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).     

Complete Genome Sequence of the Representative γ-Hexachlorocyclohexane-Degrading Bacterium Sphingobium japonicum UT26

Sphingobium japonicum strain UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a man-made chlorinated pesticide that causes serious environmental problems due to its toxicity and long persistence, as a sole source of carbon and energy. Here, we report the complete genome sequence of UT26, which consists of two chromosomes and three plasmids. The 15 lin genes involved in γ-HCH degradation are dispersed on the two chromosomes and one of the three plasmids.γ-Hexachlorocylohexane (γ-HCH) is a man-made insecticide that has caused serious environmental problems worldwide (9). Although only several decades have passed since the initial release of γ-HCH into the environment, many γ-HCH-degrading bacterial strains have been isolated (9), suggesting that γ-HCH-degrading ability was acquired by such strains within a short period (14). Sphingobium japonicum UT26 was isolated from upland γ-HCH-contaminated soil in Japan and utilizes γ-HCH as its sole source of carbon and energy (8, 16). In UT26, 15 lin genes are involved in γ-HCH utilization (2, 11, 12). Our clarification of the complete genome sequence of this strain is expected to provide insights into the mechanisms by which bacteria adapt to xenobiotics.One S. japonicum UT26 colony was designated UT26S (NBRC 101211), and its complete genome sequence was determined by a whole-genome shotgun sequencing strategy using the Sanger method (10, 15, 17). The sequences of ca. 92,400 reads were assembled by the Phrap and CONSED assembly tools (4, 5, 7), and the gaps between contigs were closed by sequencing PCR products which were amplified from genomic DNA using the appropriate primers. The prediction and annotation of open reading frames (ORFs) were performed using Glimmer3 (1), BLASTP, the In Silico Molecular Cloning software suite (In Silico Biology, Inc.), and the GenomeMatcher software (13). Nontranslating genes were predicted using the Rfam, tRNAscan-SE, and ARAGORN programs.The genome of S. japonicum UT26S consists of two circular chromosomes (Chr), Chr 1 (3,514,822 bp, 64.8% G+C, 3,529 ORFs) and Chr 2 (681,892 bp, 65.9% G+C, 589 ORFs), and three circular plasmids, pCHQ1 (190,974 bp, 63.0% G+C, 224 ORFs), pUT1 (31,776 bp, 63.7% G+C, 44 ORFs), and pUT2 (5,398 bp, 61.0% G+C, 8 ORFs). Chr 1 and Chr 2 have one and two copies, respectively, of rRNA operons. Fifty-one and 4 tRNA genes were located on Chr 1 and Chr 2, respectively. One hundred ninety-six out of 206 bacterial essential genes proposed by Gil et al. (6) were all located on Chr 1, indicating that this is clearly a “main” chromosome. The 15 lin genes for γ-HCH degradation are dispersed on Chr 1, Chr 2, and pCHQ1. Comparison of the UT26S genome with those of five other sphingomonad (TM1) strains revealed that the lin genes (linA, linB, linC, linRED, and linF) specific for the conversion of γ-HCH to β-ketoadipate are located in the DNA regions unique to the UT26S genome. On the other hand, linGHIJ for the β-ketoadipate pathway (12) and linKLMN for the ABC transporter system (3) are located in conserved genomic regions of these sphingomonads. Based on these results, we propose a model in which UT26S was established by recruiting the specific lin genes into an ancestral strain having core functions of sphingomonads.     

Horizontal Chromosome Transfer,a Mechanism for the Evolution and Differentiation of a Plant-Pathogenic Fungus

The tomato pathotype of Alternaria alternata produces host-specific AAL toxin and causes Alternaria stem canker on tomato. A polyketide synthetase (PKS) gene, ALT1, which is involved in AAL toxin biosynthesis, resides on a 1.0-Mb conditionally dispensable chromosome (CDC) found only in the pathogenic and AAL toxin-producing strains. Genomic sequences of ALT1 and another PKS gene, both of which reside on the CDC in the tomato pathotype strains, were compared to those of tomato pathotype strains collected worldwide. This revealed that the sequences of both CDC genes were identical among five A. alternata tomato pathotype strains having different geographical origins. On the other hand, the sequences of other genes located on chromosomes other than the CDC are not identical in each strain, indicating that the origin of the CDC might be different from that of other chromosomes in the tomato pathotype. Telomere fingerprinting and restriction fragment length polymorphism analyses of the A. alternata strains also indicated that the CDCs in the tomato pathotype strains were identical, although the genetic backgrounds of the strains differed. A hybrid strain between two different pathotypes was shown to harbor the CDCs derived from both parental strains with an expanded range of pathogenicity, indicating that CDCs can be transmitted from one strain to another and stably maintained in the new genome. We propose a hypothesis whereby the ability to produce AAL toxin and to infect a plant could potentially be distributed among A. alternata strains by horizontal transfer of an entire pathogenicity chromosome. This could provide a possible mechanism by which new pathogens arise in nature.Fungi produce a huge variety of secondary metabolites. Some plant-pathogenic fungi, especially necrotrophic pathogens that kill plant cells during invasion, produce phytotoxic metabolites to impair host tissue functions (20, 30, 42, 47). Phytotoxins produced by fungal plant pathogens are generally low-molecular-weight secondary metabolites that exert toxic effects on host plants. Among these phytotoxins, host-specific toxins (HSTs) are critical determinants of pathogenicity or virulence in several plant-pathogen interactions (13, 30, 33, 40, 42, 47, 49).Recent advances in molecular biological techniques for fungi have led to the identification of fungal genes involved in pathogenesis, as exemplified by those used in the biosynthesis of toxic secondary metabolites, such as HSTs. Genes involved in the biosynthesis of secondary metabolites are typically clustered in filamentous fungi, including plant pathogens (20, 24, 44). The origins and evolutionary processes of these gene clusters, however, are largely unknown. Analysis of the arrangement and sequences of genes in the clusters would shed light on how the clusters themselves and their ability to produce toxic secondary metabolites evolved (20, 24, 44).The involvement of horizontal gene transfer (HGT) in the evolution of fungal secondary-metabolite gene clusters has been discussed (34, 44). HGT events are well known in prokaryotes (21, 29), and the genomic regions that have undergone HGT are referred to as pathogenicity or genomic islands (7). In prokaryotes, the mechanisms of HGT are also associated with conjugation, transformation, and transduction (21, 29). Although these transfer mechanisms are generally unknown in eukaryotes such as fungi, interspecific transfer of a virulence gene encoding the production of a critical toxin has been reported in Pyrenophora tritici-repentis (14). There is also clear evidence of recent lateral gene transfer of the ToxA gene from Stagonospora nodorum to P. tritici-repentis (14, 30).In Alternaria alternata plant pathogens (37), we have shown that all strains of the A. alternata pathotypes harbor small extra chromosomes of less than 1.7 Mb, whereas nonpathogenic isolates do not have these small chromosomes (5). A cyclic peptide synthetase gene, AMT, which is involved in host-specific AM toxin biosynthesis of the apple pathotype of A. alternata, was located on a small chromosome of 1.1 to 1.7 Mb, depending on the strain (22, 23). The AF toxin biosynthesis gene cluster was also present on a single small chromosome of 1.05 Mb in the strawberry pathotype of A. alternata (18). Based on biological and pathological observations, those small chromosomes were regarded as supernumerary chromosomes, or conditionally dispensable chromosomes (CDCs) (10, 18, 22). Fungal supernumerary chromosomes, which are not important for normal growth but confer advantages for colonizing an ecological niche, such as infecting host plants, are regarded as CDCs (21). The functions and pathological roles of CDCs have been studied in the pea pathogen Nectria haematococca (11, 17, 25, 32, 43, 46).The origin and evolution of CDCs have been intriguing issues in the study of plant-microbe interactions. The supernumerary chromosomes of certain strains of N. haematococca have been suggested to have a different evolutionary history than essential chromosomes (ECs) in the same genome, and they might have been introduced into the genome by horizontal transfer from another strain (10, 12, 36). In Colletotrichum gloeosporioides, the 2-Mb supernumerary chromosome was transferred from a biotype A strain to a vegetative incompatible biotype B strain (19, 31). Transfer of the chromosome, however, did not affect the pathogenicity of the recipient fungus, perhaps because it did not harbor pathogenicity genes (19, 31). These results suggest that supernumerary chromosomes of fungi might have the capacity for horizontal transfer across an incompatibility barrier between two distinct strains.AAL toxins are HSTs produced by the tomato pathotype of A. alternata (synonym A. alternata f. sp. lycopersici, synonym Alternaria arborescens), the causal agent of Alternaria stem canker disease in tomatoes, which causes severe necrosis of susceptible tomato cultivars (15, 26, 35). AAL toxins and fumonisins of the maize pathogen Gibberella moniliformis are structurally related to sphinganine and termed sphinganine-analogue mycotoxins. AAL toxins and fumonisins are sphinganine-analogue mycotoxins, which are toxic to some plant species and mammalian cells (16, 48). They cause apoptosis in susceptible tomato cells and mammalian cells by inhibiting ceramide biosynthesis (9, 41, 45). In the tomato pathotype of A. alternata-tomato interactions, a major factor in pathogenicity is the production of host-specific AAL toxins capable of inducing cell death only in susceptible cultivars (3, 9, 48).In this study, we describe evidence showing that the ability to produce the host-specific AAL toxin and to infect host tomato plants could potentially be distributed among a population of strains of the A. alternata tomato pathotype by horizontal transfer of an entire pathogenicity chromosome of the pathogen.     

Genetic Characterization of Vibrio vulnificus Strains from Tilapia Aquaculture in Bangladesh

Outbreaks of Vibrio vulnificus wound infections in Israel were previously attributed to tilapia aquaculture. In this study, V. vulnificus was frequently isolated from coastal but not freshwater aquaculture in Bangladesh. Phylogenetic analyses showed that strains from Bangladesh differed remarkably from isolates commonly recovered elsewhere from fish or oysters and were more closely related to strains of clinical origin.Vibrio vulnificus causes severe wound infections and life-threatening septicemia (mortality, >50%), primarily in patients with underlying chronic diseases (10, 19, 23) and primarily from raw oyster consumption (21). This Gram-negative halophile is readily recovered from oysters (27, 35, 43) and fish (14) and was initially classified into two biotypes (BTs) based on growth characteristics and serology (5, 18, 39). Most human isolates are BT1, while BT2 is usually associated with diseased eels (1, 39). An outbreak of wound infections from aquacultured tilapia in Israel (6) revealed a new biotype (BT3). Phenotypic assays do not consistently distinguish biotypes (33), but genetic analyses have helped resolve relationships (20). A 10-locus multilocus sequence typing (MLST) scheme (8, 9) and a similar analysis of 6 loci (13) segregated V. vulnificus strains into two clusters. BT1 strains were in both clusters, while BT2 segregated into a single cluster and BT3 was a genetic mosaic of the two lineages. Significant associations were observed between MLST clusters and strain origin: most clinical strains (BT1) were in one cluster, and the other cluster was comprised mostly of environmental strains (some BT1 and all BT2). Clinical isolates were also associated with a unique genomic island (13).The relationship between genetic lineages and virulence has not been determined, and confirmed virulence genes are universally present in V. vulnificus strains from both clinical and environmental origins (19, 23). However, segregation of several polymorphic alleles agreed with the MLST analysis and correlated genotype with either clinical or environmental strain origin. Alleles include 16S rRNA loci (15, 26, 42), a virulence-correlated gene (vcg) locus (31, 41, 42), and repetitive sequence in the CPS operon (12). DiversiLab repetitive extrageneic palindromic (rep-PCR) analysis also confirmed these genetic distinctions and showed greater diversity among clinical strains (12).Wound infections associated with tilapia in Israel implicated aquaculture as a potential source of V. vulnificus in human disease (6, 40). Tilapia aquaculture is increasing rapidly, as shown by a 2.8-fold increase in tons produced from 1998 to 2007 (Food and Agriculture Organization; http://www.fao.org/fishery/statistics/en). Therefore, presence of V. vulnificus in tilapia aquaculture was examined in Bangladesh, a region that supports both coastal and freshwater sources of industrial-scale aquaculture. V. vulnificus strains were recovered from market fish, netted fish, and water samples, and the phylogenetic relationship among strains was examined relative to clinical and environmental reference strains collected elsewhere.     

Functional and Spatial Regulation of Mitotic Centromere- Associated Kinesin by Cyclin-Dependent Kinase 1

Mitotic centromere-associated kinesin (MCAK) plays an essential role in spindle formation and in correction of improper microtubule-kinetochore attachments. The localization and activity of MCAK at the centromere/kinetochore are controlled by Aurora B kinase. However, MCAK is also abundant in the cytosol and at centrosomes during mitosis, and its regulatory mechanism at these sites is unknown. We show here that cyclin-dependent kinase 1 (Cdk1) phosphorylates T537 in the core domain of MCAK and attenuates its microtubule-destabilizing activity in vitro and in vivo. Phosphorylation of MCAK by Cdk1 promotes the release of MCAK from centrosomes and is required for proper spindle formation. Interfering with the regulation of MCAK by Cdk1 causes dramatic defects in spindle formation and in chromosome positioning. This is the first study demonstrating that Cdk1 regulates the localization and activity of MCAK in mitosis by directly phosphorylating the catalytic core domain of MCAK.Chromosomes are properly attached to the mitotic spindles, and chromosome movement is tightly linked to the structure and dynamics of spindle microtubules during mitosis. Important regulators of microtubule dynamics are the kinesin-13 proteins (37). This kinesin superfamily is defined by the localization of the conserved kinesin core motor domain in the middle of the polypeptide (19). Kinesin-13 proteins induce microtubule depolymerization by disassembling tubulin subunits from the polymer end (6). Among them, mitotic centromere-associated kinesin (MCAK) is the best-characterized member of the family. It depolymerizes microtubules in vitro and in vivo, regulates microtubule dynamics, and has been implicated in correcting misaligned chromosomes (12, 14, 16, 24). In agreement with these observations, both overexpression and inhibition of MCAK result in a disruption of microtubule dynamics, leading further to improper spindle assembly and errors in chromosome alignment and segregation (7, 11, 15, 22, 33). The importance of MCAK in ensuring the faithful segregation of chromosomes is consistent with the observation that MCAK is highly expressed in several types of cancer and thus is likely to be involved in causing aneuploidy (25, 32).While MCAK is found both in the cytoplasm and at the centromeres throughout the cell cycle, it is highly enriched on centrosomes, the centromeres/kinetochores, and the spindle midzone during mitosis (18, 21, 36, 38). In accordance with its localizations, MCAK affects many aspects throughout mitosis, from spindle assembly and maintenance (3, 10, 36) to chromosome positioning and segregation (14, 21, 35). Thus, the precise control of the localization and activity of MCAK is crucial for maintaining genetic integrity during mitosis. Regulation of MCAK on the centromeres/kinetochores by Aurora B kinase in mitosis has been intensively investigated (1, 28, 29, 43). The data reveal that MCAK is phosphorylated on several serine/threonine residues by Aurora B, which inhibits the microtubule-destabilizing activity of MCAK and regulates its localization on chromosome arms/centromeres/kinetochores during mitosis (1, 18, 28). Moreover, in concert with Aurora B, ICIS (inner centromere KinI stimulator), a protein targeting the inner centromeres in an MCAK-dependent manner, may regulate MCAK at the inner centromeres and prevent kinetochore-microtubule attachment errors in mitosis by stimulating the activity of MCAK (27). Interestingly, hSgo2, a recently discovered inner centromere protein essential for centromere cohesion, has been reported to be important in localizing MCAK to the centromere and in spatially regulating its mitotic activity (13). These data highlight that the activity and localization of MCAK on the centromeres/kinetochores during mitosis are tightly controlled by Aurora B and its cofactors. Remarkably, MCAK concentrates at spindle poles from prophase to telophase during mitosis (18); however, only a few studies have been done to deal with that issue. Aurora A-depleted prometaphase cells delocalize MCAK from spindle poles but accumulate the microtubule-stabilizing protein ch-TOG at poles (5), implying that Aurora A might influence the centrosomal localization of MCAK in mitosis. Aurora A is also found to be important for focusing microtubules at aster centers and for facilitating the transition from asters to bipolar spindles in Xenopus egg extracts (42). In addition, it has been revealed that Ca²⁺/calmodulin-dependent protein kinase II gamma (CaMKII gamma) suppresses MCAK''s activity, which is essential for bipolar spindle formation in mitosis (11). More work is required to gain insight into the regulatory mechanisms of MCAK at spindle poles during mitosis.Deregulated cyclin-dependent kinases (Cdks) are very often linked to genomic and chromosomal instability (20). Cyclin B1, the regulatory subunit of Cdk1, is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment (2, 4). We observed that knockdown of cyclin B1 induces defects in chromosome alignment and mitotic spindle formation (N.-N. Kreis, M. Sanhaji, A. Krämer, K. Sommor, F. Rödel, K. Strebhardt, and J. Yuan, submitted for publication). Yet, how Cdk1/cyclin B1 carries out these functions is not very well understood. In this context, it is extremely interesting to investigate the relationship between the essential mitotic kinase Cdk1 and the microtubule depolymerase MCAK in human cells.     

Clonal Relationship among Atypical Enteropathogenic Escherichia coli Strains Isolated from Different Animal Species and Humans

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.Enteropathogenic Escherichia coli (EPEC) strains are among the major causes of infantile diarrhea in developing countries (71) and can be classified as typical and atypical, depending on the presence or absence of the E. coli adherence factor plasmid (pEAF), respectively (39).The pathogenesis of EPEC resides in the ability to cause the attaching and effacing (A/E) lesion in the gut mucosa of human or animal hosts, leading to diarrheal illness (40). The genes responsible for the A/E lesion formation are located in a chromosomal pathogenicity island of ∼35 kb, known as the locus of enterocyte effacement (LEE) (23, 47). LEE encodes an adhesin called intimin (38), its translocated receptor (Tir) (42), components of a type III secretion system (36), and effector molecules, named E. coli-secreted proteins (Esp proteins) (41). These virulence factors have a crucial role in A/E lesion formation, and their detection in EPEC strains is an indicator of their potential to produce these lesions (19, 56).Atypical EPEC strains have been associated with diarrhea outbreaks in developed countries (31, 73, 77) and with sporadic cases of diarrhea in developing and developed countries (1, 12, 26, 52, 55). At present, the prevalence of atypical EPEC is higher than that of typical EPEC in several countries (1, 12, 26, 52, 55, 65).Different from the situation in developed countries, where atypical EPEC outbreaks and sporadic infections are associated with children and adults, atypical EPEC infection in Brazil is mainly associated with children''s illnesses (32, 71).Typical EPEC strains are rarely isolated from animals, and humans are the major natural reservoir for these pathogens (14, 32, 53, 71). In contrast, atypical EPEC strains are present in both healthy and diseased animals (dog, monkey, cats, and bovines) and humans (4, 6, 18, 28, 71). Some studies have associated pets and farm and wild animals as reservoirs and infection sources of atypical EPEC strains for humans (32). However, these studies did not compare atypical EPEC strains isolated from humans and animals by gold-standard molecular methods like multilocus sequence typing (MLST) or pulsed-field gel electrophoresis (PFGE) (15, 35, 43, 53). For this reason, there are some doubts about whether atypical EPEC strains isolated from animals represent risks for human health and whether animals really play the role of reservoirs of atypical EPEC.The aim of this study was to compare atypical EPEC strains isolated from humans and different animals, including pets (cats and dogs), farm animals (bovines, ovines, and rabbits), and wild animals (monkeys), by molecular phylogenetic techniques to verify the role of animals as reservoirs of and sources of infection with atypical EPEC in humans.     

African 1, an Epidemiologically Important Clonal Complex of Mycobacterium bovis Dominant in Mali,Nigeria, Cameroon,and Chad

We have identified a clonal complex of Mycobacterium bovis present at high frequency in cattle in population samples from several sub-Saharan west-central African countries. This closely related group of bacteria is defined by a specific chromosomal deletion (RDAf1) and can be identified by the absence of spacer 30 in the standard spoligotype typing scheme. We have named this group of strains the African 1 (Af1) clonal complex and have defined the spoligotype signature of this clonal complex as being the same as the M. bovis BCG vaccine strain but with the deletion of spacer 30. Strains of the Af1 clonal complex were found at high frequency in population samples of M. bovis from cattle in Mali, Cameroon, Nigeria, and Chad, and using a combination of variable-number tandem repeat typing and spoligotyping, we show that the population of M. bovis in each of these countries is distinct, suggesting that the recent mixing of strains between countries is not common in this area of Africa. Strains with the Af1-specific deletion (RDAf1) were not identified in M. bovis isolates from Algeria, Burundi, Ethiopia, Madagascar, Mozambique, South Africa, Tanzania, and Uganda. Furthermore, the spoligotype signature of the Af1 clonal complex has not been identified in population samples of bovine tuberculosis from Europe, Iran, and South America. These observations suggest that the Af1 clonal complex is geographically localized, albeit to several African countries, and we suggest that the dominance of the clonal complex in this region is the result of an original introduction into cows naïve to bovine tuberculosis.Mycobacterium bovis causes bovine tuberculosis (TB), an important disease of domesticated cattle that has a major economic and health impact throughout the world (61, 64, 65). The pathogen is a member of the Mycobacterium tuberculosis complex, which includes many species and subspecies that cause similar pathologies in a variety of mammalian hosts. The most notable member of the complex is M. tuberculosis, the most important bacterial pathogen of humans. In contrast to M. tuberculosis, which is largely host restricted to humans, M. bovis is primarily maintained in bovids, in particular, domesticated cattle, although the pathogen can frequently be recovered from other mammals, including humans (61). Bovine TB is found in cattle throughout the world and has been reported on every continent where cattle are farmed (3).Bovine TB has been reduced or eliminated from domestic cattle in many developed countries by the application of a test-and-cull policy that removes infected cattle (3, 8, 16, 17, 61, 64, 65). However, in Africa, although bovine TB is known to be common in both cattle and wildlife, control policies have not been enforced in many countries due to cost implications, lack of capacity, and infrastructure limitations (8, 16, 17, 57). In 1998, Cosivi et al. reported of bovine TB, “Of all nations in Africa, only seven apply disease control measures as part of a test-and-slaughter policy and consider bovine TB a notifiable disease; the remaining 48 control the disease inadequately or not at all” (16). In the intervening years, the situation is not thought to have improved (8); however, preliminary surveys of bovine TB have been carried out in some African countries (4, 7, 12, 37, 44, 49, 53, 54, 56).The most common epidemiological molecular-typing method applied to strains of M. bovis is spoligotyping. This method identifies polymorphism in the presence of spacer units in the direct-repeat (DR) region in strains of the M. tuberculosis complex (36, 67). The DR is composed of multiple, virtually identical 36-bp regions interspersed with unique DNA spacer sequences of similar size (direct variant repeat [DVR] units). Spacer sequences are unique to the DR region, and copies are not located elsewhere in the chromosome (68). The DR region may contain over 60 DVR units; however, 43 of the spacer units were selected from the spacer sequences of the M. tuberculosis reference strain H37Rv and M. bovis BCG strain P3 and are used in the standard application of spoligotyping to strains of the M. tuberculosis complex (29, 36). The DR region is polymorphic because of the loss (deletion) of single or multiple spacers, and each spoligotype pattern from strains of M. bovis is given an identifier (http://www.Mbovis.org).Several studies of the DR regions in closely related strains of M. tuberculosis have concluded that the evolutionary trend for this region is primarily loss of single DVRs or multiple contiguous DVRs (22, 29, 68); duplication of DVR units or point mutations in spacer sequences were found to be rare. The loss of discrete units observed by Groenen et al. (29) led them to suggest that the mechanism for spacer loss was homologous recombination between repeat units. However, a study by Warren et al. (69) suggested that for strains of M. tuberculosis, insertion of IS6110 sequences into the DR region and recombination between adjacent IS6110 elements were more important mechanisms for the loss of spacer units.The population structure of the M. tuberculosis group of organisms is apparently highly clonal, without any transfer and recombination of chromosomal sequences between strains (15, 30, 60, 61). In a strictly clonal population, the loss by deletion of unique chromosomal DNA cannot be replaced by recombination from another strain, and the deleted region will act as a molecular marker for the strain and all its descendants. Deletions of specific chromosomal regions (regions of difference [RDs] or large sequence polymorphisms) have been very successful at identifying phylogenetic relationships in the M. tuberculosis complex (11, 25, 26, 35, 48, 50, 61, 62, 66). However, because the loss of spoligotype spacer sequences is so frequent, identical spoligotype patterns can occur independently in unrelated lineages (homoplasy), and therefore, the deletion of spoligotype spacers may be an unreliable indicator of phylogenetic relationship (61, 69).In samples of M. bovis strains from Cameroon, Nigeria, Chad, and Mali, spoligotyping was used to show that many of the strains had similar spoligotype patterns that lacked spacer 30, and it has been suggested that strains from these four countries are phylogenetically related (12, 18, 49, 53). We have extended the previous observations of spoligotype similarities between strains from these countries and confirmed the existence of a unique clonal complex of M. bovis, all descended from a single strain in which a specific deletion of chromosomal DNA occurred. We have named this clonal complex of M. bovis strains African 1 (Af1), and we show that this clonal complex is dominant in these four west-central African countries but rare in eastern and southern Africa. Extended genotyping, using variable-number tandem repeats (VNTR), of strains with the most common spoligotype patterns suggests that each of these four west-central African countries has a unique population structure. Evolutionary scenarios that may have led to the present day distribution of the Af1 clonal complex are discussed.     

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.     

Polymorphic Mutation Frequencies of Clinical and Environmental Stenotrophomonas maltophilia Populations

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

Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of Cronobacter Genomospecies (Enterobacter sakazakii) in Powdered Infant Formula

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.Cronobacter strains were originally described as Enterobacter sakazakii (12), but they are now known to comprise a novel genus consisting of six separate genomospecies (20, 21). These opportunistic pathogens are ubiquitous in the environment and various types of food and are occasionally found in the normal human flora (11, 12, 16, 32, 47). Based on case reports, Cronobacter infections in adults are generally less severe than Cronobacter infections in newborn infants, with which a high fatality rate is associated (24).The ability to detect Cronobacter and trace possible sources of infection is essential as a means of limiting the impact of these organisms on neonatal health and maintaining consumer confidence in powdered infant formula (PIF). Conventional methods, involving isolation of individual colonies followed by biochemical identification, are more time-consuming than molecular methods, and the reliability of some currently proposed culture-based methods has been questioned (28). Recently, several PCR-based techniques have been described (23, 26, 28-31, 38). These techniques are reported to be efficient even when low levels of Cronobacter cells are found in a sample (0.36 to 66 CFU/100 g). However, PCR requires DNA extraction and does not allow direct, in situ visualization of the bacterium in a sample.Fluorescence in situ hybridization (FISH) is a method that is commonly used for bacterial identification and localization in samples. This method is based on specific binding of nucleic acid probes to particular DNA or RNA target regions (1, 2). rRNA has been regarded as the most suitable target for bacterial FISH, allowing differentiation of potentially viable cells. Traditionally, FISH methods are based on the use of conventional DNA oligonucleotide probes, and a commercial system, VIT-E sakazakii (Vermicon A.G., Munich, Germany), has been developed based on this technology (25). However, a recently developed synthetic DNA analogue, peptide nucleic acid (PNA), has been shown to provide improved hybridization performance compared to DNA probes, making FISH procedures easier and more efficient (41). Taking advantage of the PNA properties, FISH using PNA has been successfully used for detection of several clinically relevant microorganisms (5, 15, 17, 27, 34-36).     

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.