. coli protein, providing insights into the role of the G288D mutation in increasing drug efflux and extending our understanding of the mechanisms underlying antibiotic resistance.

Outer membrane protein size and LPS O-antigen define protective antibody targeting to the Salmonella surface.

Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.

The mycolic acid reductase Rv2509 has distinct structural motifs and is essential for growth in slow-growing mycobacteria.

The final step in mycolic acid biosynthesis in Mycobacterium tuberculosis is catalysed by mycolyl reductase encoded by the Rv2509 gene. Sequence analysis and homology modelling indicate that Rv2509 belongs to the short-chain fatty acid dehydrogenase/reductase (SDR) family, but with some distinct features that warrant its classification as belonging to a novel family of short-chain dehydrogenases. In particular, the predicted structure revealed a unique α-helical C-terminal region which we demonstrated to be essential for Rv2509 function, though this region did not seem to play any role in protein stabilisation or oligomerisation. We also show that unlike the M. smegmatis homologue which was not essential for growth, Rv2509 was an essential gene in slow-growing mycobacteria. A knockdown strain of the BCG2529 gene, the Rv2509 homologue in Mycobacterium bovis BCG, was unable to grow following the conditional depletion of BCG2529. This conditional depletion also led to a reduction of mature mycolic acid production and accumulation of intermediates derived from 3-oxo-mycolate precursors. Our studies demonstrate novel features of the mycolyl reductase Rv2509 and outline its role in mycobacterial growth, highlighting its potential as a new target for therapies.

Identification of binding residues between periplasmic adapter protein (PAP) and RND efflux pumps explains PAP-pump promiscuity and roles in antimicrobial resistance.

Active efflux due to tripartite RND efflux pumps is an important mechanism of clinically relevant antibiotic resistance in Gram-negative bacteria. These pumps are also essential for Gram-negative pathogens to cause infection and form biofilms. They consist of an inner membrane RND transporter; a periplasmic adaptor protein (PAP), and an outer membrane channel. The role of PAPs in assembly, and the identities of specific residues involved in PAP-RND binding, remain poorly understood. Using recent high-resolution structures, four 3D sites involved in PAP-RND binding within each PAP protomer were defined that correspond to nine discrete linear binding sequences or "binding boxes" within the PAP sequence. In the important human pathogen Salmonella enterica, these binding boxes are conserved within phylogenetically-related PAPs, such as AcrA and AcrE, while differing considerably between divergent PAPs such as MdsA and MdtA, despite overall conservation of the PAP structure. By analysing these binding sequences we created a predictive model of PAP-RND interaction, which suggested the determinants that may allow promiscuity between certain PAPs, but discrimination of others. We corroborated these predictions using direct phenotypic data, confirming that only AcrA and AcrE, but not MdtA or MsdA, can function with the major RND pump AcrB. Furthermore, we provide functional validation of the involvement of the binding boxes by disruptive site-directed mutagenesis. These results directly link sequence conservation within identified PAP binding sites with functional data providing mechanistic explanation for assembly of clinically relevant RND-pumps and explain how Salmonella and other pathogens maintain a degree of redundancy in efflux mediated resistance. Overall, our study provides a novel understanding of the molecular determinants driving the RND-PAP recognition by bridging the available structural information with experimental functional validation thus providing the scientific community with a predictive model of pump-contacts that could be exploited in the future for the development of targeted therapeutics and efflux pump inhibitors.

Structure-Guided Identification of a Nonhuman Morbillivirus with Zoonotic Potential.

Morbilliviruses infect a broad range of mammalian hosts, including ruminants, carnivores, and humans. The recent eradication of rinderpest virus (RPV) and the active campaigns for eradication of the human-specific measles virus (MeV) have raised significant concerns that the remaining morbilliviruses may emerge in so-called vacated ecological niches. Seeking to assess the zoonotic potential of nonhuman morbilliviruses within human populations, we found that peste des petits ruminants virus (PPRV)-the small-ruminant morbillivirus-is restricted at the point of entry into human cells due to deficient interactions with human SLAMF1-the immune cell receptor for morbilliviruses. Using a structure-guided approach, we characterized a single amino acid change, mapping to the receptor-binding domain in the PPRV hemagglutinin (H) protein, which overcomes this restriction. The same mutation allowed escape from some cross-protective, human patient, anti-MeV antibodies, raising concerns that PPRV is a pathogen with zoonotic potential. Analysis of natural variation within human and ovine SLAMF1 also identified polymorphisms that could correlate with disease resistance. Finally, the mechanistic nature of the PPRV restriction was also investigated, identifying charge incompatibility and steric hindrance between PPRV H and human SLAMF1 proteins. Importantly, this research was performed entirely using surrogate virus entry assays, negating the requirement for in situ derivation of a human-tropic PPRV and illustrating alternative strategies for identifying gain-of-function mutations in viral pathogens.IMPORTANCE A significant proportion of viral pandemics occur following zoonotic transmission events, where animal-associated viruses jump species into human populations. In order to provide forewarnings of the emergence of these viruses, it is necessary to develop a better understanding of what determines virus host range, often at the genetic and structural levels. In this study, we demonstrated that the small-ruminant morbillivirus, a close relative of measles, is unable to use human receptors to enter cells; however, a change of a single amino acid in the virus is sufficient to overcome this restriction. This information will be important for monitoring this virus's evolution in the field. Of note, this study was undertaken in vitro, without generation of a fully infectious virus with this phenotype.

ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function.

Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein), as well as cells lacking the outer membrane factor (OMF) TolC (Tolerance to colicins). Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV), however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency.

A cytosolic copper storage protein provides a second level of copper tolerance in Streptomyces lividans.

Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P1-type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Δccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance.

Structural and Functional Analysis of the Escherichia coli Acid-Sensing Histidine Kinase EvgS.

The EvgS/EvgA two-component system of Escherichia coli is activated in response to low pH and alkali metals and regulates many genes, including those for the glutamate-dependent acid resistance system and a number of efflux pumps. EvgS, the sensor kinase, is one of five unconventional histidine kinases (HKs) in E. coli and has a large periplasmic domain and a cytoplasmic PAS domain in addition to phospho-acceptor, HK and dimerization, internal receiver, and phosphotransfer domains. Mutations that constitutively activate the protein at pH 7 map to the PAS domain. Here, we built a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide mutagenesis of potential key residues in this region. We show that histidine 226 is required for induction and that it is structurally colocated with a proline residue (P522) at the top of the predicted transmembrane helix that is expected to play a key role in passing information to the cytoplasmic domains. We also show that the constitutive mutations in the PAS domain can be further activated by low external pH. Expression of the cytoplasmic part of the protein alone also gives constitutive activation, which is lost if the constitutive PAS mutations are present. These findings are consistent with a model in which EvgS senses both external and internal pH and is activated by a shift from a tight inactive to a weak active dimer, and we present an analysis of the purified cytoplasmic portion of EvgS that supports this.IMPORTANCE One of the ways bacteria sense their environment is through two-component systems, which have one membrane-bound protein to do the sensing and another inside the cell to turn genes on or off in response to what the membrane-bound protein has detected. The membrane-bound protein must thus be able to detect the stress and signal this detection event to the protein inside the cell. To understand this process, we studied a protein that helps E. coli to survive exposure to low pH, which it must do before taking up residence in the gastrointestinal tract. We describe a predicted structure for the main sensing part of the protein and identify some key residues within it that are involved in the sensing and signaling processes. We propose a mechanism for how the protein may become activated and present some evidence to support our proposal.