Diversity and evolution of the small multidrug resistance protein family

<p>Abstract</p> <p>Background</p> <p>Members of the small multidrug resistance (SMR) protein family are integral membrane proteins characterized by four α-helical transmembrane strands that confer resistance to a broad range of antiseptics and lipophilic quaternary ammonium compounds (QAC) in bacteria. Due to their short length and broad substrate profile, SMR proteins are suggested to be the progenitors for larger α-helical transporters such as the major facilitator superfamily (MFS) and drug/metabolite transporter (DMT) superfamily. To explore their evolutionary association with larger multidrug transporters, an extensive bioinformatics analysis of SMR sequences (> 300 Bacteria taxa) was performed to expand upon previous evolutionary studies of the SMR protein family and its origins.</p> <p>Results</p> <p>A thorough annotation of unidentified/putative SMR sequences was performed placing sequences into each of the three SMR protein subclass designations, namely small multidrug proteins (SMP), suppressor of <it>groEL </it>mutations (SUG), and paired small multidrug resistance (PSMR) using protein alignments and phylogenetic analysis. Examination of SMR subclass distribution within Bacteria and Archaea taxa identified specific Bacterial classes that uniquely encode for particular SMR subclass members. The extent of selective pressure acting upon each SMR subclass was determined by calculating the rate of synonymous to non-synonymous nucleotide substitutions using Syn-SCAN analysis. SUG and SMP subclasses are maintained under moderate selection pressure in comparison to integron and plasmid encoded SMR homologues. Conversely, PSMR sequences are maintained under lower levels of selection pressure, where one of the two PSMR pairs diverges in sequence more rapidly than the other. SMR genomic loci surveys identified potential SMR efflux substrates based on its gene association to putative operons that encode for genes regulating amino acid biogenesis and QAC-like metabolites. SMR subclass protein transmembrane domain alignments to Bacterial/Archaeal transporters (BAT), DMT, and MFS sequences supports SMR participation in multidrug transport evolution by identifying common TM domains.</p> <p>Conclusion</p> <p>Based on this study, PSMR sequences originated recently within both SUG and SMP clades through gene duplication events and it appears that SMR members may be evolving towards specific metabolite transport.</p

Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

<div><p>Background</p><p>The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence.</p><p>Methodology/Principal Findings</p><p>The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more <i>tatC</i> loci and half of these classes possessed only <i>tatC</i> and <i>tatA</i> genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli <i>tatCd</i>/<i>tatCy</i> isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to <i>tatC</i> loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the <i>Escherichia coli</i> TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial <i>tatC</i> homologues determined that <i>tatC</i> sequence variation differs dramatically between various classes and suggests TatC specialization in these species.</p><p>Conclusions/Significance</p><p>TatC proteins appear to be diversifying within particular bacterial classes and its specialization may be driven by the substrates it transports and the environment of its host.</p></div

The bar plot distribution of <i>tatC</i> sequences in bacterial genomes.

<p>Bacterial species surveyed from 44 different classes are provided on the y-axis and the total number of species genomes surveyed is provided on the x-axis. Bars indicate the total number of surveyed species with one or more <i>tatC</i> sequences (black) and surveyed species that lacked <i>tatC</i> homologues (unfilled) within a given bacterial class. Numbers provided in each bar plot indicate the total number of species having one or more <i>tatC</i> sequence is reported for each bacterial class. Bacterial species with more than one <i>tatC</i> encoded within its genome is summarized on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078742#pone-0078742-t002" target="_blank">Table 2</a>. At the time of the survey (July 2012) a total of 1018 bacterial species from 44 classes were examined and 782 bacterial species genomes in total possessed one or more <i>tatC</i> locus. Data for this distribution is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078742#pone.0078742.s001" target="_blank">Figure S1</a>.</p

Phylogenetic analysis of bacilli TatCd and TatCy proteins and <i>tatC</i> operon conservation.

<p>Bayesian analysis was performed to generate the dendrogram provided on the left hand side of the panel and shows the homology between 57 representative bacilli TatC sequences including characterized <i>B. subtilis</i> TatCd and TatCy seed sequences (underlined). Nodes of importance are indicated by labelled black circles based on its relationship to either TatCd (d1–d3) or TatCy (y1, y2, y3a, y3b- y7). <i>Bacillus</i> species marked by an asterisk ‘Tn’ indicate that its corresponding <i>tatC</i> operon was adjacent to transposons or integron sequences. The arrow and box line diagrams shown on the right hand side of the dendrogram summarize <i>tatC</i> operon conservation for the indicated species. White filled arrows/boxes represent conserved genes located upstream and downstream from <i>tatA</i> (grey arrows) and <i>tatC</i> (black filled arrows). The direction of each arrow represents the open read frame direction relative to <i>tatC</i> and boxes represent genes present in either reading frame. Boxes and arrows with dotted lines indicate genes with moderate conservation 50–80% of <i>tatC</i> loci and solid lines indicate ≥80% gene conservation. The triangle symbol located on the dendrogram points out the division between the TatCd and TatCy clades. The symbol (≠) after <i>L. spaericus</i> indicates that it shares a similar <i>tatC</i> operon arrangement to that shown for sequences in the distal node of y1. Full names for abbreviated genes are listed as follows: alkaline phosphatase (<i>phoD</i>), pyrrolidone-carboxylate peptidase (<i>pcp</i>), domain of unknown function protein (DUF839), lipid esterase A (<i>lipA</i>), hypothetical lipid binding phosphotransferase (<i>degV</i>), domain of unknown function protein (DUF2535), arabinose family transcriptional regulator (<i>araC</i>), dihydrofolate reductase A (<i>dfrA</i>), thymidylate synthase A (<i>thyA</i>), periplasmic binding protein domain 3 (<i>pbp</i>), azoreductase (<i>yvaB</i>), 1-acyl-sn-glycerol 3-phosphate acyltransferase (<i>plsC</i>), 5-formyltetrahydrofolate cyclo ligase (5′FTHF), riboendonuclease liver perchloric acid-soluble protein (L-PSP) (<i>yjgF</i>), molybdenum cofactor biosynthesis protein C (<i>moaC</i>), molybdopterin biosynthesis protein (<i>mogA</i>/<i>moeA</i>), small and large subunit chaperones (<i>groES</i>/groEL), mercury resistant transcriptional regulator (<i>merR</i>), gluconate 2 dehydrogenase subunit 3 (GADH), gluconate 2 dehydrogenase flavoprotein (<i>betA</i>), ABC transporter ATP-binding protein- Elongation factor 3 (<i>uup</i>), redox sensing transcriptional repressor (<i>rex</i>), abortive infection CAAX protease protein (<i>abi</i>), iron dependent DyP-type peroxidase (<i>efeB/ywbN</i>), DUF4305 a 53 amino acid lipoprotein (<i>ydkI</i>), glycoprotein endopeptidase peptidase M22 (<i>yeaZ</i>), transcriptional regulator of sugar metabolism (<i>glpR</i>), high-affinity Fe²⁺/Pb²⁺ permease (FTR1/<i>efeU</i>).</p

A summary of Tat translocase subunits (A/E, B, C) present in each bacterial class surveyed for this study and the most common arrangement of these genes in <i>tat</i> operons.

§<p><i>tat</i> genes are listed as single letters A to C and combinations written together indicate their presence at the same <i>tat</i> operon locus. Bidirectional ↔ arrows indicate that the Tat component genes are not found in the same operon (separate loci). <i>tat</i> genes (A, B or C) found in parentheses indicate an optional occurrence in the <i>tatC</i> locus.</p

Deletion Variants of Neurospora Mitochondrial Porin: Electrophysiological and Spectroscopic Analysis

Mitochondrial porins are predicted to traverse the outer membrane as a series of β-strands, but the precise structure of the resulting β-barrel has remained elusive. Toward determining the positions of the membrane-spanning segments, a series of small deletions was introduced into several of the predicted β-strands of the Neurospora crassa porin. Overall, three classes of porin variants were identified: i), those producing large, stable pores, indicating deletions likely outside of β-strands; ii), those with minimal pore-forming ability, indicating disruptions in key β-strands or β-turns; and iii), those that formed small unstable pores with a variety of gating and ion-selectivity properties. The latter class presumably results from a subset of proteins that adopt an alternative barrel structure upon the loss of stabilizing residues. Some variants were not sufficiently stable in detergent for structural analysis; circular dichroism spectropolarimetry of those that were did not reveal significant differences in the overall structural composition among the detergent-solubilized porin variants and the wild-type protein. Several of the variants displayed altered tryptophan fluorescence profiles, indicative of differing microenvironments surrounding these residues. Based on these results, modifications to the existing models for porin structure are proposed

Phylogenetic analysis of TatC protein sequences from diverse bacterial taxa.

<p>The unrooted phylogenetic tree is based on a Bayesian analysis of 232<i>Vulcanisaeta distributa</i> (AC: YP_003902595.1), is labelled as “<i>V.distributa</i>”. Supporting values at each node and individual labels, genus and species names, of all TatC sequences are not shown in this tree for clarity but are provided in the rooted version of the tree in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078742#pone.0078742.s002" target="_blank">Figure S2</a>. Taxa are described according to their bacterial class, except for classes Verrucomicrobiae and Opitutae (collectively referred to as “Verrucomicrobia phylum”) and classes Chlorobia, Ignavibacteria, Cytophagia, Bacteroidia, Flavobacteria and Sphingobacteria (collectively referred to as “Bacteroidetes/Chlorobi phyla”). Taxa marked with asterisks are detailed as follows: Actinobacteria* includes TatC homologues from class Coriobacteria; Deinococci* includes TatC homologues from classes Deinococci, Thermodesulfobacteria, Nitrospira and Rubrobacteriia; δ-proteobacteria (*) includes TatC homologues from δ-proteobacterial orders Desulfovibrionales and Desulfobacterales; δ-proteobacteria (**) includes TatC homologues from δ-proteobacterial order Myxococcales; δ-proteobacteria (***) includes TatC homologues from δ-proteobacterial order Desulfuromonadales. Symbols adjacent to taxa indicates multiple TatC copies from the same genomes discussed in the Results and Discussion section and the corresponding species are detailed as follows: <i>Cytophaga hutchinsonii</i> (•); <i>Geobacter lovely</i> (••); <i>Colwellia psychrerythraea</i> (•••); <i>Xylanimonas cellulosilytica</i> (x); <i>Streptomyces hygroscopicus</i> (xx); <i>Candidatus Solibacter usitatus</i> (xxx); <i>Nakamurella multipartite</i> (¥); <i>Candidatus Nitrospira defluvii</i> (¥¥); <i>Sulfobacillus acidophilus</i> (Sa); <i>Thermodesulfobium narugense</i> (Tn); <i>Thermodesulfatator indicus</i> (Ti). Labeled nodes are discussed in the Results and Discussion section.</p