Comparison of the Complete Genome Sequences of Bifidobacterium animalis subsp. lactis DSM 10140 and Bl-04

Bifidobacteria are important members of the human gut flora, especially in infants. Comparative genomic analysis of two Bifidobacterium animalis subsp. lactis strains revealed evolution by internal deletion of consecutive spacer-repeat units within a novel clustered regularly interspaced short palindromic repeat locus, which represented the largest differential content between the two genomes. Additionally, 47 single nucleotide polymorphisms were identified, consisting primarily of nonsynonymous mutations, indicating positive selection and/or recent divergence. A particular nonsynonymous mutation in a putative glucose transporter was linked to a negative phenotypic effect on the ability of the variant to catabolize glucose, consistent with a modification in the predicted protein transmembrane topology. Comparative genome sequence analysis of three Bifidobacterium species provided a core genome set of 1,117 orthologs complemented by a pan-genome of 2,445 genes. The genome sequences of the intestinal bacterium B. animalis subsp. lactis provide insights into rapid genome evolution and the genetic basis for adaptation to the human gut environment, notably with regard to catabolism of dietary carbohydrates, resistance to bile and acid, and interaction with the intestinal epithelium. The high degree of genome conservation observed between the two strains in terms of size, organization, and sequence is indicative of a genomically monomorphic subspecies and explains the inability to differentiate the strains by standard techniques such as pulsed-field gel electrophoresis.Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes are dominant microbial phyla widely distributed in diverse ecosystems on the planet (10, 13, 20, 23, 33, 40, 51). Metagenomic analyses of the microbial landscape inhabiting various mammalian environments, notably the human gastrointestinal tract (GIT) and skin, have specifically identified Actinobacteria as an important and occasionally dominant phylum (18, 21, 33). Among the members of the large, diverse, and dynamic microbial community residing in the human GIT, Bifidobacterium is a dominant genus considered beneficial to humans and includes probiotic strains (live microorganisms which, when administered in adequate amounts, confer a health benefit on the host) (11). The population of bifidobacteria in the human intestine varies over time. Following vaginal delivery, the GIT of healthy newborns is typically colonized by bifidobacteria, especially in breast-fed infants, during the first few days of life (12). Interindividual variation, however, is remarkable in the human infant intestinal flora (41), and dominant genera are not always consistent across metagenomic analyses of the human gut flora (18, 30, 33, 41). Over time, the infant intestinal ecosystem becomes more complex as the diet becomes more diverse, with bifidobacteria typically remaining dominant until weaning (30).Bifidobacterium animalis subsp. lactis is a gram-positive lactic acid bacterium commonly found in the guts of healthy humans and has been identified in the infant gut biota, particularly in ileal, fecal, and mucosal samples (52, 56). Some strains of B. animalis subsp. lactis are able to survive in the GIT, to adhere to human epithelial cells in vitro, to modify fecal flora, to modulate the host immune response, or to prevent microbial gastroenteritis and colitis (4, 15, 20, 40, 52, 56). Additionally, B. animalis subsp. lactis has been reported to utilize nondigestible oligosaccharides, which may contribute to the organism''s ability to compete in the human gut. Carbohydrates resistant to enzymatic degradation and not absorbed in the upper intestinal tract are a primary source of energy for microbes residing in the large intestine. The benefits associated with probiotic strains of B. animalis subsp. lactis have resulted in their inclusion in the human diet via formulation into a large array of dietary supplements and foods, including dairy products such as yogurt. Deciphering the complete genome sequences of such microbes will provide additional insight into the genetic basis for survival and residence in the human gut, notably with regard to the ability to survive gastric passage and utilize available nutrients. Also, these genomes provide reference sequences for ongoing metagenomic analyses of the human environment, including the gut metagenome.Bifidobacterium animalis subsp. lactis is the most common bifidobacterium utilized as a probiotic in commercial dairy products in North America and Europe (22, 38). However, despite this commercial and probiotic significance, strain-level differentiation of B. animalis subsp. lactis strains has been hindered by the high genetic similarity of these organisms, as determined by pulsed-field gel electrophoresis and other nucleic acid-based techniques (6, 55, 56), and the lack of available genomic sequence information. The genome sequence of strain BB-12 (17) is not currently publicly available, and only a draft genome sequence in 28 contigs is available for strain HN019 (GenBank project 28807). The complete B. animalis subsp. lactis genome for strain AD011 (28) was only recently (2009) published. While this was an important first step, a single genome does not allow identification of unique targets for strain differentiation or comparative analyses within the subspecies.The objectives of this study were to determine the complete genome sequences of two B. animalis subsp. lactis strains, the type strain and a widely used commercial strain, to provide insights into the functionality of this species and into species identification and strain specialization.     

Strain-Specific Genotyping of Bifidobacterium animalis subsp. lactis by Using Single-Nucleotide Polymorphisms,Insertions, and Deletions

Several probiotic strains of Bifidobacterium animalis subsp. lactis are widely supplemented into food products and dietary supplements due to their documented health benefits and ability to survive within the mammalian gastrointestinal tract and acidified dairy products. The strain specificity of these characteristics demands techniques with high discriminatory power to differentiate among strains. However, to date, molecular approaches, such as pulsed-field gel electrophoresis and randomly amplified polymorphic DNA-PCR, have been ineffective at achieving strain separation due to the monomorphic nature of this subspecies. Previously, sequencing and comparison of two B. animalis subsp. lactis genomes (DSMZ 10140 and Bl-04) confirmed this high level of sequence similarity, identifying only 47 single-nucleotide polymorphisms (SNPs) and four insertions and/or deletions (INDELs) between them. In this study, we hypothesized that a sequence-based typing method targeting these loci would permit greater discrimination between strains than previously attempted methods. Sequencing 50 of these loci in 24 strains of B. animalis subsp. lactis revealed that a combination of nine SNPs/INDELs could be used to differentiate strains into 14 distinct genotypic groups. In addition, the presence of a nonsynonymous SNP within the gene encoding a putative glucose uptake protein was found to correlate with the ability of certain strains to transport glucose and to grow rapidly in a medium containing glucose as the sole carbon source. The method reported here can be used in clinical, regulatory, and commercial applications requiring identification of B. animalis subsp. lactis at the strain level.Probiotics are currently defined as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (12). Many of the organisms studied for their probiotic potential are members of lactic acid bacteria and the genus Bifidobacterium, which has resulted in their inclusion in a large variety of dietary supplements and food products. Relative to most bifidobacterial species of human origin, Bifidobacterium animalis subsp. lactis is less sensitive to stressful conditions (bile, acid, and oxygen) which might be encountered in the mammalian gastrointestinal tract or in fermented or acidified dairy products (7, 26, 28, 31, 37). B. animalis subsp. lactis is widely added to commercial products because it is better able to withstand the adverse conditions of starter culture and product manufacture and to maintain viability and stability during product shelf-life (30). Therefore, strains of B. animalis, specifically B. animalis subsp. lactis, have been found in the majority of probiotic-supplemented dairy products surveyed in North America (the United States and Canada) and Europe (Great Britain, France, Italy, and Germany) (6, 13-15, 21, 22, 28, 29, 32, 49).When selecting a probiotic microorganism to add to supplements or foods, the strain must be identified at the genus, species, and strain levels (40). Proper characterization of a strain is important for safety and quality assurance, for identifying and differentiating putative probiotic strains, and for understanding the interactions among members of gut microbiota. In addition, proper characterization is important to maintain consumer confidence. Product labels often list invalid names of organisms or misidentify the species the product contains, leading to consumer confusion (6, 16, 20, 28, 29, 35, 38, 49). In the case of Bifidobacterium, most dairy products sold in the United States do not identify species, and many only refer to the invalid name “Bifid” or “Bifidus.” At the very least, added microorganisms should be accurately identified to the species level on product labels.According to the FAO/WHO guidelines for probiotic use, specific health benefits observed in research using a specific strain cannot be extrapolated to other, closely related strains (12). Although most clinical studies of probiotic strains compare strains of different genera or different species, few studies have assessed the actual variability of expected health benefits within species or subspecies. However, it is reasonable to consider that health effects, like the phenotypic traits exhibited by strains within a species, are strain specific. Therefore, reliable techniques for the identification of probiotic organisms at the strain level are required.Characterization to the strain level has several important potential applications. Understanding the complex interactions among microorganisms in the intestinal ecosystem requires methods of differentiating a strain of interest from other strains of the same species contained in the autochthonous microbiota. Strain differentiation techniques also aid in assessing survival of a probiotic organism through the gastrointestinal system, which is particularly important for clinical trials and regulatory purposes (17). The ability to uniquely identify a strain also lends credibility to statements made about the potential health benefits of consuming a particular product containing a strain with demonstrated probiotic effects and supports the licensing or intellectual property rights of the manufacturer.The high degree of genome conservation observed between strains of B. animalis subsp. lactis in terms of size, organization, and sequence is indicative of a genomically monomorphic subspecies (2, 25; also HN019 GenBank project 28807). As an example, comparison of the complete genome sequences of two B. animalis subsp. lactis strains, DSMZ 10140 (the type strain) and Bl-04 (a commercial strain, also known as RB 4825) (2), identified 47 single-nucleotide polymorphisms (SNPs) in nonrepetitive elements, as well as 443 bp distributed among four INDEL sites: a 121-bp tRNA-encoding sequence, a 54-bp region within the long-chain fatty acid-coenzyme A ligase gene, a 214-bp region within the CRISPR (clustered regularly interspaced short palindromic repeats) locus, and a 54-bp intergenic sequence. Overall, this 99.975% genome identity explains the inability to differentiate these strains by techniques such as the sequencing of housekeeping genes, multilocus sequence typing, and pulsed-field gel electrophoresis (PFGE) (3, 9, 23, 39, 44-46, 50).The strain specificity of reported health benefits of probiotics and the frequent use of B. animalis subsp. lactis as a probiotic in food products and supplements demands techniques with greater discriminatory power to identify and differentiate among strains within this highly homogeneous group. Unfortunately, strain level differentiation of B. animalis subsp. lactis presents several challenges. Although Ventura and Zink were able to differentiate strains of B. animalis subsp. lactis by sequencing the 16S-23S internal transcribed sequence (ITS) region (47), analysis of the four ITS operons between DSMZ 10140 and Bl-04 indicated complete identity (2). However, SNPs and INDELs do have potential for strain differentiation. According to Achtman, focusing on polymorphic SNPs is a desirable approach for the typing of monomorphic species (1). Therefore, the objective of the present study was to exploit the previously identified SNP and INDEL sites to develop a technique capable of differentiating among a collection of B. animalis subsp. lactis strains obtained from culture collections and commercial starter culture companies.     

Complete Genome Sequence of Probiotic Bifidobacterium animalis subsp. lactis Strain V9

Bifidobacterium animalis subsp. lactis strain V9 is a Chinese commercial bifidobacteria with several probiotic functions. It was isolated from a healthy Mongolian child in China. We present here the complete genome sequence of V9 and compare it to 3 other published genome sequences of B. animalis subsp. lactis strains. The result indicates the lack of polymorphism among strains of this subspecies from different continents.Bifidobacterium animalis subsp. lactis strain V9 was isolated from the feces of a healthy Mongolian child in China (5). It has shown a high level of tolerance to gastric acid and bile acids (5). This strain has been implemented in the industrial production of dairy starter cultures by Inner Mongolia Yili Industrial Group Company Limited, the largest dairy corporation in China.Whole-genome sequencing of B. animalis subsp. lactis V9 was performed with a combined strategy of 454 sequencing (8) and Solexa paired-end sequencing technology (2). Genomic libraries containing 7-kb inserts were constructed, and 325,824 paired-end reads and 67,177 single-end reads were generated using the GS FLX system, giving 36.0-fold coverage of the genome. A total of 96.0% of the reads were assembled into four large scaffolds, including 163 nonredundant contigs, using the 454 Newbler assembler (454 Life Sciences, Branford, CT). A total of 8,953,102 reads (2-kb library) were generated to reach a depth of 335-fold coverage with an Illumina Solexa Genome Analyzer IIx and mapped to the scaffolds using the Burrows-Wheeler Alignment (BWA) tool (7). The gaps between scaffolds were filled by sequencing PCR products using an ABI 3730 capillary sequencer. The analysis of the genome was performed as described previously (3, 4).The complete genome sequence of V9 contains a circular 1,944,050-bp chromosome, with a GC content of 60.5%. The genome size is slightly larger than the sequenced genome sizes of B. animalis subsp. lactis strains DSM 10140^T (1), Bl-04 (1), and AD011 (6) due to a unique insertion of 4,037 bp. The V9 genome contains 1,636 genes in total, including 1,572 coding genes, 4 rRNA operons, and 52 tRNAs.Comparison of the four B. animalis subsp. lactis genomes revealed nearly perfect synteny. AD011 is the most diverged strain, with more single nucleotide polymorphisms (SNPs) and indels than the other three strains. There are 197 SNPs in AD011, with 70 synonymous and 16 nonsynonymous SNPs, which means that there is only 1 SNP per 10 kb, indicating the high consistency within this subspecies. The other three strains are almost identical, with only 25 SNPs in V9, 13 SNPs in Bl-04, and 44 SNPs in DSM 10140^T. Strain V9 was isolated from the feces of a Mongolian child in Inner Mongolia, China, where traditional fermented milk has been consumed for thousands of years, and the other three strains were originally isolated from fecal samples (1, 6) or yogurt (1) in the United States of America, France, and Korea. The result indicated the lack of polymorphism among multiple lineages from different continents (1).Interestingly, compared to the other three sequenced B. animalis subsp. lactis strains, V9 has a large insertion, which encodes one putative transposase (BalV_1091) and two sugar metabolism-related proteins, an alpha-1,4-glucosidase (BalV_1092) and an ABC transporter solute-binding protein (BalV_1093). This insertion is a copy of the region at positions 1,860,164 to 1,864,073, which is commonly shared by all four B. animalis subsp. lactis strains.     

Identification and Typing of Lactococcus lactis by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Currently, the genus Lactococcus is classified into six species: Lactococcus chungangensis, L. garvieae, L. lactis, L. piscium, L. plantarum, and L. raffinolactis. Among these six species, L. lactis is especially important because of its use in the manufacture of probiotic dairy products. L. lactis consists of three subspecies: L. lactis subsp. cremoris, L. lactis subsp. hordniae, and L. lactis subsp. lactis. However, these subspecies have not yet been reliably discriminated. To date, mainly phenotypic identification has been used, with a few genotypic identifications. We discriminated species or subspecies in the genus Lactococcus not only by proteomics identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) but also by phenotypic and genotypic identification. The proteomics identification using differences in the mass spectra of ribosomal proteins was nearly identical to that by genotypic identification (i.e., by analyses of 16S rRNA and recA gene sequences and amplified fragment length polymorphism). The three ribosomal subunits 30S/L31, 50S/L31, and 50S/L35 were the best markers for discriminating L. lactis subsp. cremoris from L. lactis subsp. lactis. Proteomics identification using MALDI-TOF MS was therefore a powerful method for discriminating and identifying these bacteria. In addition, this method was faster and more reliable than others that we examined.Lactococci are lactic acid bacteria (LAB) that are important contributors to the production of fermented dairy products, and some species produce antimicrobial compounds. Most species in the genus Lactococcus have been isolated from food-related sources and plants and are generally regarded as safe. Probiotic foods use these LAB, and there have been various studies of the relationship between these foods and the maintenance of human intestinal health (32). Lactococcus was first established as a genus distinct from the genus Streptococcus in 1985 (29).Currently, six species and three subspecies in the genus Lactococcus have been validated. Lactococcus plantarum has been isolated mainly from plants; L. garvieae has been isolated from fish, animals, and milk, and L. piscium has been isolated from salmon. Lactococcus lactis is most commonly found in raw milk, cheese, and other dairy products; L. raffinolactis has been found in raw milk and cheese, and L. chunagangensis has been isolated from wastewater. Among the six species, L. lactis is considered one of the most important in food production because it is used to manufacture fermented milk, butter, and cheese. Because of this importance, the whole genomes of three strains of L. lactis—L. lactis subsp. cremoris SK11 (10), L. lactis subsp. cremoris MG 1363 (37), and L. lactis subsp. lactis IL1403 (2)—have been sequenced.Since L. lactis was first described by Orla-Jensen in 1919 (21), there have been various classifications. To date, L. lactis has been classified into three subspecies: L. lactis subsp. cremoris, L. lactis subsp. hordniae, and L. lactis subsp. lactis. However, this classification was based on only a few phenotypic characteristics and is considered imperfect because of its inherent disadvantages of sensitivity to culture conditions or bacterial growth phase. Discriminating between L. lactis subsp. cremoris and L. lactis subsp. lactis is particularly difficult but is very important in industrial applications, because the activities of the two subspecies in cheese manufacture differ. In addition, when newly isolated bacterial strains are registered in public culture collections, these strains have to be identified and discriminated at the subspecies level. Normally, these two subspecies are identified on the basis of the following phenotypic features: (i) the ability to ferment maltose and ribose, (ii) growth in 4% NaCl (pH 9.2) at 40°C, (iii) the ability to produce ammonia from arginine, and (iv) the presence of glutamate decarboxylase activity (18-20). However, determining the results of the phenotypic identification is difficult because they are sometimes ambiguous and time sensitive, as demonstrated by the sugar fermentation tests described below, which gave different results over time. In addition, the results of phenotypic identifications in previous reports were not identical each other (9, 28, 35).From an evolutionary viewpoint, it is reasonable to classify subspecies by using the divergence of housekeeping genes that are well preserved at the genus or species level. 16S rRNA gene sequencing is the most common technique currently used to identify species. At the subspecies level, however, 16S rRNA gene sequence identity is often very high, and these sequences therefore cannot be used for identification purposes (14, 24, 27, 36). Recently, for LAB, the partial sequences of the recA (recombinase A), pheS (phenylalanyl tRNA synthetase alpha subunit), and rpoA (DNA-directed RNA polymerase alpha chain) genes have been effectively used for species or subspecies identification (5, 7, 17), and the analysis of 16S rRNA gene sequences in combination with housekeeping gene sequences has been used to identify subspecies.In recent years, a number of important experiments have used matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for rapid bacterial identification, including clostridia (15), LAB (34), Listeria (1), mycobacteria (12), salmonellae (6), viridans group streptococci (8), and other nonfermenting bacteria (16). In these studies, MALDI-TOF MS spectra were obtained from intact cells without biomarker purification or chromatographic separation. MALDI-TOF MS is a good tool for the analysis of biopolymers because of its soft ionization, and it plays a central role in proteomic research. Because of their simplicity, speed, and accuracy, MS methods have been successfully applied to biomarker discovery and the characterization of various bacterial agents. Although DNA sequencing is the current standard for molecular characterization of bacteria, molecular methods cannot be easily applied for rapid classification and identification.Our aim was to examine whether a proteomic approach using MALDI-TOF MS was effective for rapid bacterial identification, especially of two of the subspecies of L. lactis.     

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).     

Broad Conservation of Milk Utilization Genes in Bifidobacterium longum subsp. infantis as Revealed by Comparative Genomic Hybridization

Human milk oligosaccharides (HMOs) are the third-largest solid component of milk. Their structural complexity renders them nondigestible to the host but liable to hydrolytic enzymes of the infant colonic microbiota. Bifidobacteria and, frequently, Bifidobacterium longum strains predominate the colonic microbiota of exclusively breast-fed infants. Among the three recognized subspecies of B. longum, B. longum subsp. infantis achieves high levels of cell growth on HMOs and is associated with early colonization of the infant gut. The B. longum subsp. infantis ATCC 15697 genome features five distinct gene clusters with the predicted capacity to bind, cleave, and import milk oligosaccharides. Comparative genomic hybridizations (CGHs) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations. Multilocus sequence typing provided taxonomic subspecies designations and grouped the strains between B. longum subsp. infantis and B. longum subsp. longum. CGH analysis determined that HMO utilization gene regions are exclusively conserved across all B. longum subsp. infantis strains capable of growth on HMOs and have diverged in B. longum subsp. longum strains that cannot grow on HMOs. These regions contain fucosidases, sialidases, glycosyl hydrolases, ABC transporters, and family 1 solute binding proteins and are likely needed for efficient metabolism of HMOs. Urea metabolism genes and their activity were exclusively conserved in B. longum subsp. infantis. These results imply that the B. longum has at least two distinct subspecies: B. longum subsp. infantis, specialized to utilize milk carbon, and B. longum subsp. longum, specialized for plant-derived carbon metabolism.The newborn infant not only tolerates but requires colonization by commensal microbes for its own development and health (3). The relevance of the gut microbiome in health and disease is reflected by its influence in a number of important physiological processes, from physical maturation of the developing immune system (28) to the altered energy homeostasis associated with obesity (51, 52).Human milk provides all the nutrients needed to satisfy the neonate energy expenditure and a cadre of molecules with nonnutritional but biologically relevant functions (6). Neonatal health is likely dependent on the timely and complex interactions among bioactive components in human milk, the mucosal immune system, and specialized gut microbial communities (30). Human milk contains complex prebiotic oligosaccharides that stimulated the growth of select bifidobacteria (24, 25) and are believed to modulate mucosal immunity and protect the newborn against pathogens (23, 33, 41). These complex oligosaccharides, which are abundantly present in human milk (their structures are reviewed by Ninonuevo et al. [31] and LoCascio et al. [24]), arrive intact in the infant colon (5) and modulate the composition of neonatal gastrointestinal (GI) microbial communities.Bifidobacteria and, frequently, Bifidobacterium longum strains often predominate the colonic microbiota of exclusively breast-fed infants (10, 11). Among the three subspecies of B. longum, only B. longum subsp. infantis grows robustly on human milk oligosaccharides (HMOs) (24, 25). The availability of the complete genome sequences of B. longum subsp. infantis ATCC 15697 (40) and two other B. longum subsp. longum strains (22, 39) made possible the analysis of whole-genome diversity across the B. longum species. Analysis of the B. longum subsp. infantis ATCC 15697 genome has identified regions predicted to enable the metabolism of HMOs (40); however, their distribution across the B. longum spp. remains unknown. We predict that these regions are exclusively conserved in B. longum strains adapted to colonization of the infant gut microbiome and are therefore capable of robust growth on HMOs. In this work, whole-genome microarray comparisons (comparative genomic hybridizations [CGHs]) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations.     

Distribution of In Vitro Fermentation Ability of Lacto-N-Biose I,a Major Building Block of Human Milk Oligosaccharides,in Bifidobacterial Strains

This study investigated the potential utilization of lacto-N-biose I (LNB) by individual strains of bifidobacteria. LNB is a building block for the human milk oligosaccharides, which have been suggested to be a factor for selective growth of bifidobacteria. A total of 208 strains comprising 10 species and 4 subspecies were analyzed for the presence of the galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) gene (lnpA) and examined for growth when LNB was used as the sole carbohydrate source. While all strains of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, B. breve, and B. bifidum were able to grow on LNB, none of the strains of B. adolescentis, B. catenulatum, B. dentium, B. angulatum, B. animalis subsp. lactis, and B. thermophilum showed any growth. In addition, some strains of B. pseudocatenulatum, B. animalis subsp. animalis, and B. pseudolongum exhibited the ability to utilize LNB. With the exception for B. pseudocatenulatum, the presence of lnpA coincided with LNB utilization in almost all strains. These results indicate that bifidobacterial species, which are the predominant species found in infant intestines, are potential utilizers of LNB. These findings support the hypothesis that GLNBP plays a key role in the colonization of bifidobacteria in the infant intestine.Bifidobacteria are gram-positive anaerobic bacteria that naturally colonize the human intestinal tract and are believed to be beneficial to human health (21, 30). Breastfeeding has been shown to be associated with an infant fecal microbiota dominated by bifidobacteria, whereas the fecal microbiota of infants who are consuming alternative diets has been described as being mixed and adult-like (12, 21). It has been suggested that the selective growth of bifidobacteria observed in breast-fed newborns is related to the oligosaccharides and other factors that are contained in human milk (human milk oligosaccharides [HMOs]) (3, 4, 10, 11, 16, 17, 34). Kitaoka et al. (15) have recently found that bifidobacteria possess a unique metabolic pathway that is specific for lacto-N-biose I (LNB; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc). LNB is a building block for the type 1 HMOs [such as lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), and lacto-N-difucohexaose I (Fucα1-2Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4Glc)], and GNB is a core structure of the mucin sugar that is present in the human intestine and milk (18, 27). The GNB/LNB pathway, as previously illustrated by Wada et al. (33), involves proteins/enzymes that are required for the uptake and degradation of disaccharides such as the GNB/LNB transporter (29, 32), galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP; LnpA) (15, 24) (renamed from lacto-N-biose phosphorylase after the finding of phosphorylases specific to GNB [23] and LNB [22]), N-acetylhexosamine 1-kinase (NahK) (25), UDP-glucose-hexose 1-phosphate uridylyltransferase (GalT), and UDP-galactose epimerase (GalE). Some bifidobacteria have been demonstrated to be enzymatically equipped to release LNB from HMOs that have a type 1 structure (lacto-N biosidase; LnbB) (33) or GNB from the core 1-type O-glycans in mucin glycoproteins (endo-α-N-acetylgalatosaminidase) (6, 13, 14). It has been suggested that the presence of the LnbB and GNB/LNB pathways in some bifidobacterial strains could provide a nutritional advantage for these organisms, thereby increasing their populations within the ecosystem of these breast-fed newborns (33).The species that predominantly colonize the infant intestine are the bifidobacterial species B. breve, B. longum subsp. infantis, B. longum subsp. longum, and B. bifidum (21, 28). On the other hand, strains of B. adolescentis, B. catenulatum, B. pseudocatenulatum, and B. longum subsp. longum are frequently isolated from the adult intestine (19), and strains of B. animalis subsp. animalis, B. animalis subsp. lactis, B. thermophilum and B. pseudolongum have been shown to naturally colonize the guts of animals (1, 2, 7, 8). However, it is unclear whether there is a relationship between the differential colonization of the bifidobacterial species and the presence of the GNB/LNB pathway. In the present study, we investigated the ability of individual bifidobacterial strains in the in vitro fermentation of LNB and in addition, we also tried to determine whether or not the GLNBP gene (lnpA), which is a key enzyme of the GNB/LNB pathway, was present.     

The MG1363 and IL1403 Laboratory Strains of Lactococcus lactis and Several Dairy Strains Are Diploid

Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of Lactococcus lactis, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division.In contrast to higher eukaryotes, bacteria are haploid (6, 19); i.e., they store their genetic information in a single chromosome, which is then duplicated during the cell cycle. If the growth rate is sufficiently low, bacteria are born with a single copy of the chromosome, which will then be duplicated before the bacterium divides.There are a few reports about bacteria that have more than one genome per cell, i.e., that are polyploid. Deinococcus radiodurans has been shown to have 4 to 10 copies of its genome (13, 14). The diplococcal bacterium Neisseria gonorrhoeae was found to be diploid per coccal unit (31). Azotobacter vinelandii bacteria amplify the genome during growth in rich medium more than 40 times (20, 24, 27). The giant bacterium Epulopiscium fishelsoni has been shown to amplify its genome into a polytene chromosome of 3,000-fold ploidy (2). In addition, noncomplementing diploid bacteria have been isolated from protoplast fusions in Bacillus subtilis (11) and, as a result of zygogenesis, in Escherichia coli (10). A few other bacteria with two to six different chromosomes have been reported (15, 30).The normal cell cycle is divided into three periods: (i) the B period from cell division until initiation of replication, (ii) the C period in which the cell replicates its DNA, and (iii) the D period from termination of productive replication until cell division. The D period thus includes processes such as proofreading and deconcatenation. The B period is found only in cells whose generation times exceed the length of the combined C and D periods. If the generation times become shorter than the combined lengths of the C and D periods, then the initiations of replication move into previous cell cycles (16). Fast-growing bacteria will therefore have more than one ongoing round of DNA replication at the same time; they might have 4, 8, or even 16 origins of replication (4). Normal haploid cells are born with one chromosome, either replicating or nonreplicating, and always with one terminus of replication. Not until the replication has ended do the cells have two termini. If the D period becomes longer than the generation time, which happens at high growth rates, the cells will be born with two termini as a result of the overlapping cell cycles. Long D periods are discussed further in the Discussion.We have examined the cell cycle of Lactococcus lactis subsp. cremoris MG1363 in order to determine the cell cycle periods. To our surprise, we found that slow-growing cultures of these bacteria were born with two complete chromosomes, which were replicated into four chromosomes during the C period. This strain thus fulfills the criterion of being diploid without overlapping chromosomal replication cycles. Comparison with other L. lactis strains showed that both of the subspecies, L. lactis subsp. cremoris and L. lactis subsp. lactis, had members that were either diploid, like MG1363, or haploid, like most bacteria.     

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.     

Insertion and Deletion Events That Define the Pathogen Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium comprises genetically related yet phenotypically distinct subspecies. Consistent with their common origin, whole-genome sequence comparisons have revealed extensive synteny among M. avium organisms. However, the sequenced strains also display numerous regions of heterogeneity that likely contribute to the diversity of the individual subspecies. Starting from a phylogenetic framework derived by multilocus sequence analysis, we examined the distribution of 25 large sequence polymorphisms across a panel of genetically defined M. avium strains. This distribution was most variable among M. avium subsp. hominissuis isolates. In contrast, M. avium subsp. paratuberculosis strains exhibited a characteristic profile, with all isolates containing a set of genomic insertions absent from other M. avium strains. The emergence of the pathogen from its putative M. avium subsp. hominissuis ancestor entailed the acquisition of approximately 125 kb of novel genetic material, followed by a second phase, characterized by reductive genomics. One genomic deletion is common to all isolates while additional deletions distinguish two major lineages of M. avium subsp. paratuberculosis. For the average strain, these losses total at least 38 kb (sheep lineage) to 90 kb (cattle lineage). This biphasic pattern of evolution, characterized by chromosomal gene acquisition with subsequent gene loss, describes the emergence of M. avium subsp. paratuberculosis and may serve as a general model for the origin of pathogenic mycobacteria.Mycobacterium avium organisms are nontuberculous mycobacteria prevalent in environmental and clinical settings. M. avium infections result in diverse diseases, including avian tuberculosis, Johne''s disease, and Lady Windermere''s syndrome. Isolates are phenotypically different and were historically classified as separate species. However, current taxonomy, based on molecular analyses, recognizes a single species, M. avium, which is divided into distinct subgroups (21, 22).At present, M. avium subsp. hominissuis denotes environmental organisms associated with opportunistic infections in humans and swine (13, 23). M. avium subsp. avium is the classical agent of tuberculosis in birds and, along with M. avium subsp. silvaticum, represents a distinct lineage of bird pathogens (22). M. avium subsp. paratuberculosis causes Johne''s disease (Paratuberculosis), a chronic granulomatous intestinal disease (5). Although primarily associated with livestock, the bacterium may infect a wide range of mammalian hosts. A number of studies, using molecular testing for the M. avium subsp. paratuberculosis-specific insertion element IS900, have found an association between the presence of M. avium subsp. paratuberculosis and Crohn''s disease in humans (1, 9).Previous studies, including bigenomic comparisons of the sequenced strains M. avium subsp. hominissuis 104 and M. avium subsp. paratuberculosis K-10 (11), have revealed inter- and intrasubspecies differences (6, 12, 15, 16, 18, 19, 26). The phenotypic heterogeneity of M. avium strains may stem from genomic differences, but in the absence of a phylogenetic framework it has been difficult to define the key variations associated with the emergence of an individual subspecies. Recently, we proposed a phylogeny for M. avium based on multilocus sequence analysis (MLSA) of 10 genes and 56 M. avium isolates. This phylogeny is consistent with the current taxonomy and indicates that M. avium subsp. paratuberculosis is a distinct, clonal lineage of M. avium (22). To better understand the evolution of this subspecies, we have now examined the distribution of large sequence polymorphisms among a genetically defined panel of M. avium strains. Our findings reveal a characteristic genomic profile for M. avium subsp. paratuberculosis and provide insight into the biphasic evolution of this successful pathogen.