Cellular Localization of Predicted Transmembrane and Soluble Chemoreceptors in Sinorhizobium meliloti

Bacterial chemoreceptors primarily locate in clusters at the cell pole, where they form large sensory complexes which recruit cytoplasmic components of the signaling pathway. The genome of the soil bacterium Sinorhizobium meliloti encodes seven transmembrane and two soluble chemoreceptors. We have investigated the localization of all nine chemoreceptors in vivo using genome-encoded fusions to a variant of the enhanced green fluorescent protein and to monomeric red fluorescent protein. Six of the transmembrane (McpT to McpX and McpZ) and both soluble (McpY and IcpA) receptors localize to the cell pole. Only McpS, encoded from the symbiotic plasmid pSymA, is evenly distributed in the cell. While the synthesis of all polar localized receptors is confined to exponential growth correlating with the motility phase of cells, McpS is only weakly expressed throughout cell culture growth. Therefore, motile S. meliloti cells form one major chemotaxis cluster that harbors all chemoreceptors except for McpS. Colocalization and deletion analysis demonstrated that formation of polar foci by the majority of receptors is dependent on other chemoreceptors and that receptor clusters are stabilized by the presence of the chemotaxis proteins CheA and CheW. The transmembrane McpV and the soluble IcpA localize to the pole independently of CheA and CheW. However, in mutant strains McpV formed delocalized polar caps that spread throughout the cell membrane while IcpA exhibited increased bipolarity. Immunoblotting of fractionated cells revealed that IcpA, which lacks any hydrophobic domains, nevertheless is associated to the cell membrane.The chemosensory machinery of Escherichia coli and other bacteria is arranged in large protein clusters (22, 28, 43, 49). One individual signaling unit is formed by a ternary assembly of chemoreceptor dimers, the histidine kinase CheA, and the so-called adaptor protein CheW. E. coli cells contain 20,000 receptor molecules (22). Recent studies suggest that the stoichiometry of such chemosensory complexes is flexible (17, 32). Allosteric interactions among receptors in a chemosensory cluster facilitate amplification and integration of chemotactic stimuli (20, 21, 41, 42).In contrast to E. coli, which has a single set of che genes and only five receptors, some species from the alpha subgroup of the proteobacteria, such as Pseudomonas aeruginosa, Rhodobacter sphaeroides, and Sinorhizobium meliloti, encode multiple chemotaxis-like systems, reflecting their complex lifestyle. The opportunistic pathogen P. aeruginosa possesses four chemotaxis systems that together have 26 known receptor genes (47), while the nonsulfur bacterium R. sphaeroides has three separate che operons with 13 known receptor-like genes (27).The symbiotic soil bacterium S. meliloti possesses eight methyl-accepting chemotaxis proteins (MCPs), McpS to McpZ, and one transducer-like-protein, IcpA, which lacks the conserved Glu or Gln residues that serve as methyl-accepting sites (29). Seven of the MCP proteins are localized in the cytoplasmic membrane via two membrane-spanning regions, whereas McpY and IcpA lack such hydrophobic regions. The S. meliloti mcpS gene is the third gene of the che2 operon located on the symbiotic plasmid pSymA (4). The icpA gene is the first gene of the chromosomal che operon comprising a total of 10 genes (9). This operon is part of the flagellar gene cluster with 56 chemotaxis, motor, and flagellar genes residing on one contiguous 51.4-kb chromosomal region (7, 46). For bacteria with numerous chemoreceptor genes, it is not unusual to find most of them located outside chemotaxis operons. This is the case with six monocistronic S. meliloti mcp genes which are scattered throughout the genome. The remaining mcpW gene is cotranscribed with a putative cheW gene. In this study, we examined the localization of the nine receptor gene products in the S. meliloti cell by fluorescence microscopy in wild-type and various deletion strains. The cellular localization of the two soluble receptors, McpY and IcpA, was also analyzed in vitro using an immunoblot assay on fractionated cell components. Furthermore, timing of chemoreceptor gene expression during exponential and stationary phase was determined.     

BacA Is Essential for Bacteroid Development in Nodules of Galegoid,but not Phaseoloid,Legumes

BacA is an integral membrane protein, the mutation of which leads to increased resistance to the antimicrobial peptides bleomycin and Bac7_1-35 and a greater sensitivity to SDS and vancomycin in Rhizobium leguminosarum bv. viciae, R. leguminosarum bv. phaseoli, and Rhizobium etli. The growth of Rhizobium strains on dicarboxylates as a sole carbon source was impaired in bacA mutants but was overcome by elevating the calcium level. While bacA mutants elicited indeterminate nodule formation on peas, which belong to the galegoid tribe of legumes, bacteria lysed after release from infection threads and mature bacteroids were not formed. Microarray analysis revealed almost no change in a bacA mutant of R. leguminosarum bv. viciae in free-living culture. In contrast, 45 genes were more-than 3-fold upregulated in a bacA mutant isolated from pea nodules. Almost half of these genes code for cell membrane components, suggesting that BacA is crucial to alterations that occur in the cell envelope during bacteroid development. In stark contrast, bacA mutants of R. leguminosarum bv. phaseoli and R. etli elicited the formation of normal determinate nodules on their bean host, which belongs to the phaseoloid tribe of legumes. Bacteroids from these nodules were indistinguishable from the wild type in morphology and nitrogen fixation. Thus, while bacA mutants of bacteria that infect galegoid or phaseoloid legumes have similar phenotypes in free-living culture, BacA is essential only for bacteroid development in indeterminate galegoid nodules.Bacteria of the family Rhizobiaceae are alphaproteobacteria, which form a species-specific symbiotic relationship with leguminous plants. Plants release flavonoids that typically induce the synthesis of lipochitooligosaccharides by rhizobia, which in turn initiate a signaling cascade in the plant, leading to nodule formation (34). Rhizobia become trapped by curling root hairs, which they enter via infection threads that grow and ramify into the root cortex, where newly induced meristematic cells form the nodule (34). Bacteria are released from infection threads and engulfed by a plant-derived symbiosome membrane. In galegoid legumes (a clade in the subfamily Papilionoideae, such as Medicago, Pisum, or Vicia), which form indeterminate nodules that have a persistent meristem, bacteria undergo the endoreduplication of their chromosome, resulting in dramatic increases in size, shape, and DNA content to become terminally differentiated bacteroids (32). However, in phaseoloid legumes (e.g., lotus, bean, and soybean), which form determinate nodules with a transient meristem, bacteria do not undergo endoreduplication and therefore do not enlarge substantially. These bacteroids retain a normal DNA content and can regrow after isolation from nodules (32). The endoreduplication of bacteroids is controlled by the plant, and it is believed that nodule-specific cysteine-rich (NCR) peptides, which are made in indeterminate, but not in determinate, nodules, may be responsible for inducing and maintaining bacteroid development (31, 32). Finally, mature bacteroids receive dicarboxylic acids from the plant, which they use as a carbon, reductant, and energy source for the reduction of N₂ to ammonia (38). The ammonia is secreted to the plant, where it is assimilated into amino acids or ureides, depending on the legume, for export to the shoot.Sinorhizobium meliloti BacA protein was the first bacterial factor identified to be essential for bacteroid development (15). More recently, it also has been shown to be essential for the Mesorhizobium-Astragalus symbiosis (42). S. meliloti elicits the formation of indeterminate nodules on alfalfa, and while S. meliloti bacA null mutants induce nodule formation, bacteria lyse soon after endocytosis but prior to bacteroid differentiation (15, 20). BacA is a cytoplasmic membrane protein that shares 64% identity with SbmA from Escherichia coli (15, 25). SbmA/BacA proteins belong to the ATP binding cassette (ABC) superfamily and share sequence similarity with a family of eukaryotic peroxisomal membrane proteins, including the human adrenoleukodystrophy protein, which is required for the efficient transport of very-long-chain fatty acids (VLCFAs) out of the cytoplasm (9). Consistent with this, S. meliloti BacA is required for the complete modification of lipid A with VLCFAs (9). However, since S. meliloti mutants, which are directly involved in the biosynthesis of VLCFA-modified lipid A, show bacteroid abnormalities but still can form a successful alfalfa symbiosis, the effect of BacA on lipid A VLCFA modification does not fully account for its essential role in bacteroid development (10, 11, 16). Strains mutated in bacA also have an increased resistance to the glycopeptide bleomycin, a low-level resistance to aminoglycoside antibiotics, and an increased sensitivity to ethanol, sodium dodecyl sulfate (SDS), and deoxycholate relative to the sensitivities of the parent strain (12, 18, 25). More recently it has been shown that an S. meliloti bacA null mutant has an increased resistance to a truncated form of a eukaryotic proline-rich peptide, Bac7_1-16, and was unable to accumulate a fluorescently labeled form of this peptide (28). This finding, combined with the increased resistance of an S. meliloti bacA null mutant to bleomycin, led to the hypothesis that BacA is itself a putative peptide transporter (BacA mediated) or able to alter the activity of such a transporter (BacA influenced) (11, 15, 18, 28).As the increased resistance of the S. meliloti bacA null mutant to bleomycin and Bac7_1-16 appears to be independent of the VLCFA modification of lipid A (11, 28), this suggested that either BacA-mediated or BacA-influenced peptide uptake into S. meliloti plays a role in bacteroid development. Since indeterminate galegoid nodules contain hundreds of NCR peptides, whereas determinate phaseoloid nodules lack these host peptides (31), we considered it important to assess the role of BacA in bacteroid development during the formation of both nodule types.Here, we show that bacA mutants of Rhizobium leguminosarum bv. viciae strains 3841 and A34 failed to develop bacteroids and did not fix nitrogen in indeterminate pea (Pisum sativum) nodules. However, bacA mutants of both R. leguminosarum bv. phaseoli 4292 and Rhizobium etli CE3 formed normal bacteroids and fixed nitrogen at wild-type rates in determinate bean (Phaseolus vulgaris) nodules. This is consistent with BacA being a key component of bacteroid development in indeterminate galegoid nodules that is not required for functional bacteroid formation in determinate phaseoloid nodules.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.     

The Twin Arginine Transport System Appears To Be Essential for Viability in Sinorhizobium meliloti

The twin arginine transport (Tat) system is responsible for transporting prefolded proteins to the periplasmic space. The Tat pathway has been implicated in many bacterial cellular functions, including motility, biofilm formation, and pathogenesis and symbiosis. Since the annotation of Sinorhizobium meliloti Rm1021 genome suggests that there may be up to 94 putative Tat substrates, we hypothesized that characterizing the twin arginine transport system in this organism might yield unique data that could help in the understanding of twin arginine transport. To initiate this work we attempted a targeted mutagenesis of the tat locus. Despite repeated attempts using a number of different types of media, the attempts at mutation construction were unsuccessful unless the experiment was carried out in a strain that was merodiploid for tatABC. In addition, it was shown that a plasmid carrying tatABC was stable in the absence of antibiotic selection in a tat deletion background. Finally, fluorescence microscopy and live/dead assays of these cultures show a high proportion of dead and irregularly shaped cells, suggesting that the loss of tatABC is inversely correlated with viability. Taken together, the results of this work provide evidence that the twin arginine transport system of S. meliloti appears to be essential for viability under all the conditions that we had tested.Sinorhizobium meliloti is a Gram-negative alphaproteobacterium capable of entering into a symbiotic relationship with leguminous plants such as alfalfa. Within the rhizosphere, rhizobia are capable of sensing flavones or isoflavones secreted by the host plant (4, 46, 57). In response, a cascade of events ensues that leads to the eventual attachment of the bacteria to the plant root, infection thread development, and finally release of the bacteria within the differentiated plant cells of the developing nodule structure (34, 45). It is within this tightly regulated environment that the rhizobia express the genes that encode the proteins required for nitrogen fixation and that result in the reduction of atmospheric N₂ to NH_4. In exchange for the production of nitrogen, the plant provides nutrients for the bacteria to grow and to establish the symbiotic relationship (33, 50).Protein targeting and translocation are important processes for correct cellular function within all living organisms. It is predicted in Escherichia coli that more than 450 proteins are transported across the cytoplasmic membrane (43). The vast majority of these proteins are transported through the general secretory (Sec) system, with a minority being transported by the more recently discovered twin arginine transport (Tat) pathway (43). Proteins that are targeted to the cytoplasmic membrane in Gram-negative bacteria via the Sec system rely on a core set of proteins that include SecA, a protein that has ATPase function, SecYEG, which define the minimum membrane transport apparatus, and in some cases a chaperone protein, SecB (18, 54). The translated protein is carried toward the membrane with help from the chaperone SecB and relayed to the SecYEG apparatus that threads the proteins through the membrane in a linear fashion, with the energy for transport being derived from the hydrolysis of ATP, which is provided by SecA (18).In contrast, the Tat system is believed to transport proteins that have already undergone folding and, in many cases, cofactor insertion (41, 60). In brief, following protein translation, a chaperone may be involved to help transfer the substrate to the TatBC complex, where the TatC component recognizes the twin arginine signal motif, (S/T)RRXFLK (1, 42). The TatBC complex subsequently recruits TatA oligomers that coordinately make up the membrane pore required for transport (8, 29, 31). Using the pH gradient (ΔpH), the Tat substrate protein is transported through the TatA pore in its folded state and integrated into the membrane or transported further to the periplasmic space (3, 39).Approximately 30 proteins are predicted to be transported through the Tat system in E. coli (43). The majority of these appear to be expressed or function anaerobically (43). Interestingly, bioinformatic analysis of S. meliloti and Rhizobium leguminosarum suggests that a much larger number of proteins may use the Tat system in these organisms (36). In addition, these organisms are classified as obligate aerobic organisms (12, 28, 55).Since tat mutations have been shown to affect many bacteria-host interactions (17, 25, 36, 49, 62), we set out to construct a tat mutation in S. meliloti to elucidate the role that tat may have in determining the bacteria''s ability to interact with its host plant and affect nodule development. Moreover, we reasoned that a tat mutation in S. meliloti might help characterize putative Tat substrates in a different model organism. Surprisingly, we were able to construct a tat mutation only in a merodiploid strain that contained the tatABC genes on a plasmid in trans. Using plasmid stability, transduction experiments, and live/dead assays, we show that the tat region in S. meliloti appears to be required for viability and is an essential region of the chromosome. This is the first work to show that Tat is required for viability in a bacterial species.     

Contribution of Lipoproteins and Lipoprotein Processing to Endocarditis Virulence in Streptococcus sanguinis

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (lgt) and signal peptidase II (lspA). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt, lspA, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [³H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.Streptococcus sanguinis is a member of the viridans group of streptococci and is a primary colonizer of teeth (8). The viridans species and, in particular, S. sanguinis (15, 18) are a leading cause of infective endocarditis, a serious infection of the valves or lining of the heart (48). Damage to the heart resulting from rheumatic fever or certain congenital heart defects dramatically increases the risk of developing endocarditis (48, 71). The damage is thought to result in the formation of sterile cardiac “vegetations” composed of platelets and fibrin (48) that can be colonized by certain bacteria during periods of bacteremia. This view is supported by animal studies in which formation of sterile vegetation by cardiac catheterization is required for the efficient establishment of streptococcal endocarditis (17). Prevention of infective endocarditis currently relies upon prophylactic administration of antibiotics prior to dental or other surgical procedures that are likely to produce bacteremia. The growing realization that oral bacteria such as S. sanguinis can enter the bloodstream through routine daily activities such as eating has led the American Heart Association (71) and others (57) to question the value of using antibiotic prophylaxis for dental procedures. Clearly, a better understanding of the bacterial virulence factors that contribute to endocarditis could lead to better preventive measures, such as a vaccine that could potentially afford continuous protection to high-risk patients (71).In a previous study, we used the signature-tagged mutagenesis (STM) technique to search for endocarditis virulence factors of S. sanguinis in a rabbit model (53). This study identified a number of housekeeping enzymes that contribute to endocarditis. Because these proteins are not likely to be surface localized, they hold little promise as vaccine candidates. One class of streptococcal surface proteins that is rich in both virulence factors (4, 7, 25, 33, 38, 60) and promising vaccine candidates (6, 39, 42, 51, 70) is the lipoproteins. Lipoprotein activities that have been suggested to contribute to streptococcal virulence include adhesion (4, 7, 63), posttranslational modification (25, 29, 51), and ATP-binding cassette (ABC)-mediated transport (33, 52, 60). In the last instance, lipoproteins anchored to the cell membrane by their lipid tails appear to serve the same transport function as the periplasmic substrate-binding proteins of gram-negative bacteria (66). STM studies performed with Streptococcus pneumoniae (26, 41, 55) and Streptococcus agalactiae (34) have identified multiple lipoprotein mutants among collections of reduced virulence mutants. In an attempt to determine the cumulative contribution of streptococcal lipoproteins to virulence, some investigators have created mutations in the lgt or lspA genes, encoding lipoprotein-processing enzymes (12, 25, 27, 36). The lgt gene encodes prolipoprotein diacylglyceryl transferase, which catalyzes the transfer of a diacylglycerol lipid unit to a cysteine in the conserved N-terminal “lipobox” of lipoproteins, while lspA encodes the signal peptidase II enzyme that cleaves the signal peptide of the prolipoprotein just prior to the conserved cysteine (59, 65). While mutation of these genes has been shown to be lethal in gram-negative bacteria (21, 73), many gram-positive bacterial species have been shown to tolerate such mutations, often with only minor effects on growth (3, 12, 13, 25, 27, 36, 54). Some of these studies indicated a deleterious effect on the virulence of the lgt (25, 54) or lspA (36) mutation, but others found no effect (12) or an enhancement of virulence (27). It is clear from these and other studies (3, 13) that neither the loss of acylation due to lgt inactivation nor the loss of signal peptidase II-mediated cleavage completely eliminates lipoprotein function, necessitating alternative approaches for assessing the global contribution of lipoproteins to virulence.We have used bioinformatic approaches to identify every putative lipoprotein encoded by S. sanguinis strain SK36. To determine the contribution of these lipoproteins to the endocarditis virulence of S. sanguinis, we have systematically mutagenized each of these genes, as well as the lgt and lspA genes, and evaluated these mutants for virulence by using STM in an animal model. Selected mutants were further examined for virulence in competitive index (CI) assays. A strain with a disrupted ssaB gene, which encodes a putative metal transport protein, was found to exhibit a profound defect in virulence that was far greater than that of any other strain tested, including the lgt or lspA mutant.     

Polymorphic Mutation Frequencies of Clinical and Environmental Stenotrophomonas maltophilia Populations

Mutation frequencies were studied in 174 Stenotrophomonas maltophilia isolates from clinical and nonclinical environments by detecting spontaneous rifampin-resistant mutants in otherwise-susceptible populations. The distribution of mutation frequencies followed a pattern similar to that found for other bacterial species, with a modal value of 1 × 10⁻⁸. Nevertheless, the proportion of isolates showing mutation frequencies below the modal value (hypomutators) was significantly higher for S. maltophilia than those so far reported in other organisms. Low mutation frequencies were particularly frequent among environmental S. maltophilia strains (58.3%), whereas strong mutators were found only among isolates with a clinical origin. These results indicate that clinical environments might select bacterial populations with high mutation frequencies, likely by second-order selection processes. In several of the strong-mutator isolates, functional-complementation assays with a wild-type allele of the mutS gene demonstrated that the mutator phenotype was due to the impairment of MutS activity. In silico analysis of the amino acid changes present in the MutS proteins of these hypermutator strains in comparison with the normomutator isolates suggests that the cause of the defect in MutS might be a H683P amino acid change.Stenotrophomonas maltophilia is a Gram-negative, nonfermenting environmental bacterial species often isolated from the rhizosphere and from water sources (11, 12, 63). Some S. maltophilia strains have been used for bioremediation (13, 24, 73) or bioaugmentation (37). However, besides its environmental origin and potential relevance for biotechnological purposes, S. maltophilia is also a relevant human opportunistic pathogen (44) associated with a broad spectrum of clinical syndromes, such as bacteremia (79, 81), endocarditis (18), infection in cancer patients (1), and respiratory tract infections, including those suffered by cystic fibrosis (CF) patients (72, 77). One of the most problematic characteristics of S. maltophilia is its intrinsic high resistance to several antibiotics (4). This intrinsic antibiotic resistance is at least partly due to the presence in the genome of S. maltophilia (17) of genes encoding antibiotic-inactivating enzymes (6, 9, 30, 39, 42, 58) and multidrug resistance (MDR) efflux pumps (2, 3, 43, 78). More recently, a chromosomally encoded Qnr protein that contributes to the intrinsic resistance to quinolones of S. maltophilia has been described (67, 68).A clear difference between infective (clinical) and environmental (nonclinical) S. maltophilia strains has not been reported (12, 63). However, although the available data fit the concept that opportunistic pathogens have not specifically evolved to infect humans (48), this does not mean that they do not evolve during the infective process. For most acute infections, we can presume that the time of in-host evolution is probably too short to detect relevant adaptive changes. Nevertheless, the situation might be different in chronic infections, such as those involving the bronchial compartment in CF patients. In this case, the same bacterial clone can be maintained and grow inside the host for years (62). This produces strong diversification over time and in different compartments of the lung (25, 71, 80), a process in which the acquisition of a mutator phenotype is important (52). Thus, isolates derived from an initial clone but presenting different morphotypes (47), different phenotypes of susceptibility to antibiotics (26) or in the expression of virulence determinants (14, 15, 36), or with different mutation frequencies (49, 60) are recovered from each individual patient suffering chronic infections. More recently, intraclonal diversification has also been described for Pseudomonas aeruginosa causing acute infections in intubated patients (38). Taken together, this indicates that bacteria can evolve during infection.For different bacterial species, strains isolated from CF patients with chronic lung infections show high mutation frequencies (hypermutable strains) (19, 60, 61, 66), whereas hypermutators have rarely been found in isolates from acute infections (33). An explanation for this difference could be that hypermutable strains tend to be selected for in the highly compartmentalized environment of the infected lung by intensive antibiotic therapy, as well as by the stressful conditions of the habitat. This is a second-order selection process (75, 76), in which mutations are selected because they confer an advantage in clinical environments in such a way that mutator strains are selected because they can produce more mutants (both advantageous and deleterious) for selection. In cases of chronic infections that are treated, strong and maintained selective local processes might occur, either by antibiotic treatment or by the actions of the anti-infective systems of the host. Natural out-of-host open environments obviously might have local stresses. However, the intensity of selection is expected to be lower in these habitats, and a constant replacement of potentially lost organisms by migration of neighbor populations probably mitigates the local selection of mutators and favors the enrichment of bacteria presenting low mutation frequencies. In the case of chronic infections, the replacement of mutators by neighbor normomutators is unlikely, because those infections are produced by a single clone that remains for several years in the host (62). Furthermore, although the infection process presents strong evolutionary bottlenecks for bacterial populations, the human host also provides a constant temperature, reliable nutrient supplies, and a habitat largely free from predators and competitors. Thus, while hypermutation might increase the capability of bacteria to adapt to some specific challenges in the clinical environment, the cost of hypermutation in terms of deleterious mutations might also be diminished, and these effects might be mutually reinforcing.The hypothesis explored in this paper is that S. maltophilia is adapted to deal with out-of-host fluctuating environmental variations but that once the organism enters a patient as an opportunistic pathogen, its adaptive needs significantly increase due to the actions of stressful local environmental conditions, such as the immune response and, when present, antibiotics. This enhanced stress under infective conditions might result in the selection of variants with increased mutation frequencies in a second-order selection process (75, 76). To test this hypothesis, the mutation frequencies of S. maltophilia clinical isolates (obtained from CF and non-CF patients) and from the environment (nonclinical origin) were compared. Most works that have been published on the different mutation frequencies in bacterial populations have focused on the detection of strains showing a high mutation frequency (mutators). In our work, we describe for the first time the presence of mutators in clinical isolates of S. maltophilia and demonstrate that hypermutation in several of those isolates is due to defects in MutS.Nevertheless, our main goal has been the analysis of the global distribution of mutation frequencies in an ample number of samples from clinical and nonclinical environments. Our results indicate not only that mutators are more frequent in clinical S. maltophilia isolates, but also that the overall distribution of mutation frequencies is different in S. maltophilia populations with environmental or clinical origins, with a tendency toward mutation frequencies lower than the modal mutation value (hypomutators) in the environmental isolates.     

Characterization of Lassa Virus Glycoprotein Oligomerization and Influence of Cholesterol on Virus Replication

Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.Lassa virus (LASV) is a member of the family Arenaviridae, of which Lymphocytic choriomeningitis virus (LCMV) is the prototype. Arenaviruses comprise more than 20 species, divided into the Old World and New World virus complexes (19). The Old World arenaviruses include the human pathogenic LASV strains, Lujo virus, which was first identified in late 2008 and is associated with an unprecedented high case fatality rate in humans, the nonhuman pathogenic Ippy, Mobala, and Mopeia viruses, and the recently described Kodoko virus (10, 30, 49). The New World virus complex contains, among others, the South American hemorrhagic fever-causing viruses Junín virus, Machupo virus, Guanarito virus, Sabiá virus, and the recently discovered Chapare virus (22).Arenaviruses contain a bisegmented single-stranded RNA genome encoding the polymerase L, matrix protein Z, nucleoprotein NP, and glycoprotein GP. The bipartite ribonucleoprotein of LASV is surrounded by a lipid envelope derived from the plasma membrane of the host cell. The matrix protein Z has been identified as a major budding factor, which lines the interior of the viral lipid membrane, in which GP spikes are inserted (61, 75). The glycoprotein is synthesized as precursor protein pre-GP-C and is cotranslationally cleaved by signal peptidase into GP-C and the signal peptide, which exhibits unusual length, stability, and topology (3, 27, 28, 33, 70, 87). Moreover, the arenaviral signal peptide functions as trans-acting maturation factor (2, 26, 33). After processing by signal peptidase, GP-C of both New World and Old World arenaviruses is cleaved by the cellular subtilase subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) into the distal subunit GP-1 and the membrane-anchored subunit GP-2 within the secretory pathway (5, 52, 63). For LCMV, it has been shown that GP-1 subunits are linked to each other by disulfide bonds and are noncovalently connected to GP-2 subunits (14, 24, 31). GP-1 is responsible for binding to the host cell receptor, while GP-2 mediates fusion between the virus envelope and the endosomal membrane at low pH due to a bipartite fusion peptide near the amino terminus (24, 36, 44). Sequence analysis of the LCMV GP-2 ectodomain revealed two heptad repeats that most likely form amphipathic helices important for this process (34, 86).In general, viral class I fusion proteins have triplets of α-helical structures in common, which contain heptad repeats (47, 73). In contrast, class II fusion proteins are characterized by β-sheets that form dimers in the prefusion status and trimers in the postfusion status (43). The class III fusion proteins are trimers that, unlike class I fusion proteins, were not proteolytically processed N-terminally of the fusion peptide, resulting in a fusion-active membrane-anchored subunit (39, 62). Previous studies with LCMV described a tetrameric organization of the glycoprotein spikes (14), while more recent data using a bacterially expressed truncated ectodomain of the LCMV GP-2 subunit pointed toward a trimeric spike structure (31). Due to these conflicting data regarding the oligomerization status of LCMV GP, it remains unclear to which class of fusion proteins the arenaviral glycoproteins belong.The state of oligomerization and the correct conformation of viral glycoproteins are crucial for membrane fusion during virus entry. The early steps of infection have been shown for several viruses to be dependent on the cholesterol content of the participating membranes (i.e., either the virus envelope or the host cell membrane) (4, 9, 15, 20, 21, 23, 40, 42, 53, 56, 76, 78, 79). In fact, it has been shown previously that entry of both LASV and LCMV is susceptible to cholesterol depletion of the target host cell membrane using methyl-β-cyclodextrin (MβCD) treatment (64, 71). Moreover, cholesterol not only plays an important role in the early steps during entry in the viral life cycle but also is critical in the virus assembly and release process. Several viruses of various families, including influenza virus, human immunodeficiency virus type 1 (HIV-1), measles virus, and Ebola virus, use the ordered environment of lipid raft microdomains. Due to their high levels of glycosphingolipids and cholesterol, these domains are characterized by insolubility in nonionic detergents under cold conditions (60, 72). Recent observations have suggested that budding of the New World arenavirus Junin virus occurs from detergent-soluble membrane areas (1). Assembly and release from distinct membrane microdomains that are detergent soluble have also been described for vesicular stomatitis virus (VSV) (12, 38, 68). At present, however, it is not known whether LASV requires cholesterol in its viral envelope for successful virus entry or whether specific membrane microdomains are important for LASV assembly and release.In this study, we first investigated the oligomeric state of the premature and mature LASV glycoprotein complexes. Since it has been shown for several membrane proteins that the oligomerization and conformation are dependent on cholesterol (58, 59, 76, 78), we further analyzed the dependence of the cholesterol content of the virus envelope on glycoprotein oligomerization and virus infectivity. Finally, we characterized the lipid membrane areas from which LASV is released.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

The Rhodococcus opacus PD630 Heparin-Binding Hemagglutinin Homolog TadA Mediates Lipid Body Formation

Generally, prokaryotes store carbon as polyhydroxyalkanoate, starch, or glycogen. The Gram-positive actinomycete Rhodococcus opacus strain PD630 is noteworthy in that it stores carbon in the form of triacylglycerol (TAG). Several studies have demonstrated that R. opacus PD630 can accumulate up to 76% of its cell dry weight as TAG when grown under nitrogen-limiting conditions. While this process is well studied, the underlying molecular and biochemical mechanisms leading to TAG biosynthesis and subsequent storage are poorly understood. We designed a high-throughput genetic screening to identify genes and their products required for TAG biosynthesis and storage in R. opacus PD630. We identified a gene predicted to encode a putative heparin-binding hemagglutinin homolog, which we have termed tadA (triacylglycerol accumulation deficient), as being important for TAG accumulation. Kinetic studies of TAG accumulation in both the wild-type (WT) and mutant strains demonstrated that the tadA mutant accumulates 30 to 40% less TAG than the parental strain (WT). We observed that lipid bodies formed by the mutant strain were of a different size and shape than those of the WT. Characterization of TadA demonstrated that the protein is capable of binding heparin and of agglutinating purified lipid bodies. Finally, we observed that the TadA protein localizes to lipid bodies in R. opacus PD630 both in vivo and in vitro. Based on these data, we hypothesize that the TadA protein acts to aggregate small lipid bodies, found in cells during early stages of lipid storage, into larger lipid bodies and thus plays a key role in lipid body maturation in R. opacus PD630.While the majority of eubacteria (24, 33), and indeed many archaea (22, 33), store carbon as polyhydroxyalkanoate (PHA), a small subset of organisms, primarily actinomycetes, are capable of storing carbon in the form of triacylglycerol (TAG). TAG biosynthesis and storage has been observed in members of the genera Mycobacterium, Rhodococcus, Streptomyces, Nocardia, and others (4, 6, 11, 12, 19, 20, 36). Of these organisms, TAG biosynthesis and storage has been most extensively studied for the Gram-positive, non-spore-forming actinomycete Rhodococcus opacus, strain PD630 (1-6, 11, 12, 19, 20, 25, 36, 38-41).Several studies have demonstrated that R. opacus PD630 is capable of accumulating up to 76% of its cell dry weight (CDW) as TAG (summarized in reference 3). As is the case for PHA biosynthesis, TAG accumulation occurs during nitrogen starvation when carbon is in excess (1-3, 27, 41). Paralleling PHA biosynthesis further, TAG is stored in R. opacus PD630 in distinct inclusion bodies, termed lipid bodies (2, 3, 25, 38, 40). While several studies have sought to identify the underlying molecular and biochemical mechanisms behind TAG biosynthesis and storage in the form of lipid bodies, very little is known concerning these processes.We sought to identify genes and their products that are essential for lipid metabolism in R. opacus PD630. Utilizing a forward genetic approach, we identified a conserved hypothetical gene, termed herein tadA (triacylglycerol accumulation deficient), which is predicted to encode a protein with sequence similarity to the heparin-binding hemagglutinin (HbhA) family of proteins from the genus Mycobacterium. The tadA::Tn5 mutant accumulates 30 to 40% less TAG than the parental strain. We demonstrate that this deficiency is most likely the result of altered lipid body formation and morphology. Through biochemical studies, we further demonstrate that the predicted heparin-binding activity of this protein is essential for its activity both in vivo and in vitro. To our knowledge, this is the first protein shown to regulate lipid body assembly and maturation in prokaryotes.