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).     

Host-Interactive Genes in Amerindian Helicobacter pylori Diverge from Their Old World Homologs and Mediate Inflammatory Responses

Helicobacter pylori is the dominant member of the gastric microbiota and has been associated with an increased risk of gastric cancer and peptic ulcers in adults. H. pylori populations have migrated and diverged with human populations, and health effects vary. Here, we describe the whole genome of the cag-positive strain V225d, cultured from a Venezuelan Piaroa Amerindian subject. To gain insight into the evolution and host adaptation of this bacterium, we undertook comparative H. pylori genomic analyses. A robust multiprotein phylogenetic tree reflects the major human migration out of Africa, across Europe, through Asia, and into the New World, placing Amerindian H. pylori as a particularly close sister group to East Asian H. pylori. In contrast, phylogenetic analysis of the host-interactive genes vacA and cagA shows substantial divergence of Amerindian from Old World forms and indicates new genotypes (e.g., VacA m3) involving these loci. Despite deletions in CagA EPIYA and CRPIA domains, V225d stimulates interleukin-8 secretion and the hummingbird phenotype in AGS cells. However, following a 33-week passage in the mouse stomach, these phenotypes were lost in isolate V225-RE, which had a 15-kb deletion in the cag pathogenicity island that truncated CagA and eliminated some of the type IV secretion system genes. Thus, the unusual V225d cag architecture was fully functional via conserved elements, but the natural deletion of 13 cag pathogenicity island genes and the truncation of CagA impaired the ability to induce inflammation.Helicobacter pylori is a microaerophilic bacterium of the Epsilonproteobacteria that has colonized the stomach since early in human evolution (45) and diverged with ancient human migrations (24, 45, 92). Thus, several major H. pylori populations, such as hpAfrica1, hpEurope, hspEAsia, and hspAmerind, whose names indicate their original geographic associations (45, 51), have been defined. In particular, similarities between the hspAmerind and hspEAsia populations suggest that the first colonizers of the New World brought H. pylori with them (24, 28). With recent mixing of human groups, H. pylori populations are also mixing and competing, with an apparent dominance by the hpEurope population at least in Latin America (19).H. pylori usually does not cause illness, but colonization with strains bearing the cag (cytotoxin-associated gene) pathogenicity island (cag PAI) (3, 7, 25, 52, 57, 61, 63) is associated with an increased risk of noncardia gastric adenocarcinoma and peptic ulcer disease (56, 64). Nonetheless, a high prevalence of cag-positive H. pylori strains occurs concurrently with low gastric cancer rates in Africa (40) and some regions in Latin America, such as the Venezuelan savannas and Amazonas (29, 53). Moreover, clinical and epidemiological data provide evidence for an inverse relationship between H. pylori colonization and the prevalence of certain metabolic disorders, esophageal diseases, asthma and allergic disorders, and acute infectious diseases, as well as a direct relationship with improved nutritional status of rural children (3, 14, 34, 37, 49, 68). That the host interaction with an indigenous gastric microbe provides some health benefits to the host is not unexpected given the well-established role of gastrointestinal microflora in maintaining gastroenteric homeostasis (8).The most thoroughly studied H. pylori proteins that interact with human cells are CagA and VacA. CagA is an effector protein injected into gastric epithelial cells by a type IV secretion system encoded by the cag PAI (10, 12, 15, 83). VacA is initially secreted from the bacterial cell by an autotransporter mechanism (16). Both proteins have multiple effects on host cells. Inside the host cell, phosphorylation of CagA on EPIYA repeats in the phosphotyrosine (PY) region (73) induces cellular elongation known as the hummingbird phenotype (72). CagA may also induce secretion of interleukin-8 (IL-8) (11), a process commonly attributed to NF-κB, and disrupt the barrier function of the tight junctions in polarized epithelial cells, leading to a loss of adhesion (1, 5). Other motifs in the PY region promote phosphorylation-independent effects (79). In addition, cagA may be considered an oncogene (60), since transgenic expression of cagA in mice leads to gastric epithelial hyperplasia through aberrant epithelial cell signaling and gastric carcinogenesis (60, 62). In contrast, VacA is a multifunctional protein with several activities in epithelial and immune cells (16). VacA induces cell vacuolation (43), alters mitochondrial membrane permeability (27, 41, 90), and increases epithelial monolayer permeability. VacA also activates several signal transduction pathways that are important in immune and epithelial cells, including the mitogen-activated protein (MAP) kinase and p38/ATF-2-mediated signal pathways (9, 55).Genomic analysis provides insights into the evolution of H. pylori strains and their relation with their human hosts and may be useful for the development of diagnostic tools and novel therapies. To date, there are six published complete H. pylori genomes, mostly from the hpEurope population (see Table SA1 in the supplemental material). Here, we report the whole genome of a newly characterized hspAmerind strain, V225d, and assess its genetic structure in comparison to those of Old World H. pylori strains through a comprehensive multiprotein phylogenetic analysis, as well as through single-gene examination of cagA and vacA, revealing clues to the evolution and migration of this strain into the New World and the implications for human health. We also present the results of functional and genomic studies using gastric epithelial cells demonstrating that V225d can induce an inflammatory host response, an effect that was lost following passage through the mouse stomach.     

Abundance of Novel and Diverse tfdA-Like Genes,Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases,in Soil

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAα). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 × 10⁶ to 65 × 10⁶ per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.Phenoxyalkanoic acid (PAA) herbicides such as MCPA (4-chloro-2-methyl-phenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are widely used to control broad-leaf weeds in agricultural as well as nonagricultural areas (19, 77). Degradation occurs primarily under oxic conditions in soil, and microorganisms play a key role in the degradation of such herbicides in soil (62, 64). Although relatively rapidly degraded in soil (32, 45), both MCPA and 2,4-D are potential groundwater contaminants (10, 56, 70), accentuating the importance of bacterial PAA herbicide-degrading bacteria in soils (e.g., references 3, 5, 6, 20, 41, 59, and 78).Degradation can occur cometabolically or be associated with energy conservation (15, 54). The first step in the degradation of 2,4-D and MCPA is initiated by the product of cadAB or tfdA-like genes (29, 30, 35, 67), which constitutes an α-ketoglutarate (α-KG)- and Fe²⁺-dependent dioxygenase. TfdA removes the acetate side chain of 2,4-D and MCPA to produce 2,4-dichlorophenol and 4-chloro-2-methylphenol, respectively, and glyoxylate while oxidizing α-ketoglutarate to CO₂ and succinate (16, 17).Organisms capable of PAA herbicide degradation are phylogenetically diverse and belong to the Alpha-, Beta-, and Gammproteobacteria and the Bacteroidetes/Chlorobi group (e.g., references 2, 14, 29-34, 39, 60, 68, and 71). These bacteria harbor tfdA-like genes (i.e., tfdA or tfdAα) and are categorized into three groups on an evolutionary and physiological basis (34). The first group consists of beta- and gammaproteobacteria and can be further divided into three distinct classes based on their tfdA genes (30, 46). Class I tfdA genes are closely related to those of Cupriavidus necator JMP134 (formerly Ralstonia eutropha). Class II tfdA genes consist of those of Burkholderia sp. strain RASC and a few strains that are 76% identical to class I tfdA genes. Class III tfdA genes are 77% identical to class I and 80% identical to class II tfdA genes and linked to MCPA degradation in soil (3). The second group consists of alphaproteobacteria, which are closely related to Bradyrhizobium spp. with tfdAα genes having 60% identity to tfdA of group 1 (18, 29, 34). The third group also harbors the tfdAα genes and consists of Sphingomonas spp. within the alphaproteobacteria (30).Diverse PAA herbicide degraders of all three groups were identified in soil by cultivation-dependent studies (32, 34, 41, 78). Besides CadAB, TfdA and certain TfdAα proteins catalyze the conversion of PAA herbicides (29, 30, 35). All groups of tfdA-like genes are potentially linked to the degradation of PAA herbicides, although alternative primary functions of group 2 and 3 TfdAs have been proposed (30, 35). However, recent cultivation-independent studies focused on 16S rRNA genes or solely on group 1 tfdA sequences in soil (e.g., references 3-5, 13, and 41). Whether group 2 and 3 tfdA-like genes are also quantitatively linked to the degradation of PAA herbicides in soils is unknown. Thus, tools to target a broad range of tfdA-like genes are needed to resolve such an issue. Primers used to assess the diversity of tfdA-like sequences used in previous studies were based on the alignment of approximately 50% or less of available sequences to date (3, 20, 29, 32, 39, 47, 58, 73). Primers specifically targeting all major groups of tfdA-like genes to assess and quantify a broad diversity of potential PAA degraders in soil are unavailable. Thus, the objectives of this study were (i) to develop primers specific for all three groups of tfdA-like genes, (ii) to establish quantitative kinetic PCR (qPCR) assays based on such primers for different soil samples, and (iii) to assess the diversity and abundance of tfdA-like genes in soil.     

Diversity of Dissimilatory Sulfite Reductase Genes (dsrAB) in a Salt Marsh Impacted by Long-Term Acid Mine Drainage

Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered dsrAB sequences from three sites sampled along an AMD flow path indicated the dominance of a single Desulfovibrio species. Other major sequence clades were related most closely to Desulfosarcina, Desulfococcus, Desulfobulbus, and Desulfosporosinus species. The presence of metal sulfides with low δ³⁴S values relative to δ³⁴S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of ∼2), although possibly within less acidic microenvironments. Interestingly, δ³⁴S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments.Salt marshes exhibit high primary production rates (1, 101) and form biogeochemical “transition zones” for nutrient production, transport, and cycling between terrestrial and coastal marine environments (41, 66, 100). These zones also serve to reduce the flux of potentially toxic metals in contaminated groundwater to estuaries (12, 99, 106). Both functions depend strongly on microbial activity, especially that of sulfate-reducing bacteria (SRB) (42, 62, 67). SRB recycle much of the sedimentary organic carbon pool in marsh sediments (42-44) and indirectly inhibit production of the greenhouse gas methane (37, 71). They can restrict the mobility of dissolved contaminant metals by inducing precipitation of poorly soluble metal sulfides, and studies have examined their use in constructed wetlands to bioremediate acid mine drainage (AMD) and other metalliferous waste streams (11, 35, 40, 46, 50, 76, 90, 94, 104). However, the high acidity and metal concentrations inherent to AMD can inhibit SRB growth (15, 88, 98), and preferential growth of iron- and sulfur-oxidizing bacteria over SRB has been observed in some treatment wetlands (39).For natural salt marshes, 16S ribosomal nucleic acid- and phospholipid fatty acid (PLFA)-based analyses have shown that SRB commonly comprise a significant fraction of the microbial community (13, 24, 31, 34, 51, 58). Studies of salt marsh dissimilatory sulfite reductase genes (dsrAB), a highly conserved functional phylogenetic marker of prokaryotic sulfate reducers (49, 57, 102, 103, 107), have revealed both novel and deeply branching clades (3). Studies of mining-impacted sites at pH 2.0 to 7.8 (5, 7, 39, 70, 72, 77, 84), of soils and geothermal settings at a pH of ∼4 (55, 68), of metal-contaminated estuaries at pH 6.8 to 7.2 (65), and of hypersaline lakes at pH 7.5 (56) further outline the distribution and tolerance of specific groups and species of SRB under geochemically stringent conditions. Other findings point toward the existence of deltaproteobacteria in environments at a pH of ∼1 (10), although it is unknown if these include SRB. SRB diversity in salt marshes under long-term contamination by AMD has not been well investigated. Such studies may provide useful information for bioremediation projects in estuarine environments, as well as general insights into relationships between SRB physiology and the geochemistry of AMD.We studied the diversity of SRB, based on phylogenetic analysis of recovered DsrAB gene sequences (∼1.9 kb), in natural salt marsh sediments of the San Francisco Bay impacted by AMD for over 100 years. Sulfur isotope ratio and concentration measurements of pore water sulfate and metal sulfide minerals provided information about the spatial and temporal extent of active bacterial sulfate reduction (BSR) in sediment cores taken from specific sites along an AMD flow path. Collectively, the results revealed a tidal marsh system characterized by rapidly cycling bacterial sulfate reduction and sulfide reoxidation associated with oscillating tidal inundation and groundwater infiltration.     

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.     

Functional Identification of the Proteus mirabilis Core Lipopolysaccharide Biosynthesis Genes

In this study, we report the identification of genes required for the biosynthesis of the core lipopolysaccharides (LPSs) of two strains of Proteus mirabilis. Since P. mirabilis and Klebsiella pneumoniae share a core LPS carbohydrate backbone extending up to the second outer-core residue, the functions of the common P. mirabilis genes was elucidated by genetic complementation studies using well-defined mutants of K. pneumoniae. The functions of strain-specific outer-core genes were identified by using as surrogate acceptors LPSs from two well-defined K. pneumoniae core LPS mutants. This approach allowed the identification of two new heptosyltransferases (WamA and WamC), a galactosyltransferase (WamB), and an N-acetylglucosaminyltransferase (WamD). In both strains, most of these genes were found in the so-called waa gene cluster, although one common core biosynthetic gene (wabO) was found outside this cluster.Gram-negative motile and frequently swarming bacteria of the genus Proteus and the family Enterobacteriaceae are opportunistic human pathogens (33). Currently, the genus consists of five species (Proteus mirabilis, P. penneri, P. vulgaris, P. myxofaciens, and P. hauseri) and three genomospecies (4, 5, and 6) (33, 35). P. mirabilis is a common uropathogen that causes urinary tract infections especially in individuals with functional or anatomical abnormalities of the urinary tract (52) and elderly persons undergoing long-term catheterization (53) but less frequently in normal hosts (43). Potentially serious complications arising from P. mirabilis infections include bladder and kidney stone formation, catheter obstruction due to the formation of encrusting biofilms, and bacteremia (reviewed in reference 2). This bacterium is found more frequently than Escherichia coli in kidney infections (14) and may be associated with rheumatoid arthritis (38). Studies aimed at the identification of P. mirabilis virulence factors showed that flagella and fimbriae (MR/P and PMF) are required for entry into and colonization of the bladder, respectively (reviewed in reference 12). Other important virulence factors are urease, hemolysin, and iron acquisition (12). More recently, an extracellular metalloprotease (37) and several putative DNA binding regulatory, cell-envelope related, and plasmid-encoded proteins have been identified by signature-tagged mutagenesis (8, 21).The lipopolysaccharide (LPS), as in other members of the family Enterobacteriaceae, consists of three domains, an endotoxic glycolipid (lipid A), an O-polysaccharide (O-PS) chain or O-antigen, and an intervening core oligosaccharide (OS) region. The O-antigen is the major surface antigen, and its serological O specificity, in contrast to that of other Gram-negative bacteria (31), is defined by the structure of the O-PS chain and that of the core OS (51). On the basis of immunospecificity, 60 O serogroups (28, 36) have been recognized in P. mirabilis and P. vulgaris, and several new Proteus O serogroups have been proposed for P. penneri (27, 55). The LPS is a potential Proteus virulence factor (42), and recently two mutants deficient in a glycosyltransferase and with attenuated virulence have been isolated and it has been speculated that this glycosyltransferase could be involved in LPS biosynthesis (21). LPS plays a significant role in the resistance of P. mirabilis to antimicrobial peptides (32), and LPS charge alterations may influence the swarming motility of the bacterium (3, 32). In addition, the core LPS is a charged OS which plays an important role in the biological activities of the LPS and the function of the bacterial outer membrane (10). In Proteus, the core OS structures of up to 34 strains of different O serogroups have been determined (51). These structures revealed that Proteus core OSs share a heptasaccharide fragment that includes a 3-deoxy-α-d-manno-oct-2-ulosonic acid (Kdo) disaccharide, an l-glycero-α-d-manno-heptose (l,d-Hep) trisaccharide, and one residue each of d-glucose (d-Glc), d-galacturonic acid (d-GalA), and either d-glucosamine (d-GlcN) or d-galactosamine (d-GalN) (51). This common fragment is also found in the core LPSs of Klebsiella pneumoniae and Serratia marcescens (11, 41, 50). The rest of the Proteus core OS is quite variable, and it is possible to recognize up to 37 and 11 different structures in the genus and P. mirabilis, respectively (51). Some P. mirabilis core OS structures are characterized by the presence of unusual residues, such as, for instance, quinovosamine; an open-chain form of N-acetylgalactosamine (GalNAc); or unusual amino acids (51). In contrast, little is known about the genes encoding enzymes involved in core LPS biosynthesis in P. mirabilis, which makes detailed genetic analysis of the role of LPS in P. mirabilis pathogenesis difficult. Thus, we decided to identify these genes by using P. mirabilis strains R110 and 51/57, the whole structures of whose core LPSs are known (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Chemical structures of the core LPSs of P. mirabilis strains R110 and 51/57 (51), K. pneumoniae types 1 (50) and 2 (41), and S. marcescens N28b (11).     

Pathway Evolution by Horizontal Transfer and Positive Selection Is Accommodated by Relaxed Negative Selection upon Upstream Pathway Genes in Purple Bacterial Carotenoid Biosynthesis

Horizontal gene transfer and selection are major forces driving microbial evolution. However, interactions between them are rarely studied. Phylogenetic analyses of purple bacterial carotenoid biosynthesis genes suggest two lineages: one producing spheroidenone and the other producing spirilloxanthin. Of the latter lineage, Rubrivivax gelatinosus S1 and Hoeflea phototrophica DFL-43 also or instead produce spheroidenone. Evolution of the spheroidenone pathway from that producing spirilloxanthin theoretically requires changes in the substrate specificity of upstream pathway enzymes and acquisition of a terminal ketolase (CrtA). In R. gelatinosus and likely also in H. phototrophica, CrtA was acquired from the Bacteroidetes, in which it functions as a hydroxylase. Estimation of nonsynonymous and synonymous mutations using several pairwise methods indicated positive selection upon both genes, consistent with their functional changes from hydroxylases to ketolases. Relaxed negative selection upon all other carotenoid biosynthetic genes in these organisms was also apparent, likely facilitating changes in their substrate specificities. Furthermore, all genes responsible for terminal carotenoid biosynthetic pathway steps were under reduced negative selection compared to those known to govern biosynthetic pathway specificity. Horizontal transfer of crtA into R. gelatinosus and H. phototrophica has therefore likely been promoted by (i) the apparent selective advantage of spheroidenone production relative to spirilloxanthin production, (ii) reduced negative selection upon other carotenoid biosynthetic genes, facilitating changes in their substrate specificities, and (iii) preexisting low enzyme substrate specificities due to relaxed negative selection. These results highlight the importance and complexity of selection acting upon both a horizontally transferred gene and the biochemical network into which it is integrating.Biochemical pathway evolution has been examined extensively, particularly regarding mechanisms by which novel functions can be generated, diversified, and maintained (9, 12). Best studied in this regard is the role of gene duplication followed by divergence, resulting in paralog families that, despite sharing a common evolutionary ancestor, possess different functions (10). In clonally reproducing organisms, such as bacteria and archaea, this type of diversification is further compounded by horizontal gene transfer (18), whereby a divergent ortholog from one organism is introduced into the metabolic network of another, thereby becoming a “xenolog” (29). Horizontal gene transfer between distantly related organisms is especially diversifying due to the likelihood of altering the genome structure or biochemical and regulatory networks of the recipient, in contrast to recombination between close relatives, which may promote genetic cohesion (33).Selection controls phenotypic diversity as a function of evolutionary fitness. Three scenarios can be detected from patterns of nucleotide substitutions (23): (i) positive selection, by which advantageous functionally divergent mutants are further optimized by increased mutational sampling of phenotypic space; (ii) negative (purifying) selection, by which deleterious mutations are purged; and (iii) neutral mutation, in which mutations that do not affect the selected phenotype accumulate, resulting in genetic drift. Considering horizontal transfer, selection will favor fixation of a horizontally transferred gene if its phenotype is advantageous and will disfavor it when either the gene product or the alterations that it causes in the host network are deleterious. Successful horizontal gene transfer resulting in gene fixation is the result of net evolutionary benefit for the host, due both to the horizontally transferred gene itself and to minimal suboptimal alteration of the host metabolic and genetic networks into which it is integrating. Genetic parasites such as plasmids, transposons, and integrated phages are exceptional in directly promoting their own retention.Carotenoids are isoprenoid pigments produced by many bacteria and fungi and all photosynthetic eukaryotes (7, 8). They are typically colored red, orange, and yellow due to their extensively conjugated polyene chains (7). In all photosynthetic organisms, carotenoids facilitate the assembly of the photosynthetic reaction center and interact with it as auxiliary light-harvesting pigments and antioxidant molecules (13-15). Indeed, the niche in which a particular phototroph lives is defined, at least in part, by the absorption spectrum of its pigments, including light-harvesting carotenoids (55). Carotenoids also modulate membrane fluidity and permeability (19); these functions remain poorly understood.In this study, the evolution of carotenoid biosynthesis by horizontal gene transfer and selection is evaluated in the purple bacteria, anoxygenic phototrophic Proteobacteria that can use reduced sulfur compounds as electron sources. Whereas phylogenetically most purple bacteria belong to the Alphaproteobacteria, some belonging to the Betaproteobacteria and Gammaproteobacteria have evolved by horizontal transfer of alphaproteobacterial photosynthetic superoperons, which include carotenoid biosynthetic genes (24, 37). Carotenoid biosynthesis in the purple bacteria (Fig. ​(Fig.1)1) begins with the condensation of two molecules of geranylgeranyl pyrophosphate by the phytoene synthase CrtB, forming phytoene. The phytoene desaturase CrtI then desaturates phytoene either three or four times, producing neurosporene or lycopene, respectively. Both of these intermediates are subsequently hydroxylated at the 1 position by the hydroxylase CrtC, desaturated at the 3 and 4 positions by the CrtI homolog CrtD, methylated at the 1-hydroxyl group by the methyltransferase CrtF, and, in spheroidenone- and 2,2′-diketospirilloxanthin-producing organisms, ketolated at the 2 position by the ketolase CrtA (Fig. ​(Fig.1).1). Considerable subpathway diversity also exists because of the potential for asymmetry between carotenoid ends.Open in a separate windowFIG. 1.Carotenoid biosynthetic pathway in purple bacteria. For simplicity, not all subpathways are shown. Carbon numbers for the ψ end group are shown for phytoene.Carotenoid biosynthesis has been well studied biochemically and genetically in the spheroidene-producing organisms Rhodobacter capsulatus (3, 4, 16, 45) and Rhodobacter sphaeroides (1, 31, 32) and the spirilloxanthin-producing organisms Bradyrhizobium sp. strain ORS278 (17), Thiocapsa roseopersicina (30), and Rubrivivax gelatinosus (16, 21, 22, 40, 41, 43, 52-54). The latter organism produces 2,2′-diketospirilloxanthin (lycopene derived), spheroidenone (neurosporene derived), and their precursors using the same enzymes. Pathway utilization in R. gelatinosus is determined primarily by the substrate specificities of CrtC, CrtD, and CrtI and by the rate of metabolic flux (52-54). These extensive biochemical and genetic studies provide a solid framework for sequence-based evolutionary analyses, making this pathway a valuable model with which to study biochemical pathway evolution.     

Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.