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.     

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.     

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).     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

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

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

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.