Evolutionary Dynamics of Insertion Sequences in Relation to the Evolutionary Histories of the Chromosome and Symbiotic Plasmid Genes of Rhizobium etli Populations

Insertion sequences (IS) are mobile genetic elements that are distributed in many prokaryotes. In particular, in the genomes of the symbiotic nitrogen-fixing bacteria collectively known as rhizobia, IS are fairly abundant in plasmids or chromosomal islands that carry the genes needed for symbiosis. Here, we report an analysis of the distribution and genetic conservation of the IS found in the genome of Rhizobium etli CFN42 in a collection of 87 Rhizobium strains belonging to populations with different geographical origins. We used PCR to generate presence/absence profiles of the 39 IS found in R. etli CFN42 and evaluated whether the IS were located in consistent genomic contexts. We found that the IS from the symbiotic plasmid were frequently present in the analyzed strains, whereas the chromosomal IS were observed less frequently. We then examined the evolutionary dynamics of these strains based on a population genetic analysis of two chromosomal housekeeping genes (glyA and dnaB) and three symbiotic sequences (nodC and the two IS elements). Our results indicate that the IS contained within the symbiotic plasmid have a higher degree of genomic context conservation, lower nucleotide diversity and genetic differentiation, and fewer recombination events than the chromosomal housekeeping genes. These results suggest that the R. etli populations diverged recently in Mexico, that the symbiotic plasmid also had a recent origin, and that the IS elements have undergone a process of cyclic infection and expansion.Insertion sequences (IS) are the smallest transposable elements found in prokaryotes (usually less than 3 kb in size). They encode a transposase and may also encode small hypothetical proteins (4, 9). IS are distinguished by their ability to move within prokaryotic replicons, including both the chromosome and plasmids, and to copy themselves into various genomic sites. In this manner, IS elements can inactivate or alter the expression of adjacent genes (4). When IS occur in two or more identical copies within a genome, they can participate in various types of genetic rearrangements (e.g., duplications, inversions, and deletions), suggesting that IS may play an important role in the evolution of their hosts by promoting genomic plasticity (34). Due to these evolutionary dynamics, the diversity and distribution of IS elements differ greatly between taxa and even within strains of the same species (27).Various theories have been proposed to explain the evolution of IS elements in laboratory model strains and environmental bacterial populations (8, 18, 25, 29). Two main hypotheses seek to explain how these elements are maintained over the long term in their host genomes. The first proposes that they occasionally generate beneficial mutations and therefore may represent a selective advantage to their hosts (34). The second suggests that IS elements are genomic parasites that are maintained by their high rate of transposition and might be disseminated among different bacterial lineages by horizontal gene transfer (HGT). Data supporting the second hypothesis have shown that some IS elements may transpose at high rates upon entering a new host (42). After the initial infection, however, purifying selection may continuously remove these elements from the genome. Thus, IS may undergo an infection-expansion-extinction cycle that allows them to remain in different bacterial populations within the gene pool (42). These two hypotheses are not contradictory, and the evolutionary dynamics and distribution of IS may differ greatly depending on several factors, including (most notably) the rate of transposition and HGT, as well as selective pressures, population size, and the host''s habitat (6, 18, 21, 25, 27, 29).In the nitrogen-fixing symbiotic bacteria of the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium (of the alphaproteobacteria), Cupriavidus, and Burkholderia (of the betaproteobacteria), IS are particularly abundant in symbiotic plasmids (pSym) and symbiotic chromosomal islands (SI) (2, 12, 14, 19, 20, 43). SI and pSym include most of the genes needed to establish symbiosis in the roots of leguminous plants through nodule formation and nitrogen fixation (11). It is generally believed that these elements entered the rhizobial genomes through HGT (39, 40). Comparative genomic analyses have shown that both pSym and SI are highly variable, with the exception of a common set of genes encoding factors critical to nitrogen fixation (nif) and nodulation (nod) (5, 14). SI and pSym have been found to have lower GC contents and different codon usages than the corresponding chromosomal and nonsymbiotic plasmid sequences, suggesting that they were recently acquired by HGT.Some of these symbiotic elements, such as in pSym of Rhizobium etli CFN42 and the SI of Mesorhizobium loti, are conjugative and mobile (30, 32). Genomic analysis of R. etli CFN42 revealed the presence of 39 IS belonging to different families (14); they were found in the chromosome (11 IS); the 371-kb symbiotic plasmid (13 IS); the smaller 192-kb conjugative plasmid, p42a (13 IS); and two other plasmids, p42b and p42c (2 IS). Interestingly, this particular strain shows no evidence of IS disrupting open reading frames (ORFs) or having transpositional activity. However, another 42 incomplete IS may be found in the chromosome, pSym, and the conjugative plasmid; these incomplete sequences are truncated or contain stop codons in their coding sequences.Here, we focused on the dynamics and distribution of IS in different populations of the nitrogen-fixing symbiont R. etli. Since the maintenance of IS in bacterial species might depend on their transpositional activities and horizontal transfer rates, the identification of IS in the same genomic contexts across different strains of the same species could provide new insights into their persistence and divergence over short evolutionary periods. To examine the evolutionary dynamics of IS in natural populations of R. etli, we characterized the distributions, genomic contexts, and sequence variations of IS in isolates of R. etli from three populations with different origins, as well as in some other Rhizobium species. More specifically, we used PCR to generate presence/absence profiles of the 39 IS found in R. etli CFN42 in a collection of 87 strains representing different geographical sites and a gradient of domestication of the bacterial host, the common bean (Phaseolus vulgaris). We also evaluated whether the IS were conserved in the same genomic context relative to their position in R. etli CFN42 and determined the nucleotide sequences of two IS found in most of the isolates. Several population genetic tests applied to these IS, another pSym gene (nodC), and two chromosomal housekeeping genes (dnaB and glyA) suggest that these two IS elements have been inherited vertically and represent recent components of the R. etli gene pool. Finally, the present study strongly suggests that symbiotic plasmids have a recent origin within the R. etli populations.     

Conserved Symbiotic Plasmid DNA Sequences in the Multireplicon Pangenomic Structure of Rhizobium etli

Strains of the same bacterial species often show considerable genomic variation. To examine the extent of such variation in Rhizobium etli, the complete genome sequence of R. etli CIAT652 and the partial genomic sequences of six additional R. etli strains having different geographical origins were determined. The sequences were compared with each other and with the previously reported genome sequence of R. etli CFN42. DNA sequences common to all strains constituted the greater part of these genomes and were localized in both the chromosome and large plasmids. About 700 to 1,000 kb of DNA that did not match sequences of the complete genomes of strains CIAT652 and CFN42 was unique to each R. etli strain. These sequences were distributed throughout the chromosome as individual genes or chromosomal islands and in plasmids, and they encoded accessory functions, such as transport of sugars and amino acids, or secondary metabolism; they also included mobile elements and hypothetical genes. Sequences corresponding to symbiotic plasmids showed high levels of nucleotide identity (about 98 to 99%), whereas chromosomal sequences and the sequences with matches to other plasmids showed lower levels of identity (on average, about 90 to 95%). We concluded that R. etli has a pangenomic structure with a core genome composed of both chromosomal and plasmid sequences, including a highly conserved symbiotic plasmid, despite the overall genomic divergence.It is becoming clear that bacterial genomes of strains of the same species vary widely both in size and in gene composition (39). An unexpected degree of genomic diversity has been found by comparing whole genomes (39). For instance, in Escherichia coli strains, differences of up to 1,400 kb account for some strain-specific pathogenic traits (5, 56). The extent of intraspecies genome diversity varies in different bacterial lineages. Some species have a wide range of variation; these species include E. coli (42), Streptococcus agalactiae (53), and Haloquadratum walsbyi (34). Other bacteria display only limited gene content diversity; an example is Ureaplasma urealyticum (1, 54). Tettelin and colleagues have suggested that bacterial species can be characterized by the presence of a pangenome consisting of a core genome containing genes present in all strains and a dispensable genome consisting of partially shared and strain-specific genes (53, 54). This concept is rooted in the earlier ideas of Reanney (43) and Campbell (7) concerning the structure of bacterial populations, and it indicates both that there is a pool of accessory genetic information in bacterial species and that strains of the same or even different species can obtain this information by horizontal transfer mechanisms (7, 43).Genome size and diversity are related to bacterial lifestyle. Small genomes are typical of strict pathogens such as Rickettsia prowazekii (2) and endosymbionts such as Buchnera aphidicola (44a). In contrast, free-living bacteria, such as Pseudomonas syringae and Streptomyces coelicolor, have large genomes (4, 6). The bacteria with the largest genomes are common inhabitants of heterogeneous environments, such as soil, where energy sources are limited but diverse (32). An increase in genome size is attributable mainly to expansion of functions such as secondary metabolism, transport of metabolites, and gene regulation. All these features are common to the nitrogen-fixing symbiotic bacteria of legumes, which are collectively known as rhizobia, and their close relative the plant pathogen Agrobacterium. The genomes of such bacterial species have diverse architectures with circular chromosomes that are different sizes or linear chromosomes, like that in Agrobacterium species, and the organisms contain variable numbers of large plasmids (31, 49). Comparative genomic studies have highlighted the conservation of gene content and order among the chromosomes of some species of rhizobia (22, 23, 25, 40). Furthermore, Guerrero and colleagues (25) observed that most essential genes occur in syntenic arrangements and display a higher level of sequence identity than nonsyntenic genes. In contrast, plasmids, including symbiotic plasmids and symbiotic chromosomal islands (like those in Mesorhizobium loti and Bradyrhizobium japonicum) are poorly conserved in terms of both gene content and gene order (21). It is not clear what evolutionary advantage, if any, is provided by multipartite genomes, but some authors have speculated that such genomes may allow further accumulation of genes independent of the chromosome. Recently, Slater and coworkers (46) proposed a model for the origin of secondary chromosomes. Their idea is based on the notion of intragenomic gene transfers that might occur from primary chromosomes to ancestral plasmids of the repABC type. Observations of conservation of clusters of genes in secondary chromosomes or in large plasmids that retain synteny with respect to the main chromosome support this hypothesis (46).We have been studying Rhizobium etli as a multipartite genome model species (23). This organism is a free-living soil bacterium that is able to form nodules and fix nitrogen in the roots of bean plants. The genome of R. etli is partitioned into several replicons, a circular chromosome, and several large plasmids. In the reference strain R. etli CFN42, the genome is composed of a circular chromosome consisting of about 4,381 kb and 6 large plasmids whose total size is 2,148 kb (23). A 371-kb plasmid, termed pSym or the symbiotic plasmid, contains most of the genes required for symbiosis (21). Previous studies have described the high level of genetic diversity among geographically different R. etli isolates (41). The strains are also variable with respect to the number and size of plasmids. Nevertheless, there has been no direct measurement of diversity at the genomic level, nor have comparative studies of shared and particular genomic features of R. etli strains been reported. Therefore, to assess the degrees of genomic difference and genomic similarity in R. etli, we obtained the complete genomic sequence of an additional R. etli strain and partial genomic sequences of six other R. etli strains isolated worldwide. Our results support the concept of a pangenomic structure at the multireplicon level and show that a highly conserved symbiotic plasmid is present in divergent R. etli isolates.     

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).     

Rickettsia felis Infection in a Common Household Insect Pest,Liposcelis bostrychophila (Psocoptera: Liposcelidae)

Many species of Rickettsia are well-known mammalian pathogens transmitted by blood-feeding arthropods. However, molecular surveys are continually uncovering novel Rickettsia species, often in unexpected hosts, including many arthropods that do not feed on blood. This study reports a systematic molecular characterization of a Rickettsia infecting the psocid Liposcelis bostrychophila (Psocoptera: Liposcelidae), a common and cosmopolitan household pest. Surprisingly, the psocid Rickettsia is shown to be Rickettsia felis, a human pathogen transmitted by fleas that causes serious morbidity and occasional mortality. The plasmid from the psocid R. felis was sequenced and was found to be virtually identical to the one in R. felis from fleas. As Liposcelis insects are often intimately associated with humans and other vertebrates, it is speculated that they acquired R. felis from fleas. Whether the R. felis in psocids causes disease in vertebrates is not known and warrants further study.Many species of Rickettsia are well-known mammalian pathogens that are transmitted by blood-feeding arthropods via bites or feces and can cause mild to fatal diseases in humans (33). Some species are also considered potential bioterrorism agents (4). Most Rickettsia research has focused on pathogens that are found in two closely related species groups, the typhus and spotted fever groups, such as Rickettsia prowazekii, Rickettsia rickettsii, and Rickettsia typhi, the causal agents of epidemic typhus, Rocky Mountain spotted fever, and murine typhus, respectively (3, 4, 33). However, recent surveys suggest that Rickettsia bacteria are much more widespread than previously suspected and that they are being detected in novel hosts, the vast majority of which are arthropods, including many that do not feed on blood (29, 45).The number of new rickettsial species that cause diseases in humans is rapidly increasing (33). One such species that has been generating much interest in recent years is Rickettsia felis, the causative agent of a murine typhus-like disease (1, 2, 13, 16, 17, 28, 44). The disease is often unrecognized, and even though it is considered clinically mild, it can cause severe illness and death in older patients and in cases of delayed diagnosis (2). R. felis was identified only in 1990 (1) and has since been found worldwide in fleas, where it is maintained transovarially and can reach high infection rates (e.g., 86% to 94% in cat fleas) (2, 3, 44), as well as in ticks and mites (34). While experimental infections have confirmed that R. felis is transmitted to vertebrate hosts via blood feeding and that R. felis occurs in an infectious extracellular state (39), it is not known whether transmission can also occur through contamination of broken skin by infected vector feces, as in R. typhi (3, 34).A number of features distinguish R. felis from species in both the typhus and spotted fever groups. Lately, it has been proposed that R. felis be in its own group, allied with Rickettsia akari and Rickettsia australis, the causal agents of rickettsial pox and Queensland tick typhus, respectively, and a number of recently discovered strains infecting insects that do not feed on blood (16, 17, 29, 45). Moreover, R. felis was the first Rickettsia species shown to have a plasmid (28). While plasmids now appear to be quite widespread in the genus, the R. felis plasmid stands out with respect to its relatively large size and distinctive gene content (5, 6, 9, 14, 17).This study reports that a common and cosmopolitan insect, the psocid Liposcelis bostrychophila (Psocoptera: Liposcelidae) harbors R. felis. Liposcelids are the closest free-living relatives of parasitic lice (19) and are well-known for their close proximity to humans, particularly as pests in houses and grain storage facilities (8, 41). Through 16S rRNA gene sequencing, L. bostrychophila was recently shown to harbor a strain of Rickettsia (29, 30, 42). A systematic molecular characterization of this Rickettsia was conducted, demonstrating that it is authentic R. felis. Furthermore, the psocid symbiont plasmid was sequenced and was shown to be virtually identical to the plasmid from R. felis that infects cat fleas.     

The Mycobacterium tuberculosis DosR Regulon Assists in Metabolic Homeostasis and Enables Rapid Recovery from Nonrespiring Dormancy

Mycobacterium tuberculosis survives in latently infected individuals, likely in a nonreplicating or dormancy-like state. The M. tuberculosis DosR regulon is a genetic program induced by conditions that inhibit aerobic respiration and prevent bacillus replication. In this study, we used a mutant incapable of DosR regulon induction to investigate the contribution of this regulon to bacterial metabolism during anaerobic dormancy. Our results confirm that the DosR regulon is essential for M. tuberculosis survival during anaerobic dormancy and demonstrate that it is required for metabolic processes that occur upon entry into and throughout the dormant state. Specifically, we showed that regulon mechanisms shift metabolism away from aerobic respiration in the face of dwindling oxygen availability and are required for maintaining energy levels and redox balance as the culture becomes anaerobic. We also demonstrated that the DosR regulon is crucial for rapid resumption of growth once M. tuberculosis exits an anaerobic or nitric oxide-induced nonrespiring state. In summary, the DosR regulon encodes novel metabolic mechanisms essential for M. tuberculosis to survive in the absence of respiration and to successfully transition rapidly between respiring and nonrespiring conditions without loss of viability.Mycobacterium tuberculosis, a major human pathogen, infects nearly one-third of the people in the world and causes two million deaths per year (8). Most infections are latent, and a substantial number of new infections are transmitted by individuals in whom latent infections are being reactivated. Latency is a clinical term describing people that are infected with M. tuberculosis but lack symptoms of active disease. Traditionally, it has been thought that bacilli in latently infected individuals reside almost exclusively inside granulomas and mature tubercle lesions. Recent studies indicate that in latently infected individuals M. tuberculosis may also be found outside granulomas in places such as endothelial cells, fibroblasts, and adipose tissue (17, 28). The evidence for M. tuberculosis metabolic activity in vivo is more limited, but two studies by Lillebaek et al. are informative (24, 25). In these studies the researchers used detailed records of tuberculosis epidemiology and strain types in the fairly static population of Denmark. They found that strains isolated from patients thought to have reactivated disease (rather than a primary infection) were nearly identical to strains present 30 years earlier in the same geographic population. The near-identity of the strains and the fact that infections were attributed to reactivation suggest that bacteria in latently infected individuals experience little genetic change during years of latent infection. The researchers concluded that during latency, M. tuberculosis divides infrequently and is likely in a minimal metabolic state.One approach to study the M. tuberculosis metabolic state during latent infection is to use in vitro models that mimic conditions thought to exist in vivo. Such conditions include hypoxia produced in avascular calcified granulomas (40) and nitric oxide (NO) (27) or carbon monoxide (CO) (33) produced by activated immune cells. A widely used model is the “Wayne model” pioneered by Lawrence Wayne. In this model, a low-inoculum culture is sealed in a tube with stirring and allowed to slowly consume oxygen until the culture is anaerobic, resulting in a nonreplicating and apparently dormant state (45, 46). Another model used to look at dormant M. tuberculosis is a constant-hypoxia model that maintains a 0.2% oxygen tension in culture flasks (31).The common theme in these in vitro models used to obtain M. tuberculosis dormancy is inhibition of respiration. The DosR regulon is a set of at least 48 coregulated genes that are induced by three conditions that inhibit aerobic respiration: hypoxia, NO, and CO (42). Induction of the DosR regulon closely mirrors inhibition of respiration, indicating that control of the regulon is linked to the aerobic respiratory state of the bacilli (43). Several studies have shown that the DosR regulon is controlled by a three-component regulatory system composed of two sensor histidine kinases, DosS and DosT, and a response regulator, DosR (42). DosS and DosT both bind the respiration-impairing gases NO and CO (19, 20, 38), further supporting the hypothesis that the DosR regulon responds to, and is important during, conditions that do not allow aerobic respiration. Although the majority of the DosR-regulated genes have not been characterized, the timing of their induction combined with the conditions under which they respond suggests that they may play a role in adaptation of M. tuberculosis to its host environment. Consistent with this notion, DosR regulon genes are induced in the lungs of M. tuberculosis-infected mice (43), as well as in interferon-gamma-activated murine macrophages (34) and guinea pigs (37).Several studies have suggested that the DosR regulon plays a role in latent infection and in persistence in animal models that resemble human infection in some respects. Leyten et al. found that latently infected humans are more likely than humans with active infections to bear T cells specific for DosR regulon antigens (23), suggesting that the regulon is expressed during latency. Two recent studies confirmed that there is an immune response to DosR regulon antigens during latent infection (4, 36). Further evidence for clinical relevance in humans comes from a study showing that M. tuberculosis in sputum expresses the DosR regulon (15). The importance of this regulon for persistence in rabbit and guinea pig models was demonstrated by data showing a 2-log decrease in recovery of a DosR mutant 2 weeks (guinea pig) and 8 weeks (rabbit) after aerosol infection (11). A DosR mutant was also found to be significantly attenuated in guinea pig infection (26), further supporting the notion that the DosR regulon is required for persistence in vivo. It should be noted that in both studies showing the DosR phenotype (11, 26), full complementation and reversion to full virulence were not observed. However, it is now known that regulation of dosR expression is quite complex. Multiple regulatory sequences exist in and upstream of Rv3134c, the gene directly upstream of dosR (8). Failure to include such a regulatory sequence in a complemented strain would likely result in misregulation of dosR and poor complementation. Studies of DosR regulon mutants for murine infection have produced inconsistent findings that vary from hypervirulent (30) to attenuated (11) and not attenuated (3, 31). When animal models are compared, it is important to remember that M. tuberculosis-induced granulomas in primates, rabbits, and guinea pigs develop caseous necrosis and are hypoxic and/or anaerobic, while M. tuberculosis induced-granulomas in mice are neither hypoxic nor anaerobic (2, 21, 41). Furthermore, M. tuberculosis divides regularly in chronic murine infections (16), in contrast to the replication during latent infections, as demonstrated in the studies of Lillebaek et al. (24, 25). Such studies underscore the significant differences between models.A previous study with a DosR mutant in a closely related Mycobacterium bovis BCG strain showed that DosR expression is required for survival in an in vitro Wayne-like model of dormancy (5). Unexpectedly, two similar studies in M. tuberculosis did not show a strong survival defect for a DosR mutant (31, 43). The most recent study showed that there was only a modest survival defect in an H37Rv DosR mutant and concluded that the DosR regulon is a short-term phenomenon and is not responsible for the adaptation necessary to survive under primarily hypoxic conditions in vitro (31, 32).In this study we showed that the DosR regulon is required for M. tuberculosis survival during anaerobic dormancy. We also used a combination of genetic and biochemical approaches to demonstrate that this regulon is necessary to shift away from oxygen consumption, maintain ATP levels, and balance the redox state (NAD/NADH ratio) of the cell as oxygen becomes scarce. Furthermore, we showed that the DosR regulon is necessary for optimal transition of M. tuberculosis back to aerobic growth from an anaerobic or nitric oxide-induced nonrespiring state.     

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.     

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.     

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.