Complete Genome Sequence of Staphylococcus lugdunensis Strain HKU09-01

Staphylococcus lugdunensis is a member of the coagulase-negative staphylococci and commonly found as part of the human skin flora. It is a significant cause of catheter-related bacteremia and also causes serious infections like native valve endocarditis in previously healthy individuals. We report the complete genome sequence of this medically important bacterium.Staphylococcus lugdunensis is a member of the coagulase-negative staphylococci (CoNS) commonly colonizing the human skin and mucosal membranes. While the genus Staphylococcus contains 48 named species currently, only a few species, notably S. aureus, are coagulase positive. Thus, the phenotypic characteristic is routinely tested in the medical microbiological laboratory for rapid differentiation of the highly pathogenic S. aureus from the other staphylococci. Among the CoNS, only a few species are known to cause human disease, usually in the form of opportunistic infections only (6). However, S. lugdunensis is an important exception (3). Besides causing catheter-related bacteremia similar to other CoNS, it causes a variety of severe nosocomial and community-acquired infections, including native valve endocarditis, a devastating and potentially fatal disease that can affect previously healthy individuals. Another unusual feature are the susceptibilities of S. lugdunensis isolates to multiple antimicrobial agents even when the incidence of multiple-drug-resistant CoNS and S. aureus occurrences are increasing in both hospital and community settings (4, 5).The genome sequence of S. lugdunensis strain HKU09-01 was determined by high-throughput sequencing performed on a GS FLX system (Roche Diagnostics, Basel, Switzerland), with approximately 45-fold coverage of the genome. This clinical strain was previously isolated from the culture of pus from a skin swab. Genome assembly was performed using the Newbler assembler, resulting in 30 large contigs (>500 bp in size). The contigs were then ordered and oriented into one scaffold using OSLay (11). The genome-finishing strategy for S. lugdunensis was similar to that employed for our previously sequenced Laribacter hongkongensis genome (12). Briefly, gap closures were performed by genomic PCR followed by DNA sequencing of amplification products on an ABI 3130xl sequencer (Applied Biosystems, CA). The finished sequence was validated by genome macrorestriction analysis using multiple rare-cutting enzymes and visualization by pulsed-field gel electrophoresis. Protein coding regions were predicted with Glimmer3 (2), and automatic genome annotation was performed on the RAST server (1). Additionally, annotation of tRNA and transfer-messenger RNA (tmRNA) genes was performed using tRNAScan-SE (10) and ARAGORN (9). Identification of rRNA genes was performed using RNAmmer (8).The genome of S. lugdunensis strain HKU09-01 consists of a circular 2,658,366-bp chromosome with G+C content of 33.87%, similar to those of other staphylococci. No plasmids are present in the sequenced strain. The genome contains 61 tRNA genes for all amino acids and 2,489 predicted protein-coding genes. Eight putative genomic islands were identified, and one actually consists of a pair of duplicated 32-kb genomic regions. Similar to Staphylococcus saprophyticus (7), but different from the other staphylococci, the genome contains 6 rRNA operons, one of them having the unusual organization 5S-16S-23S-5S.With the availability of the present genome sequence, S. lugdunensis now joins other staphylococcal species with human pathogenic potential, like S. aureus, S. epidermidis, S. haemolyticus, and S. saprophyticus, to have at least one reference genome available. Further in-depth analysis will be necessary to fully elucidate the genomic differences that may explain the variation in virulence of the staphylococcal species.     

Genome Sequence of the Fleming Strain of Micrococcus luteus,a Simple Free-Living Actinobacterium

Micrococcus luteus (NCTC2665, “Fleming strain”) has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content, 73%) predicted to encode 2,403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 insertion sequence (IS) elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and 14 response regulators, a finding indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to β-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in the antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to most other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy, and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome.Micrococcus luteus, the type species of the genus Micrococcus (family Micrococcaceae, order Actinomycetales) (117), is an obligate aerobe. Three biovars have been distinguished (138). Its simple, coccoid morphology delayed the recognition of its relationship to actinomycetes, which are typically morphologically more complex. In the currently accepted phylogenetic tree of the actinobacteria, Micrococcus clusters with Arthrobacter and Renibacterium. Some other coccoid actinobacteria originally also called Micrococcus, but reclassified into four new genera (Kocuria, Nesterenkonia, Kytococcus, and Dermacoccus), are more distant relatives (121). The genus Micrococcus now includes only five species: M. luteus, M. lylae, M. antarcticus, M. endophyticus, and M. flavus (20, 69, 70, 121).We report here the genome sequence of Micrococcus luteus NCTC2665 (DSM 20030^T), a strain of historical interest, since Fleming used it to demonstrate bacteriolytic activity (due to lysozyme) in a variety of body tissues and secretions (29, 30), leading to its designation as Micrococcus lysodeikticus until its taxonomic status was clarified in 1972 (59). M. luteus has been used in a number of scientific contexts. The ease with which its cell wall could be removed made it a favored source of bacterial cell membranes and protoplasts for investigations in bioenergetics (28, 34, 89, 93). Because of the exceptionally high GC content of its DNA, M. luteus was used to investigate the relationship between codon usage and tRNA representation in bacterial genomes (51, 52, 61). Although it does not form endospores, M. luteus can enter a profoundly dormant state, which could explain why it may routinely be isolated from amber (39). Dormancy has been convincingly demonstrated under laboratory conditions (53-55, 83), and a secreted protein (Rpf) with muralytic activity is involved in the process of resuscitation (81, 82, 84, 85, 87, 125, 133).Micrococci are also of biotechnological interest. In addition to the extensive exploitation of these and related organisms by the pharmaceutical industry for testing and assaying compounds for antibacterial activity, micrococci can synthesize long-chain alkenes (1, 2, 127). They are also potentially useful for ore dressing and bioremediation applications, since they are able to concentrate heavy metals from low-grade ores (26, 66, 67, 116).Given its intrinsic historical and biological importance, and its biotechnological potential, it is perhaps surprising that the genome sequence of M. luteus was not determined previously (130). We consider here the strikingly small genome sequence in these contexts and also in relation to the morphological simplicity of M. luteus compared to many of its actinobacterial relatives, which include important pathogens as well as developmentally complex, antibiotic-producing bacteria with some of the largest bacterial genomes.     

Complete Genome Sequence of Staphylococcus aureus Strain JKD6159, a Unique Australian Clone of ST93-IV Community Methicillin-Resistant Staphylococcus aureus

Community methicillin-resistant Staphylococcus aureus (cMRSA) is an emerging issue that has resulted in multiple worldwide epidemics. We report the first complete genome sequence of an ST93-MRSA-IV clinical isolate that caused severe invasive infection and a familial outbreak of skin infection. This isolate is a representative of the most common Australian clone of cMRSA that is more distantly related to the previously sequenced genomes of S. aureus.Staphylococcus aureus is a major cause of both hospital- and community-acquired infections, with rapid emergence of antibiotic resistance, in particular methicillin resistance, adding complexity to the treatment of this organism (3). While previously a hospital problem, methicillin-resistant S. aureus (MRSA) is now being increasingly documented in healthy patients in the community, and these isolates are termed “community MRSA” (cMRSA). A number of cMRSA genomes have been sequenced; however, these are phylogenetically closely related to each other. In contrast, ST93-MRSA-IV, a unique Australian clone, is a singleton by multilocus sequence typing (MLST) eBURST analysis (4). It is now the dominant cMRSA clone in Australia and is associated with both skin infection and severe invasive infection, including necrotizing pneumonia, deep-seated abscesses, and septicemia (5, 10). JKD6159 is a representative ST93-MRSA-IV clinical isolate which caused septicemia and multifocal pulmonary and musculoskeletal abscesses in a previously well intravenous drug user and also resulted in a familial outbreak of skin infection.The genome sequence of S. aureus strain JKD6159 was determined by high-throughput whole-genome shotgun sequencing, using both Illumina GAII (Illumina, CA) and Roche GS FLX Titanium (Roche Diagnostics, Basel, Switzerland) sequencing technologies, producing approximately 164× and 32× coverage of the genome, respectively. The GS FLX Titanium reads were assembled using Newbler 2.0.01.12, resulting in 56 contigs totaling 2.8 Mbp (9). The paired GAII reads were aligned to the contigs using SHRiMP 1.3.2 to identify and correct 74 homopolymeric sequencing errors (11). Optical mapping was used to produce a high-resolution XbaI chromosome restriction map, and the contigs were ordered and oriented against this map using MapSolver 2.1.1 (Opgen). Gap closures were performed by PCR followed by Sanger sequencing of amplification products (3730S genetic analyzer sequencer; Applied Biosystems, CA). The finished sequence was validated by reference to the XbaI optical map, Roche GS FLX Titanium mate pair analysis, and Illumina paired-end-read analysis.Protein coding regions were predicted using GeneMarkS 4.6b, tRNA genes using tRNAscan-SE 1.23, and rRNA genes using RNAmmer 1.2 (2, 7, 8). Gene products were assigned using HMMER 3.0 against the Pfam database (release 23) and BLAST 2.2.23 against RefSeq Proteins (April 2010) and the Conserved Domain Database (v2.22) (1, 6). These automated analyses were followed by manual curation, including comparison with other completed S. aureus genomes.The genome of S. aureus strain JKD6159 consists of a circular 2,811,435-bp chromosome with 33% G+C content—similar to those of other staphylococci—and one circular plasmid of 20,730 bp. A total of 2,605 coding regions, 57 tRNA genes, and 5 rRNA loci were detected. Over 67% of genes were assigned to specific Clusters of Orthologous Groups (COG) Database functional groups, and 40% were assigned an enzyme classification number (12).Initial analysis of the whole-genome sequence of JKD6159 confirms that ST93-MRSA-IV is distantly related to other previously sequenced S. aureus genomes. ST93-MRSA-IV has a distinct accessory genome. There were a number of regions of difference in JKD6159 that contain coding sequences (CDS) not present in any other published S. aureus genomes. Additionally, the ssl gene cluster in JKD6159 appears distinct from other sequenced S. aureus isolates. Comparison with other S. aureus genomes also shows that although JKD6159 carries lukSF-PV (the genes encoding Panton-Valentine leukocidin), there is a relative paucity of virulence factors such as tst-1, genes encoding staphylococcal enterotoxins A to U, and the ACME locus. Further analysis of the genome is now under way to identify factors that might explain the emergence of this MRSA strain in the community.     

Virulence Factors Encoded by Legionella longbeachae Identified on the Basis of the Genome Sequence Analysis of Clinical Isolate D-4968

Legionella longbeachae causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of L. longbeachae-specific diagnostic tests. L. longbeachae displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to Legionella pneumophila, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems, L. longbeachae is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an L. longbeachae clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of L. pneumophila. The results revealed that the D-4968 genome is larger than the L. pneumophila genome and has a gene order that is different from that of the L. pneumophila genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding L. pneumophila Icm/Dot substrates have been found in the D-4968 genome. L. longbeachae encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the L. longbeachae Icm/Dot secretion system. In contrast to the L. pneumophila genome, the L. longbeachae D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of L. longbeachae to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of L. longbeachae may reflect adaptation of this species to life in soil.Isolation of Legionella longbeachae was first reported in 1981 after isolation from patients with pneumonia in the United States (11, 59). Although L. longbeachae is not a common respiratory pathogen in either North America or Europe, where Legionella pneumophila infections are predominant, it accounts for more than 50% of legionellosis cases in Australia and is also prevalent in New Zealand and Thailand (10, 12, 60, 66, 68, 77, 93, 94). Legionnaires'' disease induced by L. longbeachae infection is clinically indistinguishable from the disease caused by L. pneumophila (65). However, L. longbeachae infections have been associated with gardening and the use of potting soil, whereas the disease caused by other species is linked to freshwater sources (4, 65). L. longbeachae can survive for up to 9 months in moist potting soil at room temperature, in contrast to other Legionella species, which inhabit natural and manmade freshwater systems worldwide (34, 83, 84).In addition to the differences in habitat, L. longbeachae differs from L. pneumophila in its virulence in murine models of infection. L. longbeachae replicates in the lungs of A/J, C57BL/6, and BALB/c mice (6), whereas most inbred mice, including C57BL/6 and BALB strains, are resistant to L. pneumophila (61). These differences in murine host susceptibility are likely due to different abilities to activate caspase 1-mediated pyroptosis in macrophages. While L. pneumophila rapidly triggers pyroptosis in C57BL/6 mouse macrophages, L. longbeachae does not do this (6).Intracellular trafficking of L. longbeachae in mammalian macrophages also follows a route distinct from that of L. pneumophila. After phagocytosis, the L. pneumophila-containing vacuole (LCV) excludes early and late endosomal markers, such as early endosomal antigen 1 (EEA1), Rab5, LAMP-1, LAMP-2, and the mannose 6-phosphate receptor (M6PR) (5, 89). In L. pneumophila the Dot/Icm type IV secretion system is required for prevention of phagosome-lysosome fusion and for intracellular replication (47). Conversely, the L. longbeachae-containing vacuole acquires the early endosomal marker EEA1 and the late endosomal markers LAMP-2 and M6PR (5). It has been suggested that L. longbeachae intracellular trafficking resembles that of the facultative intracellular pathogen Brucella abortus, since a Brucella-containing vacuole also acquires early and late endosomal markers soon after infection (5). Despite the difference in intracellular trafficking between L. longbeachae and L. pneumophila, L. longbeachae rescues Dot/Icm-deficient L. pneumophila when these two organisms coinhabit LCV (5).Results of the studies cited above indicate that L. longbeachae differs from other legionellae in terms of habitat, host specificity, and intracellular trafficking. In this paper, we describe an analysis of the sequenced and annotated genome of L. longbeachae clinical isolate D-4968 compared with published genomes of L. pneumophila strains Corby, Lens, Paris, and Philadelphia-1 (16, 17, 38). Specifically, we compared genes involved in gene regulation, protein secretion systems, and motility in order to identify genes responsible for making L. longbeachae unique among the legionellae.     

Optical Mapping Reveals a Large Genetic Inversion between Two Methicillin-Resistant Staphylococcus aureus Strains

Staphylococcus aureus is a highly versatile and evolving bacterium of great clinical importance. S. aureus can evolve by acquiring single nucleotide polymorphisms and mobile genetic elements and by recombination events. Identification and location of novel genomic elements in a bacterial genome are not straightforward, unless the whole genome is sequenced. Optical mapping is a new tool that creates a high-resolution, in situ ordered restriction map of a bacterial genome. These maps can be used to determine genomic organization and perform comparative genomics to identify genomic rearrangements, such as insertions, deletions, duplications, and inversions, compared to an in silico (virtual) restriction map of a known genome sequence. Using this technology, we report here the identification, approximate location, and characterization of a genetic inversion of ∼500 kb of a DNA element between the NRS387 (USA800) and FPR3757 (USA300) strains. The presence of the inversion and location of its junction sites were confirmed by site-specific PCR and sequencing. At both the left and right junction sites in NRS387, an IS1181 element and a 73-bp sequence were identified as inverted repeats, which could explain the possible mechanism of the inversion event.Staphylococcus aureus is a gram-positive bacterium of immense clinical importance. This opportunistic pathogen is capable of causing a wide range of diseases from skin and soft-tissue infections to life-threatening bacteremia, endocarditis, and osteomyelitis (14). Approximately 75% of the S. aureus genome is composed of a core genome that is common in all strains, and 25% of the genome is composed of variable regions which can differ between different strains (4, 16, 24-26). S. aureus evolves primarily by introducing single nucleotide polymorphisms in its core genome and by acquiring mobile genetic elements (MGEs) through horizontal gene transfer. These MGEs include pathogenicity/genomic islands, plasmids, transposons, and bacteriophages that become integrated in the chromosome (4, 11, 16, 31, 32). Despite being a heterogeneous organism, genetic recombination in S. aureus is proposed to be rather rare (20, 24, 29, 35). Its clones are more likely to evolve by point mutations than by recombination events (12). The MGEs contribute to the phenotypic and genotypic diversity seen with the S. aureus population. Acquisition of the staphylococcal cassette chromosome (SCCmec) elements through site-specific recombinases has led to the epidemic of methicillin-resistant S. aureus (MRSA) strains in hospitals and communities all over the world (6, 10, 15). In recent years, the integration of arginine catabolite mobile element in the USA300 lineage of MRSA has been proposed to give the pathogen its epidemiological advantage, including traits for surviving in low-pH conditions and oxygen tension environments (11). In addition, chromosomal replacements have been observed within lineages of sequence type 34 (ST34) and ST42 (34) and ST8 and ST30 (13).Genomic rearrangements, such as inversions, have been observed with genomes of enteric bacteria, such as Salmonella enterica, Shigella flexneri, Yersinia pestis KIM, Escherichia coli (K12 and O157:H7), and group A Streptococcus pyogenes (8, 9, 18, 27, 28, 30, 37). No genomic inversions in S. aureus have been reported to date. With the use of optical mapping, large genomic rearrangements, such as inversions, that would otherwise be missed with other comparative genotyping approaches, including microarray analysis, can be identified. Optical mapping uses high-resolution restriction maps (optical maps) of a bacterial genome to determine its genomic organization (5, 21, 23, 33, 36). These optical maps can be compared to an in silico (virtual) restriction map of a known genome sequence and can be used to identify gene rearrangements and their locations. Using optical mapping in conjunction with subsequent site-specific PCR and sequencing, we report the identification, approximate location, and partial characterization of an ∼500-kb DNA element in NRS387, a USA800 strain that was found to be inverted relative to USA300FPR3757. Identification of IS1181 elements and a novel 73-bp element at both ends of the ∼500-kb element in NRS387 could suggest the possibility of an inversion event in an ancestral strain of NRS387.     

Genome Sequence of Lactobacillus crispatus ST1

Lactobacillus crispatus is a common member of the beneficial microbiota present in the vertebrate gastrointestinal and human genitourinary tracts. Here, we report the genome sequence of L. crispatus ST1, a chicken isolate displaying strong adherence to vaginal epithelial cells.Lactobacillus crispatus can persist in the vertebrate gastrointestinal tract and is among the most prevalent species of the Lactobacillus-dominated human vaginal microbiota (2, 9, 13, 14). It belongs to the so-called acidophilus group (3), which has attracted interest because some of its species are important factors in the production of fermented foods (12) and some can, at least transiently, colonize the human host (2, 9, 13, 14). Moreover, some specific strains, mainly L. acidophilus NCFM and L. johnsonii NCC 533, have received prominence as intestinal-health-promoting microbes (4). Although the genomes of seven members of the acidophilus complex have been sequenced to date (12), the genome sequences of L. crispatus and other predominant lactobacillar species in the urogenital flora have mostly remained obscure. Vaginal lactobacilli can have an important role in controlling the health of the host (2, 14). They can, for example, positively influence and stabilize the host''s vaginal microbiota via the production of compounds that are acidic or exert a direct inhibiting action toward pathogenic bacteria (2, 14). In addition to the antimicrobial compounds, the competitive exclusion of pathogens is another mechanism by which the host''s microbiota can be balanced (2). L. crispatus ST1 was originally isolated from the crop of a chicken, and PCR profiling of L. crispatus isolates has verified it to be an abundant colonizer of the chicken crop (6, 8). It also displays a strong protein-dependent adhesion to the epithelial cells of the human vagina and has been shown to inhibit the adhesion of avian pathogenic Escherichia coli (6, 7).The genome was sequenced (18× coverage) using a 454 pyrosequencer with GS FLX chemistry (Roche). The contig order was confirmed and gaps were filled by sequencing PCR fragments from the genomic DNA template using ABI 3730 and Big Dye chemistry (Applied Biosystems). Genomic data were processed using the Staden Package (11) and gsAssembler (Roche). Coding sequences (CDSs) were predicted using Glimmer3 (5) followed by manual curation of the start sites. The remaining intergenic regions were reanalyzed for missed CDSs by using BlastX (1). Annotation transfer was performed based on a BlastP search, followed by Blannotator analysis using default settings (http://ekhidna.biocenter.helsinki.fi/poxo/blannotator) and manual verification. Orthologous groups between the different lactobacillar proteomes were identified using OrthoMCL (10).The genome of L. crispatus ST1 consists of a single circular chromosome 2.04 Mbp in size, with an overall G+C content of 37%, without any plasmids. There are 64 tRNA genes, 4 rRNA operons, and 2 CRISPR loci. Out of the 2,024 predicted CDSs, a putative function was assigned to 77%, whereas 10% of the CDSs were annotated as conserved and 13% as novel. Based on the orthologous grouping, 302 (15%) of the CDSs encoded by ST1 have no detectable homologs in any of the Lactobacillus proteomes published to date.     

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.     

Complex Nature of the Genome in a Wine Spoilage Yeast,Dekkera bruxellensis

When the genome organizations of 30 native isolates belonging to a wine spoilage yeast, Dekkera (Brettanomyces) bruxellensis, a distant relative of Saccharomyces cerevisiae, were examined, the numbers of chromosomes varied drastically, from 4 to at least 9. When single gene probes were used in Southern analysis, the corresponding genes usually mapped to at least two chromosomal bands, excluding a simple haploid organization of the genome. When different loci were sequenced, in most cases, several different haplotypes were obtained for each single isolate, and they belonged to two subtypes. Phylogenetic reconstruction using haplotypes revealed that the sequences from different isolates belonging to one subtype were more similar to each other than to the sequences belonging to the other subtype within the isolate. Reanalysis of the genome sequence also confirmed that partially sequenced strain Y879 is not a simple haploid and that its genome contains approximately 1% polymorphic sites. The present situation could be explained by (i) a hybridization event where two similar but different genomes have recently fused together or (ii) an event where the diploid progenitor of all analyzed strains lost a regular sexual cycle, and the genome started to accumulate mutations.Recent achievements in genome sequencing have revealed that gene contents vary among distantly related organisms but are relatively constant among closely related species. For example, among hemiascomycete yeasts, which originated more than 250 million years ago and include well-studied yeasts such as Saccharomyces cerevisiae and Candida albicans (3, 4), an average genome contains approximately 5,000 genes. Approximately one-half of the protein-coding gene families are preserved in all of the yeasts sequenced to date. However, there is a large variation in the gene order and configuration of chromosomes among different species.Chromosome configuration is usually well preserved among populations belonging to the same species. Only rarely do geographically separated populations, for example, Mus musculus (8, 32), differ in the number and form of chromosomes. The mutability of the genome enhances the adaptability of the species, but it also decreases the viability of the new variant. In addition, these changes can preclude successful reproduction and can be a decisive factor in the emergence of new species (2; for a review, see references 6 and 7).Among closely related yeasts belonging to the Saccharomyces sensu stricto clade (including S. cerevisiae), which originated approximately 20 million years ago, the gene contents are relatively similar (13). Their genomes are almost colinear and consist of 16 chromosomes. Some inter- and intraspecific variations are observed predominantly at the chromosome ends (18, 19). Sensu stricto species are semifertile, meaning that they can successfully mate and produce F1 offspring but that the hybrids are largely sterile. It appears that this clade has still not completed the speciation process (7). The relatively low chromosome variability among Saccharomyces sensu stricto yeasts is probably promoted by regular sexual cycles. These yeasts are diploid, but heterozygosity is almost absent because of the homothallic life-style, which enables haploid spores from the same yeast cell to mate. Only for “sterile” hybrids, such as the lager brewing yeast Saccharomyces pastorianus (Saccharomyces carlsbergensis), originating upon the mating of two different species, has a pronounced heterozygosity been observed (14). The parental genomes came from S. cerevisiae and a close relative, Saccharomyces bayanus. A study of allotetraploid hybrids between a diploid S. cerevisiae strain and a diploid S. bayanus strain demonstrated that these hybrids behave essentially as diploids regarding meiosis and sporulation and had 77% spore viability (1, 22). The extent of intra- and interspecific genome variability is not well known for other yeasts, especially among distant relatives of S. cerevisiae. The only well-studied exception is a pathogen, Candida albicans, that is believed to be predominantly asexual. This yeast diverged from the S. cerevisiae lineage prior to the origin of the efficient homothallic life-style (reviewed in reference 25). The genome is diploid and shows a low level of heterozygosity (12), and large variations in the configurations of the chromosomes among different isolates have been reported (reviewed in reference 29).Dekkera bruxellensis is often isolated in wineries and is well known as a major microbial cause of wine spoilage. The lineages of D. bruxellensis and S. cerevisiae separated at approximately the same time as the lineages of S. cerevisiae and C. albicans separated, approximately 200 million years ago (40). However, D. bruxellensis and S. cerevisiae share several characteristics, such as the production of ethanol, the ability to propagate in the absence of oxygen (anaerobic growth), and petite positivity (the ability to produce offspring without mitochondrial DNA [mtDNA]), that are rarely found among other yeasts (16, 20). So far, a sexual cycle in D. bruxellensis has not been found.In this paper, we analyzed the genome structures of 30 isolates of D. bruxellensis originating from different geographical localities around the world. We show that these isolates have different numbers and sizes of chromosomes and also that the numbers of copies of several analyzed genes and their sequences vary. In addition, we could detect heterozygosity in the partial genome sequence of strain Y879.     

Complete Genome Sequence of Lactobacillus salivarius CECT 5713, a Probiotic Strain Isolated from Human Milk and Infant Feces

Lactobacillus salivarius is a homofermentative lactic acid bacterium and is frequently isolated from mucosal surfaces of healthy humans. L. salivarius CECT 5713, a strain isolated simultaneously from breast milk and infant feces of a healthy mother-infant pair, has immunomodulatory, anti-inflammatory, and anti-infectious properties, as revealed by several in vitro and in vivo assays. Here, we report its complete and annotated genome sequence.In the last years, culture-dependent and -independent analyses of the bacterial diversity of human milk and colostrum have revealed that these biological fluids are a source of live staphylococci, streptococci, lactic acid bacteria, and bifidobacteria in the infant gut (5, 6, 8, 9, 11, 13), where they play a key role in the initiation and development of the gut microbiota (12). In a previous study, we isolated L. salivarius CECT 5713 from human milk and infant feces of a mother-child pair (10). Subsequent studies revealed that this strain was a good probiotic candidate since it achieved high survival rates when exposed to the gastrointestinal tract conditions, showed a strong adherence to intestinal cells, stimulated the expression of mucin-encoding genes, produced antimicrobial compounds (lactate, acetate, and hydrogen peroxide), and displayed in vivo and in vitro immunomodulatory, anti-inflammatory, and antibacterial properties against pathogenic bacteria (2, 10, 15). Moreover, oral administration of L. salivarius CECT 5713 appears to be an efficient alternative for the treatment of infectious mastitis in lactating women (7). Similarly, studies with other L. salivarius strains in animal models and clinical trials have demonstrated their probiotic function and, particularly, their anti-inflammatory effects (3, 14, 16).In order to interrogate the genome sequence of L. salivarius CECT 5713 with regard to its probiotic properties, the complete genome sequence was determined by a whole-genome shotgun strategy using pyrosequencing technology (454 Life Sciences, Banford, CT). The initial draft assembly provided by 454 Life Sciences was based on 444,604 high-quality pyrosequencing reads, which assembled into 59 contigs. The genome sequence of L. salivarius UCC118 (1), a well-characterized probiotic strain, was used to order these contigs into large scaffolds.The genome of L. salivarius CECT 5713 consists of a circular chromosome of 1,828,169 bp, two plasmids (pHN1, 44,581 bp; pHN2, 20,426 bp), and a megaplasmid (pHN3, 242,962 bp). The overall GC content of the chromosome is 32.93%, similar to that of the megaplasmid but lower than those of the plasmids (>38%). The entire genome of CECT 5713 contains 1,558 protein-, 87 tRNA-, and 51 rRNA-encoding genes. A comparison between the genomes of L. salivarius CECT 5713 and UCC118 revealed the presence of 52 protein-encoding genes that are exclusive for CECT 5713, including genes encoding a 6-phospho-β-glucosidase and three collagen-binding proteins, which may explain the high potential for competitive exclusion of pathogens displayed by this strain. The genes responsible for the bacteriocin activity of L. salivarius CECT 5713 are located in pHN3. This megaplasmid contains six open reading frames (ORFs) closely related, but not identical, to the genes responsible for the biosynthesis of salivaricin ABP-118, a two-component class II bacteriocin (4), in L. salivarius UCC118. Globally, several features of the L. salivarius CECT 5713 genome suggest a strong probiotic potential in humans.     

Genome Sequence of the Polymyxin-Producing Plant-Probiotic Rhizobacterium Paenibacillus polymyxa E681

Paenibacillus polymyxa E681, a spore-forming, low-G+C, Gram-positive bacterium isolated from the rhizosphere of winter barley grown in South Korea, has great potential for agricultural applications due to its ability to promote plant growth and suppress plant diseases. Here we present the complete genome sequence of P. polymyxa E681. Its 5.4-Mb genome encodes functions specialized to the plant-associated lifestyle and characteristics that are beneficial to plants, such as the production of a plant growth hormone, antibiotics, and hydrolytic enzymes.Among the plant-associated microbes, some are beneficial to plants, as they antagonize various plant pathogens, induce immunity, or even promote growth (2, 21, 29). These “plant-probiotic” bacteria (15, 16, 19, 22, 23, 28) have been isolated and commercially developed for use in the biological control of plant diseases or biofertilization (7, 10). Spore-forming bacteria, in particular, members of the phylum Firmicutes and streptomycetes, are considered advantageous in product formulation and stable maintenance in soil (9).The genus Paenibacillus (1) has grown to encompass more than 110 species (http://www.bacterio.cict.fr/p/paenibacillus.html), but its genome information is severely underrepresented. Paenibacillus spp. are important members of soil- or plant-associated ecosystems (3, 8, 20), with Paenibacillus polymyxa as one of the most industrially significant bacteria (13, 17, 25, 31). P. polymyxa E681, an endospore former isolated from the rhizosphere of winter barley in South Korea (14, 27), suppresses plant diseases, produces antibiotics and a plant hormone, secretes a variety of hydrolytic enzymes, and has good root-colonizing ability (4, 26).We determined the genome sequence of a rifampin-resistant clone of E681. About 62,000 chromatograms (∼6.7-fold genome coverage) were produced from plasmid/fosmid/bacterial artificial chromosome libraries with an AB 3700/377 DNA analyzer. Base calling, fragment assembly, contig/scaffold editing, and finishing were performed with Phred/Phrap/Consed. Gaps were closed by primer walking. To improve the sequence quality, 2.4 Gb of 76-bp single-ended sequences were obtained from Illumina Genome Analyzer IIx. Errors were identified using Maq/MapView and rectified by confirmatory sequencing. Yacop-predicted coding sequences were translated and subjected to transitive annotation by searches against UniProt, COG, KEGG Genes, and TIGRFAMs.The genome is composed of one circular chromosome of 5,394,884 bp (45.8% G+C). It has as many as 12 rRNA operons. No plasmid was found. Three-quarters of the 4,805 genes were assigned putative functions. Protein-coding genes are distributed preferentially on the leading strand. Apparently to cope with an ever-changing environment in the rhizosphere, the genome hosts at least 13 extracytoplasmic function sigma factors (12). There are 19 complete/disrupted insertion sequence elements but few phage-related genes.Some antibiotic-biosynthetic genes have been characterized. Polymyxin, produced and transported by PmxA to -E (5), is a potent antimicrobial that recently attracted attention for the treatment of multidrug-resistant Gram-negative bacteria (11, 18, 30). Fusaricidin, an antifungal antibiotic consisting of six amino acids, is synthesized by a single-chain nonribosomal peptide synthetase (6). E681 may also synthesize a polyketide, a tridecaptin-like nonribosomal peptide, and a hybrid of polyketide and nonribosomal peptide. A gene cluster is responsible for the production of a novel lantibiotic.Based on sequence investigation and biochemical analysis, auxin biosynthesis via the indole-3-pyruvic acid pathway was proposed as the only possible mechanism (24). The bacterium also produces volatile compounds that may promote growth and induce resistance of plants and one or more N-acyl-l-homoserine lactonases. Genome analysis revealed a rich set of secreted enzymes that degrade various plant-derived polysaccharides. They include xylanases, pectic enzymes, cellulases, and amylases. Genes involved in nitrogen fixation were not identified.     

Complete Genome Sequence of Methanothermobacter marburgensis,a Methanoarchaeon Model Organism

The circular genome sequence of the chemolithoautotrophic euryarchaeon Methanothermobacter marburgensis, with 1,639,135 bp, was determined and compared with that of Methanothermobacter thermautotrophicus. The genomes of the two model methanogens differ substantially in protein coding sequences, in insertion sequence (IS)-like elements, and in clustered regularly interspaced short palindromic repeats (CRISPR) loci.Methanothermobacter marburgensis (DSM 2133) (formerly Methanobacterium thermoautotrophicum strain Marburg), a member of the Methanobacteriales (2), was isolated in 1978 from anaerobic sewage sludge in Marburg, Germany (5). The hydrogenotrophic methanogen grows even faster (2 h versus 3 h doubling time) and to higher cell concentrations (3 g versus 1.5 g dry mass per liter) than Methanothermobacter thermautotrophicus (DSM 1053) (formerly Methanobacterium thermoautotrophicum strain ΔH) (20) (for other differences, see references 3 and 19). Both methanogens were used in the last 35 years for the elucidation of the enzymes and coenzymes involved in CO₂ reduction to methane with H₂ (4, 16-18). The genome sequence of M. thermautotrophicus was reported in 1997 (15); that of M. marburgensis is announced here.The genome size of M. marburgensis is 1,639,135 bp (that of M. thermautotrophicus is 1,751,377 bp), the genome G+C content is 48.64% (49.54% for M. thermautotrophicus), and the part coding is 90.94% (91.02% for M. thermautotrophicus). Comparison of the sequences (13) revealed that the two genomes have 1,607 protein coding sequences (CDS) in common and 411 CDS not in common (145 CDS are found only in M. marburgensis and 266 CDS only in M. thermautotrophicus) and show a high degree of synteny. The CDS not in common could be traced back to gene splitting (15%), gene deletion (30%), gene duplication (30%), and lateral gene transfer (24%) events (percentages given are for M. marburgensis). Of the 1,607 CDS in common, approximately 40% show BLAST search expectation values of >10⁻¹⁰⁰ at the protein level, reflecting large differences in sequence divergence. Almost 470 CDS encode conserved hypothetical proteins.The genome of M. marburgensis harbors 15 insertion sequence (IS)-like elements, whereas there is no evidence for a classically organized IS-like element in M. thermautotrophicus. Consistently, a CDS for a transposase is found only in M. marburgensis.In the genome of M. marburgensis there is only one clustered regularly interspaced short palindromic repeat (CRISPR) locus with 36 repeats and only one CRISPR-associated (cas) gene (csa3), indicating that the organism is not protected from invasion by phage and plasmid DNA (7, 8, 10, 12). By comparison, in the genome of M. thermautotrophicus there are three CRISPR loci with 124, 4, and 47 repeats and 18 cas genes that encode proteins involved in adaptation and interference (http://genoweb1.irisa.fr/Serveur-GPO/outils/repeatsAnalysis/CRISPR/). The spacer sequences from locus 2 match DNA sequences found in phage ΨM1 of M. marburgensis (6, 11) and ΨM100 of M. wolfei (9), which supports the observation that M. thermautotrophicus is not lysed by those two phages. Unfortunately, there is no DNA sequence available for phage ΦF1, which is able to lyse M. thermautotrophicus (14), to compare it with the spacer sequences of the CRISPR regions. In the plasmid pM2001 (= pMTBMA4) (4,439-bp circular multicopy plasmid found only in M. marburgensis) (1, 19), no sequence identities for CRISPR spacer sequences of M. thermautotrophicus were found (14).Approximately 200 CDS were identified that are required for the synthesis of the enzymes, coenzymes, and prosthetic groups involved in CO₂ reduction to methane and in the coupling of this process with energy conservation. Some of the genes have been found only recently; others, such as those for coenzyme F₄₃₀ biosynthesis, still remain to be discovered.     

Cytoskeletal Asymmetrical Dumbbell Structure of a Gliding Mycoplasma,Mycoplasma gallisepticum,Revealed by Negative-Staining Electron Microscopy

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion. Mycoplasma gallisepticum, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of M. gallisepticum cells in detail. They glided in the direction of the bleb at an average speed of 0.4 μm/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species, Mycoplasma pneumoniae. Next, to elucidate the cytoskeletal structure of M. gallisepticum, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of M. gallisepticum were compared with those of M. pneumoniae in detail, and the possible protein components of these structures were considered.Mycoplasmas are commensal and occasionally pathogenic bacteria that lack a peptidoglycan layer (50). Several species feature a membrane protrusion at a pole; for Mycoplasma mobile, this protrusion is called the head, and for Mycoplasma pneumoniae, it is called the attachment organelle (25, 34-37, 52, 54, 58). These species bind to solid surfaces, such as glass and animal cell surfaces, and exhibit gliding motility in the direction of the protrusion (34-37). This motility is believed to be essential for the mycoplasmas'' pathogenicity (4, 22, 27, 36). Recently, the proteins directly involved in the gliding mechanisms of mycoplasmas were identified and were found to have no similarities to those of known motility systems, including bacterial flagellum, pilus, and slime motility systems (25, 34-37).Mycoplasma gallisepticum is an avian pathogen that causes serious damage to the production of eggs for human consumption (50). The cells are pear-shaped and have a membrane protrusion, consisting of the so-called bleb and infrableb (29), and gliding motility (8, 14, 22). Their putative cytoskeletal structures may maintain this characteristic morphology because M. gallisepticum, like other mycoplasma species, does not have a cell wall (50). In sectioning electron microscopy (EM) studies of M. gallisepticum, an intracellular electron-dense structure in the bleb and infrableb was observed, suggesting the existence of a cytoskeletal structure (7, 24, 29, 37, 58). Recently, the existence of such a structure has been confirmed by scanning EM of the structure remaining after Triton X-100 extraction (13), although the details are still unclear.A human pathogen, M. pneumoniae, has a rod-shaped cytoskeletal structure in the attachment organelle (9, 15, 16, 31, 37, 57). M. gallisepticum is related to M. pneumoniae (63, 64), as represented by 90.3% identity between the 16S rRNA sequences, and it has some open reading frames (ORFs) homologous to the component proteins of the cytoskeletal structures of M. pneumoniae (6, 17, 48). Therefore, the cytoskeletal structures of M. gallisepticum are expected to be similar to those of M. pneumoniae, as scanning EM images also suggest (13).The fastest-gliding species, M. mobile, is more distantly related to M. gallisepticum; it has novel cytoskeletal structures that have been analyzed through negative-staining transmission EM after extraction by Triton X-100 with image averaging (45). This method of transmission EM following Triton X-100 extraction clearly showed a cytoskeletal “jellyfish” structure. In this structure, a solid oval “bell,” about 235 nm wide and 155 nm long, is filled with a 12-nm hexagonal lattice. Connected to this bell structure are dozens of flexible “tentacles” that are covered with particles 20 nm in diameter at intervals of about 30 nm. The particles appear to have 180° rotational symmetry and a dimple at the center. The involvement of this cytoskeletal structure in the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding.In the present study, we applied this method to M. gallisepticum and analyzed its unique cytoskeletal structure, and we then compared it with that of M. pneumoniae.     

Diverse Enterotoxin Gene Profiles among Clonal Complexes of Staphylococcus aureus Isolates from the Bronx,New York

Staphylococcal enterotoxins (SE) can cause toxin-mediated disease, and those that function as superantigens are implicated in the pathogenesis of allergic diseases. The prevalence of 19 enterotoxin genes was determined by PCR in clinical S. aureus strains derived from wounds (108) and blood (99). We performed spa typing and multilocus sequence typing (MLST) to determine clonal origin, and for selected strains staphylococcal enterotoxin B (SEB) production was measured by enzyme-linked immunosorbent assay. Strains carried a median of five SE genes. For most SE genes, the prevalence rates among methicillin-resistant and methicillin-sensitive S. aureus isolates, as well as wound- and blood-derived isolates, did not differ. At least one SE gene was detected in all except two S. aureus isolates (>99%). Complete egc clusters were found in only 11% of S. aureus isolates, whereas the combination of sed, sej, and ser was detected in 24% of clinical strains. S. aureus strains exhibited distinct combinations of SE genes, even if their pulsed-field gel electrophoresis and MLST patterns demonstrated clonality. USA300 strains also showed considerable variability in SE content, although they contained a lower number of SE genes (mean, 3). By contrast, SE content was unchanged in five pairs of serial isolates. SEB production by individual strains varied up to 200-fold, and even up to 15-fold in a pair of serial isolates. In conclusion, our results illustrate the genetic diversity of S. aureus strains with respect to enterotoxin genes and suggest that horizontal transfer of mobile genetic elements encoding virulence genes occurs frequently.As a commensal, Staphylococcus aureus colonizes the nasal mucosa of 20 to 40% of humans (54), and as a pathogen it causes pyogenic diseases and toxin-mediated diseases (38). S. aureus produces many different virulence factors, including enterotoxins (SEs), which can cause defined toxic shock syndromes (4). The characterization of some of these toxins led to the discovery of superantigens (41), which bind to major histocompatibility complex class II molecules and Vβ chains of T-cell receptors, resulting in the activation of large numbers of T cells (20 to 30%) and massive cytokine production (10, 18). These superantigen-induced “cytokine storms” are responsible for the toxic effects seen in staphylococcal entertoxin B (SEB)- and toxic shock syndrome toxin (TSST)-associated shock syndromes in S. aureus infections (13, 40, 47). To date, 19 SEs have been identified based on sequence homologies, and studies have reported enterotoxin genes in up to 80% of all S. aureus strains (4, 21). Although many new enterotoxins have been identified, i.e., seg ser and seu (33, 37, 44, 49), their precise functions have not been characterized yet. The majority of experimental work with SEs is still done with SEB, toxic shock syndrome toxin 1, and SEA (27, 31), because these toxins are commercially available. Most SEs are located on mobile elements in bacterial genomes such as plasmids or pathogenicity islands and can thus be easily transferred horizontally between strains (5, 34, 35). Certain SE genes are grouped together. For instance seg, sei, sem, sen, and seo are commonly found in a gene cluster (egc) on genomic island νSAβ (34), and sel and sek are often found together with seb or sec on S. aureus pathogenicity islands. Other staphylococcal superantigen genes are encoded on plasmids (sed, sej, and ser) or are linked to the antibiotic resistance cassette SCCmec (seh) (44, 55). Phage φ3 carries either sea (strain Mu50), sep (N315), or sea sek seq (MW2) (1, 29).Although a few clinical studies have attempted to correlate shock and outcome with the presence of certain SEs in patients with S. aureus infections (17, 28), the contribution of these toxins to outcome is still unclear. Recent papers have proposed the SEs are immunomodulators and that colonization with S. aureus strains that produce SEB may contribute to the pathogenesis of asthma, chronic rhinitis, and dermatitis (2, 36, 46, 48, 56). The superantigen function of SEs in supernatants of S. aureus cultures can be neutralized by serum of colonized patients (21, 23). With new data emerging implicating SEs in the pathogenesis of chronic allergic syndromes, production of monoclonal antibodies and or vaccine strategies targeting SEs may be considered (6, 24, 26, 30) in the future. It is therefore important to characterize the prevalence of SE genes in clinical S. aureus strains.In this study, we analyzed SE content in both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains that were cultured from wounds (including USA300) and bloodstream infections of patients from a defined geographical area. In addition, SEB production was quantified by enzyme-linked immunosorbent assay (ELISA) in S. aureus strains carrying the seb gene, and spa typing confirmed clonal diversity among S. aureus isolates from different patients, as well as clonal stability in serial isolates, and multilocus sequence typing (MLST) done on a subset of less common spa types. We conclude that SE genes are abundant in S. aureus strains, albeit less abundant in USA300. SE content and combination are highly diverse and therefore more discriminatory than pulsed-field gel electrophoresis (PFGE) and MLST typing, albeit stable in serial isolates. Quantification of SEB production demonstrates that enterotoxin secretion can vary greatly among strains, even if they belong to the same S. aureus lineage. Given the complexities of SE prevalence, regulation, and possible function, we propose that the association of these toxins with chronic allergic diseases or outcome may be oversimplified at present. Precise characterizations of SE function and secretion patterns in individual S. aureus clones are warranted.     

Genome Sequences of Pelagibaca bermudensis HTCC2601T and Maritimibacter alkaliphilus HTCC2654T,the Type Strains of Two Marine Roseobacter Genera

Pelagibaca bermudensis HTCC2601^T and Maritimibacter alkaliphilus HTCC2654^T represent two marine genera in the globally significant Roseobacter clade of the Alphaproteobacteria. Here, we present the genome sequences of these organisms, isolated from the Sargasso Sea using dilution-to-extinction culturing, which offer insight into the genetic basis for the metabolic and ecological diversity of this important group.Organisms from the Roseobacter clade of the Alphaproteobacteria are numerically significant in the world''s oceans and have been found in a wide range of habitats (1, 3). Using previously described high-throughput dilution-to-extinction culturing (6, 13), the marine Roseobacter strains Pelagibaca bermudensis HTCC2601^T and Maritimibacter alkaliphilus HTCC2654^T were isolated in low-nutrient heterotrophic medium (LNHM) (4) from surface water collected at the Bermuda Atlantic Time-Series Study (BATS) site in the western Sargasso Sea (5, 9). As the type strains for two genera of this globally prolific Roseobacter group, P. bermudensis and M. alkaliphilus were selected for shotgun genome sequencing at the J. Craig Venter Institute through the Moore Foundation Microbial Genome Sequencing Project (http://www.moore.org/microgenome). Draft genomes of P. bermudensis and M. alkaliphilus, with 103 and 46 contigs, respectively, were annotated and analyzed through the Joint Genome Institute IMG/M website (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi) (10).The draft genomes of P. bermudensis and M. alkaliphilus comprise 5,425,920 and 4,529,231 bases, 5,522 and 4,764 predicted open reading frames (ORFs), and 66.44% and 64.13% G+C content, respectively. The P. bermudensis genome is predicted to contain 56 tRNA genes, five 5S rRNA genes, four 16S rRNA genes, and five 23S rRNA genes, and that of M. alkaliphilus 49 tRNA genes and one each of the 5S, 16S, and 23S rRNA genes. Both genomes have putative genes for complete glycolysis and Entner-Doudoroff pathways, a complete tricarboxylic acid cycle, and predicted metabolic pathways for the oxidation of C₁ compounds. Both have predicted genes for the synthesis of most essential amino acids and some vitamins and cofactors. Each has putative genes for the utilization of fructose, sucrose, and mannose, confirmed in physiological testing of P. bermudensis (5) but not for M. alkaliphilus (9). P. bermudensis contains a predicted complete RuBisCO complex, unique to the sequenced Roseobacter species (12, 15), a complete assimilatory nitrate reduction pathway, and several type VI secretion genes. M. alkaliphilus is predicted to have complete nitrate reduction pathways to both N₂ and ammonia and most type IV secretion genes. Both are predicted to have complete sec pathways and large numbers of ABC transporters (362 in P. bermudensis and 224 in M. alkaliphilus), similar to other Roseobacter strains (15).M. alkaliphilus was named because of its alkaline growth optimum at pH 10. Na⁺/H⁺ antiporters have been shown to be involved in conferring alkaliphilic phenotypes for a variety of organisms by increasing internal cellular H⁺ concentrations in alkaline conditions where Na⁺ is present (2, 7, 8, 14, 16, 17). As expected, the genome of M. alkaliphilus contains two putative Na⁺/H⁺ antiporters, one homologous to nhaP, important for alkaliphily in several strains (2, 16, 17), and another located adjacent to predicted ABC transporter genes for capsular polysaccharide export.     

The Spike Protein of Murine Coronavirus Regulates Viral Genome Transport from the Cell Surface to the Endoplasmic Reticulum during Infection

We observed that the nonfusogenic mouse hepatitis virus (MHV) strain MHV-2 reached a titer of ∼2 log₁₀ higher than that of the fusogenic strain A59 in astrocytoma DBT cells. To determine whether the spike protein is responsible for the difference, a recombinant virus, Penn-98-1, that contains the A59 genome with a spike from MHV-2 was used to infect DBT cells. Results showed that Penn-98-1 behaved like MHV-2, thus establishing a role for the spike protein in viral growth. The inverse correlation between viral fusogenicity and growth was further established in four different cell types and with a fusogenic mutant, the S757R mutant, derived from isogenic Penn-98-1. While both A59 and Penn-98-1 entered cells at similar levels, viral RNA and protein syntheses were significantly delayed for A59. Interestingly, when the genomic RNAs were delivered directly into the cells via transfection, the levels of gene expression for these viruses were similar. Furthermore, cell fractionation experiments revealed that significantly more genomic RNAs for the nonfusogenic MHVs were detected in the endoplasmic reticulum (ER) within the first 2 h after infection than for the fusogenic MHVs. Pretreatment of Penn-98-1 with trypsin reversed its properties in syncytium formation, virus production, and genome transport to the ER. These findings identified a novel role for the spike protein in regulating the uncoating and delivery of the viral genome to the ER after internalization.Murine coronavirus mouse hepatitis virus (MHV) is a member of the family Coronaviridae. It is an enveloped, positive-strand-RNA virus. The viral envelope contains three or four structural proteins, depending on the virus strain (21). The spike (S) protein is a glycoprotein with a molecular mass of approximately 180 kDa. For some MHV strains, such as JHM and A59, the S protein is cleaved by a furin-like proteinase into two subunits, the amino-terminal S1 and the carboxyl-terminal S2. The S1 subunit is thought to form the globular head of the spike and is responsible for the initial attachment of the virus to the receptor on the cell surface. The S2 subunit, which forms the stalk portion of the spike and which anchors the S protein to the viral envelope, facilitates the fusion between the viral envelope and the cell membrane and cell-cell fusion (4, 7, 20, 25, 39). In contrast, the S protein of some other MHV strains, such as MHV-2, does not undergo cleavage and usually does not cause cell-cell fusion (15, 34). It appears that the cleavability of the MHV S protein is associated usually, though not always, with its fusogenicity (10, 36). It has been suggested that the fusogenicity of the S protein may determine the route of virus entry, i.e., via direct fusion with plasma membranes or following endocytosis (11, 34), although the mechanism for virus-induced cell-cell fusion may differ from that for virus-cell fusion during entry (8). The S protein also elicits the induction of neutralizing antibodies and cell-mediated immunity in infected hosts (3). It is therefore an important determinant for viral infectivity, pathogenicity, and virulence (2, 5, 31, 38). The hemagglutinin-esterase (HE) protein is present only in certain MHV strains (22, 42) and may play a role in viral pathogenesis (44, 45). The small envelope (E) protein and the membrane (M) protein play a key role in virus assembly (40). The nucleocapsid (N) protein is a phosphoprotein of approximately 50 kDa and is associated with the RNA genome to form the nucleocapsid inside the envelope (21, 37).Infection of host cells by MHV is mediated through the interaction between the S protein and the cellular receptors that are members of the carcinoembryonic antigen (CEA) family of the immunoglobulin superfamily (9). This interaction then triggers fusion between the viral envelope and the plasma membrane or the endosomal membrane, the latter of which follows receptor-mediated endocytosis, thus allowing the nucleocapsid to deliver into the cytoplasm. Direct entry from the plasma membrane appears to be the predominant route for most MHV strains (19, 28), although entry by some mutant MHVs, such as OBLV60 and MHV-2, is low pH dependent, i.e., via endocytosis (11, 34). However, nothing is known about how the genomic RNA is transported to the rough endoplasmic reticulum (ER) for translation. Once on the ER, the viral genomic RNA is translated into a polymerase polyprotein from the 5′-end two open reading frames (two-thirds of the genome) via ribosomal frameshifting. The polymerase polyproteins in turn synthesize genomic and multiple species of subgenomic mRNAs. These mRNAs are then translated into nonstructural and structural proteins, the latter of which are essential for generation of progeny viruses.MHV can infect rodents, causing hepatitis, enteritis, nephritis, and central nervous system diseases. In the mouse central nervous system, some MHV strains, such as JHM and A59, are neurovirulent, causing acute encephalitis and chronic demyelination (1, 13), while others, such as MHV-2, exhibit extremely low neurovirulence, causing only meningitis without apparent encephalitis and demyelination (6, 16, 41). Extensive mutagenesis studies in combination with targeted RNA recombination have identified that the S protein is the major determinant of MHV pathogenicity in animals, although other viral genes also appear to modulate viral pathogenicity (17, 32). For example, the recombinant MHV Penn-98-1, which contains the S protein of MHV-2 in an A59 genome background, causes acute meningoencephalitis similar to that caused by A59 but does not cause demyelination similar to that observed for MHV-2 (6). It has also been shown that the amounts of antigen staining and necrosis in the liver correlate with the viral titer, which is determined largely by the S protein (29). However, how the S protein affects viral titer in cell culture and in animals is not known.In the present study, we initially observed that the levels of production of infectious viruses in an astrocytoma DBT cell line were markedly different among three MHV strains. Using the recombinant MHV Penn-98-1 and its isogenic S757R mutant, we further established that the S protein is responsible for the observed difference. The difference in virus production between A59 and Penn-98-1 was detected as early as 4 to 6 h postinfection (p.i.) and likely occurred during the early stages of the virus life cycle but after virus internalization. Interestingly, when the genomic RNAs were delivered directly into the cells via transfection, the levels of gene expression for these viruses were similar. Furthermore, cell fractionation experiments revealed that significantly more genomic RNAs for nonfusogenic MHVs were delivered to the ER within the first 2 h after infection than for fusogenic MHVs. These results demonstrate that the spike protein of MHV can regulate the intracellular transport of the viral genome to the ER following internalization. To our knowledge, this is the first study identifying a role for a coronavirus S protein in genome delivery in addition to its well-established role in receptor binding and virus-cell and cell-cell fusions during infection.