Functional Relationships between the AcrA Hairpin Tip Region and the TolC Aperture Tip Region for the Formation of the Bacterial Tripartite Efflux Pump AcrAB-TolC

Tripartite efflux pumps found in Gram-negative bacteria are involved in antibiotic resistance and toxic-protein secretion. In this study, we show, using site-directed mutational analyses, that the conserved residues located in the tip region of the α-hairpin of the membrane fusion protein (MFP) AcrA play an essential role in the action of the tripartite efflux pump AcrAB-TolC. In addition, we provide in vivo functional data showing that both the length and the amino acid sequence of the α-hairpin of AcrA can be flexible for the formation of a functional AcrAB-TolC pump. Genetic-complementation experiments further indicated functional interrelationships between the AcrA hairpin tip region and the TolC aperture tip region. Our findings may offer a molecular basis for understanding the multidrug resistance of pathogenic bacteria.The tripartite efflux pumps that are found in Gram-negative bacteria have been implicated in their intrinsic resistance to diverse antibiotics, as well as their secretion of protein toxins (10, 12, 24, 31). The bacterial efflux pump is typically assembled from three essential components: an inner membrane transporter (IMT), an outer membrane factor (OMF), and a periplasmic membrane fusion protein (MFP) (10, 12, 24, 31). The IMT provides energy for transporters, like the resistance nodulation cell division (RND) type and the ATP-binding cassette (ABC) type (18). The OMF connects to the IMT in the periplasm, providing a continuous conduit to the external medium. This conduit uses the central channel, which is opened only when in complex with other components (11, 18). The third essential component of the pump is the MFP, which is an adapter protein for the direct interaction between the IMT and OMF in the periplasm (32). The MFP consists of four linearly arranged domains: the membrane-proximal (MP) domain, the β-barrel domain, the lipoyl domain, and the α-hairpin domain (1, 6, 16, 22, 30). The MFP α-hairpin domain is known to interact with OMF, while the other domains are related to interaction with the IMT (15, 22).The Escherichia coli AcrAB-TolC pump, comprised of RND-type IMT-AcrB, MFP-AcrA, and OMF-TolC, is the major contributor to the multidrug resistance phenotype of the bacteria (7, 8, 25). The AcrAB-TolC pump, together with its homolog, the Pseudomonas aeruginosa MexAB-OprM pump (7, 13), has primarily been studied in order to elucidate the molecular mechanisms underlying the actions of the tripartite efflux pumps. Whereas the crystal structures of these proteins have revealed that RND-type IMTs (AcrB and MexB) and OMFs (TolC and OprM) are homotrimeric in their functional states (1, 6, 11, 16, 22, 30), the oligomeric state of MFP remains a topic of debate, despite the presence of crystal structures (3, 5, 17, 18, 22, 27, 30).MacAB-TolC, which was identified as a macrolide-specific extrusion pump (9), has also been implicated in E. coli enterotoxin secretion (29). While MFP-MacA shares high sequence similarity with AcrA and MexA, IMT-MacB is a homodimeric ABC transporter that uses ATP hydrolysis as the driving force (9, 14). MacA forms hexamers, and the funnel-like hexameric structure of MacA is physiologically relevant for the formation of a functional MacAB-TolC pump (30). Although the α-hairpins from AcrA and MacA are commonly involved in the interaction with TolC (30, 32), the interaction mode between AcrA and TolC remains to be elucidated. In this study, we provide experimental evidence showing that the conserved amino acid residues in the AcrA hairpin tip region is important for the action of the AcrAB-TolC efflux pump and is functionally related to the TolC aperture tip region.     

mcrA-Targeted Real-Time Quantitative PCR Method To Examine Methanogen Communities

Methanogens are of great importance in carbon cycling and alternative energy production, but quantitation with culture-based methods is time-consuming and biased against methanogen groups that are difficult to cultivate in a laboratory. For these reasons, methanogens are typically studied through culture-independent molecular techniques. We developed a SYBR green I quantitative PCR (qPCR) assay to quantify total numbers of methyl coenzyme M reductase α-subunit (mcrA) genes. TaqMan probes were also designed to target nine different phylogenetic groups of methanogens in qPCR assays. Total mcrA and mcrA levels of different methanogen phylogenetic groups were determined from six samples: four samples from anaerobic digesters used to treat either primarily cow or pig manure and two aliquots from an acidic peat sample stored at 4°C or 20°C. Only members of the Methanosaetaceae, Methanosarcina, Methanobacteriaceae, and Methanocorpusculaceae and Fen cluster were detected in the environmental samples. The three samples obtained from cow manure digesters were dominated by members of the genus Methanosarcina, whereas the sample from the pig manure digester contained detectable levels of only members of the Methanobacteriaceae. The acidic peat samples were dominated by both Methanosarcina spp. and members of the Fen cluster. In two of the manure digester samples only one methanogen group was detected, but in both of the acidic peat samples and two of the manure digester samples, multiple methanogen groups were detected. The TaqMan qPCR assays were successfully able to determine the environmental abundance of different phylogenetic groups of methanogens, including several groups with few or no cultivated members.Methanogens are integral to carbon cycling, catalyzing the production of methane and carbon dioxide, both potent greenhouse gases, during organic matter degradation in anaerobic soils and sediment (8). Methanogens are widespread in anaerobic environments, including tundra (36), freshwater lake and wetland sediments (9, 12), estuarine and marine sediments (2), acidic peatlands (4, 14), rice field soil (10, 16), animal guts (41), landfills (30), and anaerobic digesters treating animal manure (1), food processing wastewater (27), and municipal wastewater and solid waste (37, 57). Methane produced in anaerobic digesters may be captured and used for energy production, thus offsetting some or all of the cost of operation and reducing the global warming potential of methane release to the atmosphere.Methanogens are difficult to study through culture-based methods, and therefore many researchers have instead used culture-independent techniques to study methanogen populations. The 16S rRNA gene is the most widely used target for gene surveys, and a number of primers and probes have been developed to target methanogen groups (9, 11, 31, 36, 38, 40, 46, 48, 57). To eliminate potential problems with nonspecific amplification, some researchers have developed primers for the gene sequence of the α-subunit of the methyl coenzyme M reductase (mcrA) (17, 30, 49). The Mcr is exclusive to the methanogens with the exception of the methane-oxidizing Archaea (18) and shows mostly congruent phylogeny to the 16S rRNA gene, allowing mcrA analysis to be used in conjunction with, or independently of, that of the 16S rRNA gene (3, 30, 49). A number of researchers have examined methanogen communities with mcrA and have found uncultured clades quite different in sequence from cultured methanogen representatives (9, 10, 12, 14, 17, 22, 28, 47).Previous studies described methanogen communities by quantitation of different clades through the use of rRNA-targeted or rRNA gene-targeted probes with techniques such as dot blot hybridization (1, 27, 37, 38, 48) and fluorescent in situ hybridization (11, 40, 44, 57). Real-time quantitative PCR (qPCR) is an alternate technique capable of determining the copy number of a particular gene present in the DNA extracted from an environmental sample. Only a few studies have used qPCR to quantitatively examine different clades within methanogen communities, and most of these studies have exclusively targeted the 16S rRNA gene (19, 41, 42, 54-56). Far fewer researchers have used qPCR to quantify methanogen clades by targeting the mcrA (21, 34, 45), and these studies were limited to only a few phylogenetic groups.In this paper we present a methodology for determining methanogen gene copy numbers through the use of qPCR targeting the mcrA. Methanogens were quantified in total using methanogen-specific primers in SYBR green assays and also as members of nine different phylogenetic groups using TaqMan probes targeting specific subsets of methanogens.     

Widely Conserved Recombination Patterns among Single-Stranded DNA Viruses

The combinatorial nature of genetic recombination can potentially provide organisms with immediate access to many more positions in sequence space than can be reached by mutation alone. Recombination features particularly prominently in the evolution of a diverse range of viruses. Despite rapid progress having been made in the characterization of discrete recombination events for many species, little is currently known about either gross patterns of recombination across related virus families or the underlying processes that determine genome-wide recombination breakpoint distributions observable in nature. It has been hypothesized that the networks of coevolved molecular interactions that define the epistatic architectures of virus genomes might be damaged by recombination and therefore that selection strongly influences observable recombination patterns. For recombinants to thrive in nature, it is probably important that the portions of their genomes that they have inherited from different parents work well together. Here we describe a comparative analysis of recombination breakpoint distributions within the genomes of diverse single-stranded DNA (ssDNA) virus families. We show that whereas nonrandom breakpoint distributions in ssDNA virus genomes are partially attributable to mechanistic aspects of the recombination process, there is also a significant tendency for recombination breakpoints to fall either outside or on the peripheries of genes. In particular, we found significantly fewer recombination breakpoints within structural protein genes than within other gene types. Collectively, these results imply that natural selection acting against viruses expressing recombinant proteins is a major determinant of nonrandom recombination breakpoint distributions observable in most ssDNA virus families.Genetic recombination is a ubiquitous biological process that is both central to DNA repair pathways (10, 57) and an important evolutionary mechanism. By generating novel combinations of preexisting nucleotide polymorphisms, recombination can potentially accelerate evolution by increasing the population-wide genetic diversity upon which adaptive selection relies. Recombination can paradoxically also prevent the progressive accumulation of harmful mutations within individual genomes (18, 35, 53). Whereas its ability to defend high-fitness genomes from mutational decay possibly underlies the evolutionary value of sexuality in higher organisms, in many microbial species where pseudosexual genetic exchange is permissible among even highly divergent genomes, recombination can enable access to evolutionary innovations that would otherwise be inaccessible by mutation alone.Such interspecies recombination is fairly common in many virus families (8, 17, 27, 44, 82). It is becoming clear, however, that as with mutation events, most recombination events between distantly related genomes are maladaptive (5, 13, 38, 50, 63, 80). As genetic distances between parental genomes increase, so too does the probability of fitness defects in their recombinant offspring (16, 51). The viability of recombinants is apparently largely dependent on how severely recombination disrupts coevolved intragenome interaction networks (16, 32, 51). These networks include interacting nucleotide sequences that form secondary structures, sequence-specific protein-DNA interactions, interprotein interactions, and amino acid-amino acid interactions within protein three-dimensional folds.One virus family where such interaction networks appear to have a large impact on patterns of natural interspecies recombination are the single-stranded DNA (ssDNA) geminiviruses. As with other ssDNA viruses, recombination is very common among the species of this family (62, 84). Partially conserved recombination hot and cold spots have been detected in different genera (39, 81) and are apparently caused by both differential mechanistic predispositions of genome regions to recombination and natural selection disfavoring the survival of recombinants with disrupted intragenome interaction networks (38, 51).Genome organization and rolling circle replication (RCR)—the mechanism by which geminiviruses and many other ssDNA viruses replicate (9, 67, 79; see reference 24 for a review)—seem to have a large influence on basal recombination rates in different parts of geminivirus genomes (20, 33, 39, 61, 81). To initiate RCR, virion-strand ssDNA molecules are converted by host-mediated pathways into double-stranded “replicative-form” (RF) DNAs (34, 67). Initiated by a virus-encoded replication-associated protein (Rep) at a well-defined virion-strand replication origin (v-ori), new virion strands are synthesized on the complementary strand of RF DNAs (28, 73, 74) by host DNA polymerases. Virion-strand replication is concomitant with the displacement of old virion strands, which, once complete, yields covalently closed ssDNA molecules which are either encapsidated or converted into additional RF DNAs. Genome-wide basal recombination rates in ssDNA viruses are probably strongly influenced by the specific characteristics of host DNA polymerases that enable RCR. Interruption of RCR has been implicated directly in geminivirus recombination (40) and is most likely responsible for increased basal recombination rates both within genes transcribed in the opposite direction from that of virion-strand replication (40, 71) and at the v-ori (1, 9, 20, 69, 74).Whereas most ssDNA virus families replicate via either a rolling circle mechanism (the Nanoviridae, Microviridae, and Geminiviridae) (3, 23, 24, 31, 59, 67, 74) or a related rolling hairpin mechanism (the Parvoviridae) (25, 76), among the Circoviridae only the Circovirus genus is known to use RCR (45). Although the Gyrovirus genus (the other member of the Circoviridae) and the anelloviruses (a currently unclassified ssDNA virus group) might also use RCR, it is currently unknown whether they do or not (78). Additionally, some members of the Begomovirus genus of the Geminiviridae either have a second genome component, called DNA-B, or are associated with satellite ssDNA molecules called DNA-1 and DNA-Beta, all of which also replicate by RCR (1, 47, 68).Recombination is known to occur in the parvoviruses (19, 43, 70), microviruses (66), anelloviruses (40, 46), circoviruses (11, 26, 60), nanoviruses (30), geminivirus DNA-B components, and geminivirus satellite molecules (2, 62). Given that most, if not all, of these ssDNA replicons are evolutionarily related to and share many biological features with the geminiviruses (22, 31, 36), it is of interest to determine whether conserved recombination patterns observed in the geminiviruses (61, 81) are evident in these other groups. To date, no comparative analyses have ever been performed with different ssDNA virus families to identify, for example, possible influences of genome organization on recombination breakpoint distributions found in these viruses.Here we compare recombination frequencies and recombination breakpoint distributions in most currently described ssDNA viruses and satellite molecules and identify a number of sequence exchange patterns that are broadly conserved across this entire group.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

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.     

Identification of a Novel System for Boron Transport: Atr1 Is a Main Boron Exporter in Yeast

Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Δ mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1Δ cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1Δ cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.Boron has been proposed as an important micronutrient in plants and animals. Studies have shown the presence of several genes associated with boron transport and tolerance in plants (18, 25, 27); however, boron transport mechanisms in other organisms, including animals, remain unclear. In plants, boron functions as a cross-linker for rhammogalacturanon II in the cell membrane (9, 14, 21) and also as a structural component in cytoskeleton assembly (1). Arabidopsis thaliana BOR1 was the first gene shown to play a role in boron tolerance (28). Homologs of BOR1 were found in many organisms, including yeasts, plants, and mammals (22, 25, 29). A high level of boron leads to degradation of its own exporter, BOR1, in A. thaliana (27), and A. thaliana BOR1 cannot be used to produce genetically modified plants that grow in soil with high boron levels. However, transgenic plants expressing BOR4, one of six paralogs of BOR1, showed high tolerance to toxic levels of boron (18). Multicopy expression of BOT1, a BOR1 ortholog, provided boron tolerance to barley (25).The yeast Saccharomyces cerevisiae has been used as a model organism for characterization of plant boron tolerance genes (19, 20, 25, 26, 29). While 10 mM boric acid is lethal to Arabidopsis (18), yeast can grow in the presence of 80 mM boron and is considered a boron-tolerant organism (19, 20). Yeast Bor1 was characterized in detail (10). This protein is localized to the plasma membrane and functions as a boric acid exporter (26). The bor1Δ yeast strain overaccumulates boron (20, 28), and cells that overexpress BOR1 have less intracellular boron and show resistance to boron treatment (20). In addition to Bor1, two other proteins, Dur3 and Fps1, have been implicated in boron tolerance in yeast, but their functions are not clear (20). Dur3 is a plasma membrane transporter that plays a role in urea and polyamine transport (5, 31), and Fps1 is a member of the major intrinsic protein family and plays a role in glycerol, acetic acid, arsenite, and antimonite transport (16, 30, 33). Overexpression of FPS1 and DUR3 showed controversial effects on cellular boron levels. While FPS1 expression lowered the protoplasmic boron concentration, DUR3 expression led to a small increase in boron (20).The objective of this study was to identify proteins that are primarily responsible for boron transport in yeast. ATR1 was identified as a boron tolerance gene by screening a yeast DNA expression library. Yeast Atr1 is a member of the DHA2 family of drug-H⁺ antiporters with 14 predicted membrane-spanning segments (7). It was first characterized in a genetic screen as a high-copy-number suppressor of the 3-amino-1,2,4-triazole sensitivity of gcn4Δ mutants (11). It also conferred resistance to the DNA-damaging agent 4-nitroquinoline-N-oxide in a separate genetic screen (17). In this study, we demonstrated that high-copy-number expression of ATR1 conferred extreme resistance to boron and reduced intracellular levels of the element, whereas cells lacking the ATR1 gene were hypersensitive to boron and increased its intracellular levels. We analyzed changes in the global gene expression profile in response to boron and found that ATR1 is the most induced transporter gene. The Atr1-green fluorescent protein (GFP) fusion protein localized to the plasma membrane and vacuole. Taken together, our data show that Atr1 functions as a major boron efflux pump and provides tolerance of the element by pumping boron out of cells.     

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).     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.