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.     

Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange

A targeting method to insert genes at a previously characterized genetic locus to make plant transformation and transgene expression predictable is highly desirable for plant biotechnology. We report the successful targeting of transgenes to predefined soybean (Glycine max) genome sites using the yeast FLP-FRT recombination system. First, a target DNA containing a pair of incompatible FRT sites flanking a selection gene was introduced in soybean by standard biolistic transformation. Transgenic events containing a single copy of the target were retransformed with a donor DNA, which contained the same pair of FRT sites flanking a different selection gene, and a FLP expression DNA. Precise DNA cassette exchange was achieved between the target and donor DNA via recombinase-mediated cassette exchange, so that the donor DNA was introduced at the locus previously occupied by the target DNA. The introduced donor genes expressed normally and segregated according to Mendelian laws.Plant transformation has challenges such as random integration, multiple transgene copies, and unpredictable expression. Homologous recombination (Iida and Terada, 2005; Wright et al., 2005) and DNA recombinase-mediated site-specific integration (SSI) are promising technologies to address the challenges for placing a single copy of transgenes into a precharacterized site in a plant genome.Several site-specific DNA recombination systems, such as the bacteriophage Cre-lox and the yeast FLP-FRT and R-RS, have been used in SSI studies (Ow, 2002; Groth and Calos, 2003). A common feature of these systems is that each system consists of a recombinase Cre, FLP, or R and two identical or similar palindromic recognition sites, lox, FRT, or RS. Each recognition site contains a short asymmetric spacer sequence where DNA strand exchange takes place, flanked by inverted repeat sequences where the corresponding recombinase specifically binds. If two recognition sites are located in cis on a DNA molecule, the DNA segment can be excised if flanked by two directionally oriented sites or inverted if flanked by two oppositely oriented sites. If two recognition sites are located in trans on two different DNA molecules, a reciprocal translocation can happen between the two DNA molecules or the two molecules can integrate if at least one of them is a circular DNA (Ow, 2002; Groth and Calos, 2003).Single-site SSI can integrate a circular donor DNA containing one recognition site into a similar site previously placed in a plant genome. The integrated transgene now flanked by two recognition sites is vulnerable to excision. Transient Cre expression and the use of mutant lox sites to create two less compatible sites after integration helped reduce the subsequent excision in tobacco (Nicotiana tabacum; Albert et al., 1995; Day et al., 2000). A similar approach was used to produce SSI events in rice (Oryza sativa), and the transgene was proven stably expressed over generations (Srivastava and Ow, 2001; Srivastava et al., 2004; Chawla et al., 2006). Using a promoter trap to displace a cre gene in the genome with a selection gene from the donor, approximately 2% SSI was achieved in Arabidopsis (Arabidopsis thaliana; Vergunst et al., 1998).When two recognition sites located on a linear DNA molecule are similar enough to be recognized by the same recombinase but different enough to reduce or prevent DNA recombination from happening between them, the DNA segment between the two sites may not be easily excised or inverted. When a circular DNA molecule carrying the same two incompatible sites is introduced, the circular DNA can integrate by the corresponding recombinase at either site on the linear DNA to create a collinear DNA with four recognition sites, two from the original linear DNA and two from the circular DNA. DNA excision can subsequently occur between any pair of compatible sites to restore the two original DNA molecules or to exchange the intervening DNA segments between the two DNA molecules. This process, termed recombinase-mediated cassette exchange (RMCE), can be employed to integrate transgenes directionally into predefined genome sites (Trinh and Morrison, 2000; Baer and Bode, 2001).RMCE using two oppositely oriented identical RS sites, a donor containing the R recombinase gene and a third RS site to limit random integration, resulted in cassette exchange between the donor and a previously placed target in tobacco (Nanto et al., 2005). RMCE using both the Cre-lox and FLP-FRT systems improved RMCE frequency in animal cell cultures (Lauth et al., 2002). RMCE using two directly oriented incompatible FRT sites and transiently expressed FLP recombinase achieved cassette exchange between a target previously placed in the Drosophila genome and a donor introduced as a circular DNA (Horn and Handler, 2005). A gene conversion approach involving Cre-lox- and FLP-FRT-mediated SSI, RMCE, and homologous recombination was explored in maize (Zea mays; Djukanovic et al., 2006). RMCE using two oppositely oriented incompatible lox sites and transiently expressed Cre recombinase produced single-copy RMCE plants in Arabidopsis (Louwerse et al., 2007).To develop FLP-FRT-mediated RMCE in soybean (Glycine max), we created transgenic target lines containing a hygromycin resistance gene flanked by two directly oriented incompatible FRT sites via biolistic transformation. Single-copy target lines were selected and retransformed with a donor DNA containing a chlorsulfuron resistance gene flanked by the same pair of FRT sites. An FLP expression DNA was cobombarded to transiently provide FLP recombinase. RMCE events were obtained from multiple target lines and confirmed by extensive molecular characterization.     

Cre-lox-Based Method for Generation of Large Deletions within the Genomic Magnetosome Island of Magnetospirillum gryphiswaldense

Magnetosome biomineralization and magnetotaxis in magnetotactic bacteria are controlled by numerous, mostly unknown gene functions that are predominantly encoded by several operons located within the genomic magnetosome island (MAI). Genetic analysis of magnetotactic bacteria has remained difficult and requires the development of novel tools. We established a Cre-lox-based deletion method which allows the excision of large genomic fragments in Magnetospirillum gryphiswaldense. Two conjugative suicide plasmids harboring lox sites that flanked the target region were subsequently inserted into the chromosome by homologous recombination, requiring only one single-crossover event, respectively, and resulting in a double cointegrate. Excision of the targeted chromosomal segment that included the inserted plasmids and their resistance markers was induced by trans expression of Cre recombinase, which leaves behind a scar of only a single loxP site. The Cre helper plasmid was then cured from the deletant strain by relief of antibiotic selection. We have used this method for the deletion of 16.3-kb, 61-kb, and 67.3-kb fragments from the genomic MAI, either in a single round or in subsequent rounds of deletion, covering a region of approximately 87 kb that comprises the mamAB, mms6, and mamGFDC operons. As expected, all mutants were Mag⁻ and some were Mot⁻; otherwise, they showed normal growth patterns, which indicates that the deleted region is not essential for viability in the laboratory. The method will facilitate future functional analysis of magnetosome genes and also can be utilized for large-scale genome engineering in magnetotactic bacteria.Magnetosomes are unique membrane-enveloped organelles that are formed by magnetotactic bacteria (MTB) for magnetic navigation (2, 37). The mechanism of magnetosome formation is within the focus of a multidisciplinary interest and has relevance for biotechnological applications (5). It has been recognized that the biomineralization of inorganic magnetite crystals and their assembly into highly ordered magnetosome chains are under strict genetic control. Recent studies combining proteomic and bioinformatic approaches suggested that the genetic determination of magnetosome formation is complex and may potentially involve 25 to 50 gene functions (15), with unknown numbers of accessory genes and those controlling signal transduction and motility to achieve effective magnetotaxis (8, 9, 12, 26, 27, 29). However, the functional characterization of these candidate genes has been lagging behind. This is due to technical difficulties and the lack of facile tools for genetic manipulation of MTB. Allelic replacement systems have been established for Magnetospirillum magneticum (18) and Magnetospirillum gryphiswaldense (39, 40), but so far, there are only few examples of these for magnetosome genes that were functionally characterized because of the tedious and cumbersome procedures required for mutant generation (11, 19, 28, 31-32). Most genes controlling magnetosome formation in these and other MTB are located within a genomic magnetosome island (MAI) (34), which is genetically instable during stationary growth (47) and more or less conserved in other MTB (12, 13, 35). Most known magnetosome genes are organized within several conserved operons, which are interspersed with large, poorly conserved genome sections of unknown functions that have been speculated to represent genetic junk irrelevant for magnetotaxis but to cause genetic instability by their high content of repeats and transposable elements (34, 47). Thus, for large-scale functional genome analysis and rearrangements of the MAI, there is a great need for additional and more efficient genetic methods.Artificial genome recombination systems have been described for a number of bacteria. Many of them are based on the Cre-loxP system of the P1 phage (42). The Cre-loxP recombination system is a simple two-component system that is recognized as a powerful genetic tool in a multitude of eukaryotic and prokaryotic organisms (4, 6, 48). The Cre protein belongs to the integrase family of site-specific recombinases and catalyzes reciprocal site-specific recombination of DNA at 34-bp loxP sites, resulting in either excision or inversion, depending on the parallel or antiparallel orientation of the loxP sites, respectively (21). It does not require any host cofactors or accessory proteins (7). Cre-lox deletion has several advantages over other methods, such as a high efficiency and the independency of the length of DNA located between the two lox sites. The utility of Cre-lox systems has been demonstrated in a wide variety of Gram-positive and Gram-negative bacteria (17, 22-23). In several studies, it was applied for the generation of large-scale deletions, as in for example, the Gram-positive Corynebacterium glutamicum (43-46) and Bacillus subtilis (49).In M. gryphiswaldense, the functionality of a Cre-loxP antibiotic marker recycling system (25) has been previously demonstrated by deletion of a single gene based on double-crossover insertion of two loxP sites, followed by subsequent Cre-mediated excision (31). In this study, we describe a novel strategy for Cre-loxP-mediated deletion of large genomic fragments which requires only two single crossovers. The system has been validated by the generation of three large deletions, two single and one combination within the MAI, which demonstrated that the total deleted region of approximately 87 kb is not essential for viability and growth in the laboratory.     

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).     

Xer Site-Specific Recombination,an Efficient Tool To Introduce Unmarked Deletions into Mycobacteria

Genetic manipulation of mycobacteria still represents a serious challenge due to the lack of tools and selection markers. In this report, we describe the development of an intrinsically unstable excisable cassette for introduction of unmarked mutations in both Mycobacterium smegmatis and Mycobacterium tuberculosis.Mycobacterium tuberculosis causes about 2 million deaths worldwide every year (15). Over the last few years, M. tuberculosis pathogenesis characterization at a molecular level required the development of efficient genetic tools for recombination and mutagenesis. The employment of replicating temperature-sensitive and suicide plasmids (14), specialized transducing mycobacteriophages (1, 9), and a recombineering system based on two exogenous recombinases (24) improved the ability to obtain mycobacterial mutants by homologous recombination. However, the availability of only few selection markers represents a real problem when multiple knockouts are required for the study of redundant gene families in mycobacteria. A way to circumvent this problem is by the production of unmarked mutations, which can be obtained by homologous recombination and selection for sequential crossing-over events using both positive and negative markers for counterselection of the different allelic exchange events (13), or by a different approach relying on sequence-specific recombination systems allowing the excision of the positive selection marker after it has been used to select for the recombination event (19).Three different sequence-specific recombinase systems have been successfully used with mycobacteria: the TnpR/res system of the γδ transposon (1), the Flp/FRT system of Saccharomyces cerevisiae (18, 20), and the LoxP/cre systems from bacteriophage P1 (11, 19). While the Flp/FRT and the Lox/cre systems were shown to work efficiently in both slow- and fast-growing mycobacteria, the Tnp/res system was proved to be efficient only in fast-growing species. All of these systems require a first step during which the expression of an exogenous resolvase or recombinase from a replicative plasmid allows the excision of the resistant marker and a second step to eliminate the replicative plasmid, making the procedure very time-consuming, particularly when working with slow-growing mycobacteria.Recently, a new sequence-specific recombinase system based on the endogenous Xer recombinases (Xer-cise) was shown to be amenable for genetic manipulation and construction of unmarked deletion mutants in Escherichia coli and Bacillus subtilis (4). In this system, the antibiotic resistance cassette, flanked by dif sites, is intrinsically unstable since the endogenous recombinases XerC and XerD recognize and resolve the dif sites that border the cassette. This method, relying on endogenous recombinases, does not require the introduction and the subsequent removal of replicating plasmids carrying exogenous genes, making it extremely simple and practical.E. coli XerC and XerD recombinases are essential for chromosome segregation during cell division, as their role is to resolve chromosome dimers to monomers recognizing the 28-bp dif sequence present at the replication terminus region (8). The Xer site-specific recombination system is very well conserved in prokaryotes with circular chromosomes, and homologues of XerC and XerD have been identified among Gram-negative and Gram-positive bacteria (16).In this report, we adapted the Xer-cise technique to mycobacteria, demonstrating that it can be employed as a practical and efficient genetic tool for manipulating both M. tuberculosis and Mycobacterium smegmatis.     

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.     

Repeat-Associated Plasticity in the Helicobacter pylori RD Gene Family

The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two H. pylori strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the H. pylori RD gene family. Each gene of this family is composed of a conserved 3′ region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5′ region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42 H. pylori strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject''s stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by H. pylori-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding H. pylori persistence in its natural host.Helicobacter pylori, a gram-negative bacterium, is remarkable for its ability to persist in the human stomach for decades. Colonization with H. pylori increases risk for peptic ulcer disease and gastric adenocarcinoma (53, 70) and elicits a vigorous immune response (15). The persistence of H. pylori occurs in a niche in the human body previously considered inhospitable to microbial colonization: the acidic stomach replete with proteolytic enzymes.H. pylori strains exhibit substantial genetic diversity, including extensive variation in the presence, arrangement, order, and identity of genes (2, 4-7, 25, 51, 74). Furthermore, analyses of multiple single-colony H. pylori isolates from separate stomach biopsy specimens of individual patients have demonstrated diversity, both within hosts (27, 65), and over time (36). The mechanisms that generate H. pylori genetic diversity may be among the factors that enable persistence in this environment (3, 28).While the natural ability of H. pylori for transformation and recombination may explain some of the intra- and interhost genetic variation observed in this bacterium (43), point mutations and interspecies recombination alone are not sufficient for explaining the extent of the variation in H. pylori (14, 32). The initial genomic sequencing of H. pylori strains 26695 and J99 (6, 72) revealed large amounts of repetitive DNA (1, 59). DNA repeats in bacteria are associated with mechanisms of plasticity, such as phase variation (49, 67); slipped-strand mispairing (41, 46); and increased rates of recombination, deletion, and insertion (17, 60, 62). Because many of the recombination repair and mismatch repair mechanisms common in bacteria are absent or modified in H. pylori (28-30, 56, 76), this organism may be particularly susceptible to the diversifying effects of repetitive DNA. In fact, loci in the H. pylori genome containing repetitive DNA have been shown to exhibit extensive inter- and intrahost variation (9, 10, 28, 37).We hypothesized that identification of repetitive DNA hotspots in H. pylori would allow the recognition of genes whose variation could aid in persistence. To examine this hypothesis, we conducted in silico analyses to identify open reading frames (ORFs) enriched for DNA repeats and then used a combination of sequence analyses and immunoassays to examine the patterns associated with the specific repetitive DNA observed. Our approach led to the realization that a previously identified H. pylori-specific gene family (19, 52) exhibits extensive genetic variation at multiple levels.     

XRCC2 and XRCC3 Regulate the Balance between Short- and Long-Tract Gene Conversions between Sister Chromatids

Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DNA lesions associated with replication and is thought to be important for suppressing genomic instability. The mechanisms regulating the initiation and termination of SCR in mammalian cells are poorly understood. Previous work has implicated all the Rad51 paralogs in the initiation of gene conversion and the Rad51C/XRCC3 complex in its termination. Here, we show that hamster cells deficient in the Rad51 paralog XRCC2, a component of the Rad51B/Rad51C/Rad51D/XRCC2 complex, reveal a bias in favor of long-tract gene conversion (LTGC) during SCR. This defect is corrected by expression of wild-type XRCC2 and also by XRCC2 mutants defective in ATP binding and hydrolysis. In contrast, XRCC3-mediated homologous recombination and suppression of LTGC are dependent on ATP binding and hydrolysis. These results reveal an unexpectedly general role for Rad51 paralogs in the control of the termination of gene conversion between sister chromatids.DNA double-strand breaks (DSBs) are potentially dangerous lesions, since their misrepair may cause chromosomal translocations, gene amplifications, loss of heterozygosity (LOH), and other types of genomic instability characteristic of human cancers (7, 9, 21, 40, 76, 79). DSBs are repaired predominantly by nonhomologous end joining or homologous recombination (HR), two evolutionarily conserved DSB repair mechanisms (8, 12, 16, 33, 48, 60, 71). DSBs generated during the S or G₂ phase of the cell cycle may be repaired preferentially by HR, using the intact sister chromatid as a template for repair (12, 26, 29, 32, 71). Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DSBs, which has led to the proposal that SCR protects against genomic instability, cancer, and aging. Indeed, a number of human cancer predisposition genes are implicated in SCR control (10, 24, 45, 57, 75).HR entails an initial processing of the DSB to generate a free 3′ single-stranded DNA (ssDNA) overhang (25, 48, 56). This is coupled to the loading of Rad51, the eukaryotic homolog of Escherichia coli RecA, which polymerizes to form an ssDNA-Rad51 “presynaptic” nucleoprotein filament. Formation of the presynaptic filament is tightly regulated and requires the concerted action of a large number of gene products (55, 66, 68). Rad51-coated ssDNA engages in a homology search by invading homologous duplex DNA. If sufficient homology exists between the invading and invaded strands, a triple-stranded synapse (D-loop) forms, and the 3′ end of the invading (nascent) strand is extended, using the donor as a template for gene conversion. This recombination intermediate is thought to be channeled into one of the following two major subpathways: classical gap repair or synthesis-dependent strand annealing (SDSA) (48). Gap repair entails the formation of a double Holliday junction, which may resolve into either crossover or noncrossover products. Although this is a major pathway in meiotic recombination, crossing-over is highly suppressed in somatic eukaryotic cells (26, 44, 48). Indeed, the donor DNA molecule is seldom rearranged during somatic HR, suggesting that SDSA is the major pathway for the repair of somatic DSBs (26, 44, 49, 69). SDSA terminates when the nascent strand is displaced from the D-loop and pairs with the second end of the DSB to form a noncrossover product. The mechanisms underlying displacement of the nascent strand are not well understood. However, failure to displace the nascent strand might be expected to result in the production of longer gene conversion tracts during HR (36, 44, 48, 63).Gene conversion triggered in response to a Saccharomyces cerevisiae or mammalian chromosomal DSB generally results in the copying of a short (50- to 300-bp) stretch of information from the donor (short-tract gene conversion [STGC]) (14, 47, 48, 67, 69). A minority of gene conversions in mammalian cells entail more-extensive copying, generating gene conversion tracts that are up to several kilobases in length (long-tract gene conversion [LTGC]) (26, 44, 51, 54, 64). In yeast, very long gene conversions can result from break-induced replication (BIR), a highly processive form of gene conversion in which a bona fide replication fork is thought to be established at the recombination synapse (11, 36, 37, 39, 61, 63). In contrast, SDSA does not require lagging-strand polymerases and appears to be much less processive than a conventional replication fork (37, 42, 78). BIR in yeast has been proposed to play a role in LOH in aging yeast, telomere maintenance, and palindromic gene amplification (5, 41, 52). It is unclear to what extent a BIR-like mechanism operates in mammalian cells, although BIR has been invoked to explain telomere elongation in tumors lacking telomerase (13). It is currently unknown whether LTGC and STGC in somatic mammalian cells are products of mechanistically distinct pathways or whether they represent alternative outcomes of a common SDSA pathway.Vertebrate cells contain five Rad51 paralogs—polypeptides with limited sequence homology to Rad51—Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3 (74). The Rad51 paralogs form the following two major complexes: Rad51B/Rad51C/Rad51D/XRCC2 (BCDX2) and Rad51C/XRCC3 (CX3) (38, 73). Genetic deletion of any one of the rad51 paralogs in the mouse germ line produces early embryonic lethality, and mouse or chicken cells lacking any of the rad51 paralogs reveal hypersensitivity to DNA-damaging agents, reduced frequencies of HR and of sister chromatid exchanges, increased chromatid-type errors, and defective sister chromatid cohesion (18, 72, 73, 82). Collectively, these data implicate the Rad51 paralogs in SCR regulation. The purified Rad51B/Rad51C complex has been shown to assist Rad51-mediated strand exchange (62). XRCC3 null or Rad51C null hamster cells reveal a bias toward production of longer gene conversion tracts, suggesting a role for the CX3 complex in late stages of SDSA (6, 44). Rad51C copurifies with branch migration and Holliday junction resolution activities in mammalian cell extracts (35), and XRCC3, but not XRCC2, facilitates telomere shortening by reciprocal crossing-over in telomeric T loops (77). These data, taken together with the meiotic defects observed in Rad51C hypomorphic mice, suggest a specialized role for CX3, but not for BCDX2, in resolving Holliday junction structures (31, 58).To further address the roles of Rad51 paralogs in late stages of recombination, we have studied the balance between long-tract (>1-kb) and short-tract (<1-kb) SCR in XRCC2 mutant hamster cells. We found that DSB-induced gene conversion in both XRCC2 and XRCC3 mutant cells is biased in favor of LTGC. These defects were suppressed by expression of wild-type (wt) XRCC2 or XRCC3, respectively, although the dependence upon ATP binding and hydrolysis differed between the two Rad51 paralogs. These results indicate that Rad51 paralogs play a more general role in determining the balance between STGC and LTGC than was previously appreciated and suggest roles for both the BCDX2 and CX3 complexes in influencing the termination of gene conversion in mammals.     

Coexistence of Different Genotypes in the Same Bat and Serological Characterization of Rousettus Bat Coronavirus HKU9 Belonging to a Novel Betacoronavirus Subgroup

Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), a recently identified coronavirus of novel Betacoronavirus subgroup D, from Leschenault''s rousette, was previously found to display marked sequence polymorphism among genomes of four strains. Among 10 bats with complete RNA-dependent RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes sequenced, three and two sequence clades for all three genes were codetected in two and five bats, respectively, suggesting the coexistence of two or three distinct genotypes of Ro-BatCoV HKU9 in the same bat. Complete genome sequencing of the distinct genotypes from two bats, using degenerate/genome-specific primers with overlapping sequences confirmed by specific PCR, supported the coexistence of at least two distinct genomes in each bat. Recombination analysis using eight Ro-BatCoV HKU9 genomes showed possible recombination events between strains from different bat individuals, which may have allowed for the generation of different genotypes. Western blot assays using recombinant N proteins of Ro-BatCoV HKU9, Betacoronavirus subgroup A (HCoV-HKU1), subgroup B (SARSr-Rh-BatCoV), and subgroup C (Ty-BatCoV HKU4 and Pi-BatCoV HKU5) coronaviruses were subgroup specific, supporting their classification as separate subgroups under Betacoronavirus. Antibodies were detected in 75 (43%) of 175 and 224 (64%) of 350 tested serum samples from Leschenault''s rousette bats by Ro-BatCoV HKU9 N-protein-based Western blot and enzyme immunoassays, respectively. This is the first report describing coinfection of different coronavirus genotypes in bats and coronavirus genotypes of diverse nucleotide variation in the same host. Such unique phenomena, and the unusual instability of ORF7a, are likely due to recombination which may have been facilitated by the dense roosting behavior and long foraging range of Leschenault''s rousette.Coronaviruses infect a wide variety of animals in which they can cause respiratory, enteric, hepatic, and neurological diseases of various severities. Based on genotypic and serological characterization, coronaviruses were traditionally classified into three distinct groups, groups 1, 2, and 3 (3, 27, 59). Recently, the Coronavirus Study Group of the International Committee for Taxonomy of Viruses has renamed the traditional group 1, 2, and 3 coronaviruses as Alphacoronavirus, Betacoronavirus, and Gammacoronavirus, respectively (http://talk.ictvonline.org/media/p/1230.aspx). Coronaviruses are known to have a high frequency of recombination as a result of their unique mechanism of viral replication (27). Such tendency for recombination and high mutation rates may allow them to adapt to new hosts and ecological niches (24, 47, 52).The severe acute respiratory syndrome (SARS) epidemic has boosted interest in the study of coronaviruses in humans and animals (21, 34, 38, 41, 54). In the past few years, there has been a dramatic increase in the number of newly described human and animal coronaviruses (2, 4, 5, 8-10, 15-20, 23, 25, 28, 30, 32, 35, 36, 39, 43, 45, 50, 51, 53, 56, 58). Two novel human coronaviruses, human coronavirus NL63 (HCoV-NL63) and human coronavirus HKU1 (HCoV-HKU1), belonging to Alphacoronavirus and Betacoronavirus, respectively, have been discovered, in addition to the human coronavirus OC43 (HCoV-OC43), human coronavirus 229E (HCoV-229E), and SARS coronavirus (SARS-CoV) (17, 29, 45, 53, 55). We have also previously described the discovery of a diversity of novel coronaviruses in wild bats and birds in China, including SARSr-Rh-BatCoV, belonging to Betacoronavirus subgroup B, from Chinese horseshoe bats (30, 48, 56). Among these novel coronaviruses, three avian coronaviruses were found to belong to a novel subgroup of Gammacoronavirus (Gammacoronavirus subgroup C), while three bat coronaviruses were found to belong to two novel subgroups of Betacoronavirus (Betacoronavirus subgroups C and D) (48, 50). Based on the presence of the huge diversity of coronaviruses in Alphacoronavirus and Betacoronavirus among various bat species, bats are likely the reservoir for the ancestor of these two coronavirus genera (47).During our genome analysis of these novel coronaviruses, one of them, Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), belonging to Betacoronavirus subgroup D, which was identified in Leschenault''s rousette bats, was found to display marked nucleotide and amino acid sequence polymorphism among the four strains with complete genome sequences (50). In our study on HCoV-HKU1, it has been shown that such sequence polymorphisms may indicate the presence of different genotypes (52). By complete genome sequence analysis of the potentially different genotypes of HCoV-HKU1, we have demonstrated for the first time natural recombination in a human coronavirus, resulting in the generation of at least three genotypes (52). We have also recently shown that recombination between different strains of SARSr-Rh-BatCoV from different regions of China may have given rise to the emergence of civet SARSr-CoV (31). To investigate the presence of different genotypes of Ro-BatCoV HKU9, the complete RNA-dependent RNA polymerase (RdRp) (corresponding to nsp12), spike (S), and nucleocapsid (N) gene sequences of Ro-BatCoV HKU9 from 10 additional bats were determined. Since sequence analysis showed the possible coexistence of different genotypes in seven bat individuals, complete genome sequencing of these distinct genotypes from two bats was carried out to investigate for possible recombination events among the different genotypes. In addition, serological characterization of Ro-BatCoV HKU9 was also performed by Western blot and enzyme immunoassays using recombinant Ro-BatCoV HKU9 nucleocapsid proteins and recombinant nucleocapsid proteins of Betacoronavirus subgroup A, B, and C coronaviruses to determine possible cross-reactivity among the different Betacoronavirus subgroups and the seroepidemiology of Ro-BatCoV HKU9 in Leschenault''s rousette bats.     

Recombineering Using RecTE from Pseudomonas syringae

In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The ability of the pseudomonad-encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single-stranded DNA oligonucleotides and that efficient recombination of double-stranded DNA requires the expression of both the RecT and RecE homologs. Additionally, we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.There are currently more than 1,500 completed or draft bacterial genome sequences available for public access. This data resource continues to grow rapidly and provides potential insights into the roles of individual genes and regulons. However, testing hypotheses based on sequence data requires direct experimental manipulation of each genome. While many established methods for modifying bacterial DNA can assist in genetic analysis of these organisms, they are often time-consuming and limited with respect to the types of changes that can be directed.New advances in recombineering (genetic engineering by recombination) offer powerful alternative strategies for site-directed mutagenesis of genomic loci and provide methods for rapid and precise functional genomic analysis in some organisms (9, 29, 36-38, 41, 43). In these cases, recombineering is very efficient when phage-encoded recombinases are supplied, such that in vivo expression of these proteins enables direct genetic engineering of chromosomal and episomal replicons. These proteins catalyze RecA-independent recombination (21) of linear DNA substrates with homologous genomic target loci. The phage recombination functions typically involve the coordinated action of a 5′-to-3′ exonuclease (i.e., RecE or lambda Exo) and a single-stranded DNA (ssDNA)-annealing and strand invasion protein (i.e., RecT or lambda Beta), which we shall refer to as recombinases for brevity. The recombinase binds to 3′ ssDNA ends that are exposed by the action of the exonuclease, forming a protein-DNA filament, which protects the substrate DNA and promotes annealing with the homologous genomic sequence (4, 17, 19, 24). The recombinases are sufficient to facilitate recombination of ssDNA oligonucleotides, presumably because the oligonucleotides resemble the 5′-end-resected double-stranded DNA (dsDNA) substrate (11). Most of the recombinase proteins that have been shown to facilitate recombination are located in operons and are adjacent to the exonuclease-encoding genes, although there are cases where functional recombinase proteins have been identified without an accompanying exonuclease (9).Recombineering technologies have great potential in functional genomic applications and have worked exceptionally well in a few species, but adapting current systems to different bacteria is often problematic. Evidence suggests that these recombination systems have narrow species specificity such that a given system may catalyze robust recombination in one species and be essentially nonfunctional when expressed in another (9, 37). The reasons for this are not known but may be due to a requirement for specific interactions between the recombinase and host-encoded factors (9). Although there is a need to apply recombineering techniques to Pseudomonas species, only marginal success using the characterized phage recombination systems has been reported (14, 23). Most notably, recombinant strains of Pseudomonas aeruginosa were generated using long-homology substrates in the presence of plasmids expressing the lambda Red genes, but the relative influence of the Red genes was not reported (23).Here, we describe the identification of new recombineering proteins that function in a pseudomonad. The genes that encode proteins with similarity to the RecE/RecT proteins of the Rac prophage and lambda Red Exo and Beta were identified in Pseudomonas syringae pv. syringae B728a. These proteins promote efficient homologous recombination between genomic loci and linear DNA substrates introduced directly into P. syringae pv. tomato DC3000 cells by electroporation. These findings provide a foundation for more efficient site-directed mutagenesis of chromosomal loci in P. syringae and serve as a strategy for identifying similar proteins for recombineering in other bacteria.     

Xer1-Mediated Site-Specific DNA Inversions and Excisions in Mycoplasma agalactiae

Surface antigen variation in Mycoplasma agalactiae, the etiologic agent of contagious agalactia in sheep and goats, is governed by site-specific recombination within the vpma multigene locus encoding the Vpma family of variable surface lipoproteins. This high-frequency Vpma phase switching was previously shown to be mediated by a Xer1 recombinase encoded adjacent to the vpma locus. In this study, it was demonstrated in Escherichia coli that the Xer1 recombinase is responsible for catalyzing vpma gene inversions between recombination sites (RS) located in the 5′-untranslated region (UTR) in all six vpma genes, causing cleavage and strand exchange within a 21-bp conserved region that serves as a recognition sequence. It was further shown that the outcome of the site-specific recombination event depends on the orientation of the two vpma RS, as direct or inverted repeats. While recombination between inverted vpma RS led to inversions, recombination between direct repeat vpma RS led to excisions. Using a newly developed excision assay based on the lacZ reporter system, we were able to successfully demonstrate under native conditions that such Xer1-mediated excisions can indeed also occur in the M. agalactiae type strain PG2, whereas they were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1 recombinase. Unless there are specific regulatory mechanisms preventing such excisions, this might be the cost that the pathogen has to render at the population level for maintaining this high-frequency phase variation machinery.Members of the bacterial class Mollicutes, which are generally referred to as mycoplasmas, are considered among the simplest self-replicating prokaryotes carrying minimal genomes. Even having lost many biosynthetic pathways during a reductive evolution, mycoplasmas represent important pathogens of humans, animals, and plants, as they are equipped with sophisticated molecular mechanisms allowing them to spontaneously change their cell surface repertoire to persist in immunocompetent hosts (25).The important ruminant pathogen Mycoplasma agalactiae causes contagious agalactia in sheep and goats and exhibits antigenic diversity by site-specific DNA rearrangements within a pathogenicity island-like gene locus (9, 10, 26). The so-called vpma locus constitutes a family of six distinct but related genes that encode major immunodominant membrane lipoproteins, the Vpmas (variable proteins of Mycoplasma agalactiae) (10, 11). These surface-associated proteins vary in expression at an unusually high frequency, and only one vpma gene at a time is transcribed from a single promoter present in that locus, while all other genes are silent (9, 10). An open reading frame (ORF) with homology to the λ-integrase family of site-specific recombinases was found in the vicinity of the vpma locus and was predicted to mediate DNA inversions responsible for switching the promoter from an active vpma gene to a silent one, resulting in alteration of vpma expression (9, 10). This recombinase, designated Xer1, was indeed recently demonstrated to be responsible for phase variation of Vpma proteins (4). Targeted knockouts of the xer1 gene by homologous recombination prevented Vpma switching and produced Vpma phase-locked mutants (PLMs) steadily expressing a single vpma gene without any variation. Complementation of the wild-type xer1 gene in these PLMs restored Vpma phase variation (4). Similar systems generating surface diversity by DNA inversions involving site-specific recombination have been identified in other mycoplasma species (3, 18, 26).Site-specific recombination systems are widespread among bacteria, and the biological functions of these systems depend strongly on the participating recombination sites (RS) (16, 24, 27). Excision events between direct repeat RS usually resolve chromosome or plasmid dimers, which can arise through homologous recombination, ensuring proper segregation of newly replicated genetic material to daughter cells (1). Also, site-specific recombination mediates integration and excision of phage genomes into and out of the host chromosome (13). In contrast, site-specific inversion involving inverted repeat RS generates genetic diversity and often controls the expression of genes that are important for pathogenesis (21).The Xer1 recombinase of M. agalactiae belongs to the λ-integrase family of site-specific recombinases (10). Members of this family share four strongly conserved amino acid residues (R-H-R-Y) within the C-terminal half of the protein. This tetrad includes the active tyrosine residue that is directly involved in the recombination reaction (8). Recombination occurs by formation and resolution of a Holliday junction intermediate involving a covalent linkage between the recombinase and the DNA through the tyrosine residue. Since energy cofactors such as ATP are not required, such recombination events can occur in the absence of replication (16, 24).Sequence alignment of vpma genes identified a conserved 21-bp region within the 5′-untranslated region (UTR) in all vpma genes that was predicted to be involved in Xer1-mediated inversions (10). The present study clearly demonstrates that the Xer1 recombinase recognizes RS located within the 5′ UTR of vpma genes, causing cleavage and strand exchange within a conserved region of 21 bp. By placing two vpma-derived RS on a plasmid along with the xer1 gene, recombination events were demonstrated in Escherichia coli upon Xer1 induction via PCR and restriction analysis. Although the conserved 21-bp region was sufficient for inversions, additional nucleotides flanking it at the 5′ end were found to have a positive influence on the rate of recombination. An interesting outcome of these studies was that Xer1 also mediated excisions between direct repeat vpma RS in E. coli. This raised the intriguing possibility that such Xer1-mediated excisions also occur in the native M. agalactiae system. For further analysis of such excision events in the native system, we tested the feasibility of using the lacZ reporter tool in M. agalactiae, as lacZ is known to be expressed successfully in few other mycoplasma species, to study gene expression by use of promoter probe vectors (15, 19, 22, 23). We developed an excision assay based on blue-white phenotype selection to study Xer1-mediated excisions in M. agalactiae, thus displaying a novel application of the lacZ reporter gene in mycoplasmas. Successful implementation of this reporter system demonstrated Xer1-mediated excisions in the M. agalactiae type strain PG2, based on blue-white selection and PCR analysis. As expected, such excisions were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1. Excisions in the native system imply that genetic material is susceptible to loss, which might be the cost for maintaining the machinery of high-frequency gene shuffling for a greater population advantage, unless there are specific regulatory mechanisms preventing such excisions.     

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.     

Ecoepidemiology and Complete Genome Comparison of Different Strains of Severe Acute Respiratory Syndrome-Related Rhinolophus Bat Coronavirus in China Reveal Bats as a Reservoir for Acute,Self-Limiting Infection That Allows Recombination Events

Despite the identification of severe acute respiratory syndrome-related coronavirus (SARSr-CoV) in Rhinolophus Chinese horseshoe bats (SARSr-Rh-BatCoV) in China, the evolutionary and possible recombination origin of SARSr-CoV remains undetermined. We carried out the first study to investigate the migration pattern and SARSr-Rh-BatCoV genome epidemiology in Chinese horseshoe bats during a 4-year period. Of 1,401 Chinese horseshoe bats from Hong Kong and Guangdong, China, that were sampled, SARSr-Rh-BatCoV was detected in alimentary specimens from 130 (9.3%) bats, with peak activity during spring. A tagging exercise of 511 bats showed migration distances from 1.86 to 17 km. Bats carrying SARSr-Rh-BatCoV appeared healthy, with viral clearance occurring between 2 weeks and 4 months. However, lower body weights were observed in bats positive for SARSr-Rh-BatCoV, but not Rh-BatCoV HKU2. Complete genome sequencing of 10 SARSr-Rh-BatCoV strains showed frequent recombination between different strains. Moreover, recombination was detected between SARSr-Rh-BatCoV Rp3 from Guangxi, China, and Rf1 from Hubei, China, in the possible generation of civet SARSr-CoV SZ3, with a breakpoint at the nsp16/spike region. Molecular clock analysis showed that SARSr-CoVs were newly emerged viruses with the time of the most recent common ancestor (tMRCA) at 1972, which diverged between civet and bat strains in 1995. The present data suggest that SARSr-Rh-BatCoV causes acute, self-limiting infection in horseshoe bats, which serve as a reservoir for recombination between strains from different geographical locations within reachable foraging range. Civet SARSr-CoV is likely a recombinant virus arising from SARSr-CoV strains closely related to SARSr-Rh-BatCoV Rp3 and Rf1. Such frequent recombination, coupled with rapid evolution especially in ORF7b/ORF8 region, in these animals may have accounted for the cross-species transmission and emergence of SARS.Coronaviruses can infect a wide variety of animals, causing respiratory, enteric, hepatic, and neurological diseases with different degrees of severity. On the basis of genotypic and serological characteristics, coronaviruses were classified into three distinct groups (2, 20, 54). Among coronaviruses that infect humans, human coronavirus 229E (HCoV-229E) and human coronavirus NL63 (HCoV-NL63) belong to group 1 coronaviruses and human coronavirus OC43 (HCoV-OC43), and human coronavirus HKU1 (HCoV-HKU1) belong to group 2 coronaviruses, whereas severe acute respiratory syndrome-related coronavirus (SARSr-CoV) has been classified as a group 2b coronavirus, distantly related to group 2a, and the recently discovered group 2c and 2d coronaviruses (6, 8, 10, 18, 31, 38, 43, 46, 49, 50). Recently, the Coronavirus Study Group of the International Committee for Taxonomy of Viruses has proposed renaming the traditional group 1, 2, and 3 coronaviruses Alphacoronavirus, Betacoronavirus, and Gammacoronavirus, respectively (http://talk.ictvonline.org/media/p/1230.aspx).Among all coronaviruses, SARSr-CoV has caused the most severe disease in humans, with over 700 fatalities since the SARS epidemic in 2003. Although the identification of SARSr-CoV in Himalayan palm civets and raccoon dogs in live animal markets in southern China suggested that wild animals could be the origin of SARS (11), the presence of the virus in only market or farmed civets, but not civets in the wild, and the rapid evolution of SARSr-CoV genomes in market civets suggested that these caged animals were only intermediate hosts (24, 39, 42, 52). Since bats are commonly found and served in wild animal markets and restaurants in Guangdong, China (47), we have previously carried out a study of bats from the region and identified a SARSr-CoV in Rhinolophus Chinese horseshoe bats (SARSr-Rh-BatCoV) (21). Similar viruses have also been found in three other species of horseshoe bats in mainland China (25), supporting the hypothesis that horseshoe bats are a reservoir of SARSr-CoV. Recently, viruses closely related to SARSr-Rh-BatCoV in China were also reported in Chaerophon bats from Africa, although only partial RNA-dependent RNA polymerase (RdRp) sequences were available (41). In addition, more than 10 previously unrecognized coronaviruses of huge diversity have since been identified in bats from China and other countries (1, 3, 5, 9, 22, 27, 32, 33, 40, 46, 51), suggesting that bats play an important role in the ecology and evolution of coronaviruses.As a result of the unique mechanism of viral replication, coronaviruses have a high frequency of recombination (20). Such a high recombination rate, coupled with the infidelity of the polymerases of RNA viruses, may allow them to adapt to new hosts and ecological niches (12, 48). Recombination in coronaviruses was first recognized between different strains of murine hepatitis virus (MHV) and subsequently in other coronaviruses, such as infectious bronchitis virus, between MHV and bovine coronavirus, and between feline coronavirus type I and canine coronavirus generating feline coronavirus type II (12, 16, 17, 23). Recently, by complete genome analysis of 22 strains of HCoV-HKU1, we have also documented for the first time that natural recombination events in a human coronavirus can give rise to three different genotypes (48).Although previous studies have attempted to study the possible evolutionary and recombination origin of SARSr-CoV, no definite conclusion can be made on whether the viruses from bats are the direct ancestor of SARSr-CoV in civets and humans, given the paucity of available strains and genome sequences. To better define the epidemiology and evolution of SARSr-Rh-BatCoV in China and their role as a recombination origin of SARSr-CoV in civets, we carried out a 4-year study on coronaviruses in Chinese horseshoe bats in Hong Kong and Guangdong Province of southern China. Bat tagging was also performed to study the migration pattern of bats and viral persistence. The complete genomes of 10 strains of SARSr-Rh-BatCoV obtained at different time were sequenced and compared to previously sequenced genomes. With the availability of this larger set of genome sequences for more accurate analysis, recombination and molecular clock analyses were performed to elucidate the evolutionary origin and time of interspecies transmission of SARSr-CoV.