Haemophilus influenzae is an opportunistic pathogen. The emergence of virulent, non-typeable strains (NTHi) emphasizes the importance of developing new interventional targets. We screened the NTHi supragenome for genes encoding surface-exposed proteins suggestive of immune evasion, identifying a large family containing Sel1-like repeats (SLRs). Clustering identified ten SLR-containing gene subfamilies, each with various numbers of SLRs per gene. Individual strains also had varying numbers of SLR-containing genes from one or more of the subfamilies. Statistical genetic analyses of gene possession among 210 NTHi strains typed as either disease or carriage found a significant association between possession of the SlrVA subfamily (which we have termed, macrophage survival factor, msf) and the disease isolates. The PittII strain contains four chromosomally contiguous msf genes. Deleting all four of these genes (msfA1-4) (KO) resulted in a highly significant decrease in phagocytosis and survival in macrophages; which was fully complemented by a single copy of the msfA1 gene. Using the chinchilla model of otitis media and invasive disease, the KO strain displayed a significant decrease in fitness compared to the WT in co-infections; and in single infections, the KO lost its ability to invade the brain. The singly complemented strain showed only a partial ability to compete with the WT suggesting gene dosage is important in vivo. The transcriptional profiles of the KO and WT in planktonic growth were compared using the NTHi supragenome array, which revealed highly significant changes in the expression of operons involved in virulence and anaerobiosis. These findings demonstrate that the msfA1-4 genes are virulence factors for phagocytosis, persistence, and trafficking to non-mucosal sites.

There is extensive genomic diversity among Streptococcus pneumoniae isolates. Approximately half of the comprehensive set of genes in the species (the supragenome or pangenome) is present in all the isolates (core set), and the remaining is unevenly distributed among strains (distributed set). The Streptococcus pneumoniae Supragenome Hybridization (SpSGH) array provides coverage for an extensive set of genes and polymorphisms encountered within this species, capturing this genomic diversity. Further, the capture is quantitative. In this manner, the SpSGH array allows for both genomic and transcriptomic analyses of diverse S. pneumoniae isolates on a single platform. In this unit, we present the SpSGH array, and describe in detail its design and implementation for both genomic and transcriptomic analyses. The methodology can be applied to construction and modification of SpSGH array platforms, as well to other bacterial species as long as multiple whole‐genome sequences are available that collectively capture the vast majority of the species supragenome.

We previously carried out the design and testing of a custom-built Haemophilus influenzae supragenome hybridization (SGH) array that contains probe sequences to 2,890 gene clusters identified by whole genome sequencing of 24 strains of H. influenzae. The array was originally designed as a tool to interrogate the gene content of large numbers of clinical isolates without the need for sequencing, however, the data obtained is quantitative and is thus suitable for transcriptomic analyses. In the current study RNA was extracted from H. influenzae strain CZ4126/02 (which was not included in the design of the array) converted to cDNA, and labelled and hybridized to the SGH arrays to assess the quality and reproducibility of data obtained from these custom-designed chips to serve as a tool for transcriptomics. Three types of experimental replicates were analyzed with all showing very high degrees of correlation, thus validating both the array and the methods used for RNA profiling. A custom filtering pipeline for two-condition unpaired data using five metrics was developed to minimize variability within replicates and to maximize the identification of the most significant true transcriptional differences between two samples. These methods can be extended to transcriptional analysis of other bacterial species utilizing supragenome-based arrays.

Both host and viral factors are associated with HIV-1 susceptibility and disease progression. Host factors can typically be divided into three categories (Chatterjee, 2010Chatterjee K. Host genetic factors in susceptibility to HIV-1 infection and progression to AIDS.J. Genet. 2010; 89 (PubMed PMID: 20505255): 109-116Crossref PubMed Scopus (24) Google Scholar): 1) genes encoding cell-surface receptors or ligands for these proteins, including the classic CCR5 32-bp deletion; 2) genes within the human leukocyte antigens (HLA) that regulate host responses to infection; and 3) other cytokine and immune response genes, including the homozygosity at position −308 in the TNF-alpha promoter. More recently genome-wide association studies (GWAS) have detected new genetic factors associated with HIV-1 infection (van Manen et al., 2012van Manen D. van 't Wout A.B. Schuitemaker H. Genome-wide association studies on HIV susceptibility, pathogenesis and pharmacogenomics.Retrovirology. 2012; 9 (PubMed PMID: 22920050; PubMed Central PMCID: PMC3468375): 70https://doi.org/10.1186/1742-4690-9-70Crossref PubMed Scopus (42) Google Scholar). In addition, an extensive array of host physiological responses have been assessed, including MCP-1 (CCL2) (Price et al., 2007Price R.W. Epstein L.G. Becker J.T. Cinque P. Gisslen M. Pulliam L. et al.Biomarkers of HIV-1 CNS infection and injury.Neurology. 2007; 69 (PubMed PMID: 17967994): 1781-1788https://doi.org/10.1212/01.wnl.0000278457.55877.ebCrossref PubMed Scopus (70) Google Scholar). In relation to HIV-1 susceptibility, the co-receptors CXCR4 and CCR5 are classically linked to infection of T cells and cells of the monocyte–macrophage lineage (MML), respectively. CCR5-utilizing viruses are the major type of virus transmitted therefore MML cells have been thought to contribute to early disease progression. More recently it has been demonstrated that T lymphocytes may also be a major vehicle for HIV-1 transmission. Transmitted viruses preferentially use CCR5 as a co-receptor, require high levels of CD4, and are able to replicate efficiently in primary CD4+ T-cells, suggesting a CCR5-utilizing T-tropic viral phenotype (Ochsenbauer et al., 2012Ochsenbauer C. Edmonds T.G. Ding H. Keele B.F. Decker J. Salazar M.G. et al.Generation of transmitted/founder HIV-1 infectious molecular clones and characterization of their replication capacity in CD4 T lymphocytes and monocyte-derived macrophages.J. Virol. 2012; 86 (PubMed PMID: 22190722; PubMed Central PMCID: PMC3302286): 2715-2728https://doi.org/10.1128/JVI.06157-11Crossref PubMed Scopus (237) Google Scholar). Subsequently, these founder viruses can infect MML cells, resulting in the development of a wider target cell population that expresses higher levels of CCR5 than T cells (Ochsenbauer et al., 2012Ochsenbauer C. Edmonds T.G. Ding H. Keele B.F. Decker J. Salazar M.G. et al.Generation of transmitted/founder HIV-1 infectious molecular clones and characterization of their replication capacity in CD4 T lymphocytes and monocyte-derived macrophages.J. Virol. 2012; 86 (PubMed PMID: 22190722; PubMed Central PMCID: PMC3302286): 2715-2728https://doi.org/10.1128/JVI.06157-11Crossref PubMed Scopus (237) Google Scholar). Although in the classical sense, co-receptor utilization remains the most important factor in HIV-1 susceptibility, a number of other host factors are currently under investigation that also appear to play a role in cellular susceptibility. Often these host factors interact with viral proteins thereby affecting the efficiency of viral infection. HIV-1 viral protein R (Vpr) is a multifunctional virion-associated accessory protein (Cohen et al., 1990Cohen E.A. Dehni G. Sodroski J.G. Haseltine W.A. Human immunodeficiency virus vpr product is a virion-associated regulatory protein.J. Virol. 1990; 64 (PubMed PMID: 2139896; PubMed Central PMCID: PMC249501): 3097-3099PubMed Google Scholar). Vpr functions both early after entry into cells and also following integration of the proviral genome (Hrimech et al., 1999Hrimech M. Yao X.J. Bachand F. Rougeau N. Cohen E.A. Human immunodeficiency virus type 1 (HIV-1) Vpr functions as an immediate-early protein during HIV-1 infection.J. Virol. 1999; 73 (PubMed PMID: 10196306; PubMed Central PMCID: PMC104189): 4101-4109PubMed Google Scholar). Vpr is a component of the preintegration complex, and following nuclear import Vpr likely plays a major role in regulating the expression of immediate-early HIV-1 genes from the now integrated proviral genome. This mechanism is utilized prior to the transition to Tat-driven gene expression. Vpr is critical in the viral replication cycle in non-dividing cells, such as monocytes (Connor et al., 1995Connor R.I. Chen B.K. Choe S. Landau N.R. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes.Virology. 1995; 206 (PubMed PMID: 7531918): 935-944https://doi.org/10.1006/viro.1995.1016Crossref PubMed Scopus (1081) Google Scholar), whereas it has been shown to be dispensable in T-lymphocytes (Eckstein et al., 2001Eckstein D.A. Sherman M.P. Penn M.L. Chin P.S. De Noronha C.M. Greene W.C. et al.HIV-1 Vpr enhances viral burden by facilitating infection of tissue macrophages but not nondividing CD4+ T cells.J. Exp. Med. 2001; 194 (PubMed PMID: 11714748; PubMed Central PMCID: PMC2193684): 1407-1419Crossref PubMed Scopus (102) Google Scholar). In the manuscript by Chiang et al. within this issue of EBioMedicine, a new host factor is described as playing a role in HIV-1 susceptibility (Chiang et al., 2014Chiang Y.-P. et al.Large isoform of mammalian relative of DnaJ is a major determinant of human susceptibility to HIV-1 infection.EBioMedicine. 2014; 1: 126-132Summary Full Text Full Text PDF Scopus (7) Google Scholar). This host factor, MRJ-L, is the large splice isoform of DNAJB6 (a homolog of heat shock protein 40) and the authors state that it has been shown to interact with Vpr, however this data, to date has not been published and therefore this point is in need of further detailed exploration. The authors, studying cohorts of “men who had sex with men” (MSM), found that macrophage expression of MRJ-L varied among healthy subjects but was significantly and substantially higher in HIV-1-infected subjects. The macrophage MRJ-L expression level predicted the risk of HIV-1 infection with similar power as whether or not subjects engaged in unprotected sexual contact. To demonstrate a cause-and-effect relationship between MRJ-L expression and HIV-1 susceptibility, monocytes from healthy subjects were isolated and then differentiated and infected ex vivo. Interestingly, this experiment revealed that macrophages with high MRJ-L expression were more susceptible to HIV-1 infection than those with low MRJ-L expression. Furthermore, RNAi knockdown of MRJ-L in high-expressing cells decreased susceptibility to infection, while lentiviral over-expression of MRJ-L in low-expressing cells increased their susceptibility to infection. These results clearly implicate high expression of MRJ-L with macrophage susceptibility to HIV infection. The authors were able to identify a plausible mechanism, showing that MRJ-L colocalizes with Vpr and is required for localizing Vpr to the nucleus. Although the localization experiments were performed in HeLa cells, because of the importance of Vpr in macrophages, the extension can be made that MRJ-L plays a major role in the nuclear transport of this important viral protein (and possibly the whole pre-integration complex) in other cell types. The stochastic expression of MRJ-L in uninfected subjects raises the possibility that genetic factors controlling this variability are also responsible for variability in susceptibility to infection. Numerous studies have demonstrated the role of host proteins in HIV-1 susceptibility, and the strong evidence provided by Chiang et al. that MRJ-L levels impact susceptibility to infection makes it an attractive therapeutic target. However, caution needs to be taken with regard to developing a therapy to reduce MRJ-L expression. Studies have shown a link between decreased levels of MRJ-L and malignancies such as breast cancer, with MRJ-L being shown to regulate several key players in tumor formation and metastasis, and demonstrating its ability to functionally retard tumor growth (Mitra et al., 2008Mitra A. Fillmore R.A. Metge B.J. Rajesh M. Xi Y. King J. et al.Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer.Breast Cancer Res. 2008; 10 (PubMed PMID: 18328103; PubMed Central PMCID: PMC2397520): R22https://doi.org/10.1186/bcr1874Crossref PubMed Scopus (66) Google Scholar). More complete studies are needed to determine the role of MRJ-L in macrophages, as well as more directed therapeutic targeting to specific cells to avoid potentially devastating off-target consequences. The authors declared no conflicts of interest. Large Isoform of Mammalian Relative of DnaJ is a Major Determinant of Human Susceptibility to HIV-1 InfectionIndividual differences in susceptibility to human immunodeficiency virus type 1 (HIV-1) infection have been of interest for decades. We aimed to determine the contribution of large isoform of Mammalian DnaJ (MRJ-L), a HIV-1 Vpr-interacting cellular protein, to this natural variation. Expression of MRJ-L in monocyte-derived macrophages was significantly higher in HIV-infected individuals (n = 31) than their uninfected counterparts (n = 27) (p = 0.009). Fifty male homosexual subjects (20 of them are HIV-1 positive) were further recruited to examine the association between MRJ-L levels and occurrence of HIV infection. Full-Text PDF Open Access

The realization at the beginning of the current millennium that there are very substantial differences in gene content among the component strains of bacterial species, together with the observation that there exists profound phenotypic heterogeneity amongst these strains, led directly to the development of the field of comparative bacterial genomics, and the concepts of the core and supra (pan) genomes. The core genome is composed of the set of genes shared by all members of a given species (or other taxonomic grouping), and the supragenome is the set of all genes contained by the same grouping. Those genes not present in the core genome, but present in the supragenome, are referred to as distributed (accessory) genes, and it is this last group that is responsible for much of the intraspecies heterogeneity. In the current work we provide comparative genomic evidence for both the core genome hypothesis and the distributed genome hypothesis, and describe a testable means to determine if a strain or particular group of strains belongs within an extant genomically defined species (or other taxonomic) grouping.

Background Haemophilus influenzae colonizes the human nasopharynx as a commensal, and is etiologically associated with numerous opportunistic infections of the airway; it is also less commonly associated with invasive disease. Clinical isolates of H. influenzae display extensive genomic diversity and plasticity. The development of strategies to successfully prevent, diagnose and treat H. influenzae infections depends on tools to ascertain the gene content of individual isolates. Results We describe and validate a Haemophilus influenzae supragenome hybridization (SGH) array that can be used to characterize the full genic complement of any strain within the species, as well as strains from several highly related species. The array contains 31,307 probes that collectively cover essentially all alleles of the 2890 gene clusters identified from the whole genome sequencing of 24 clinical H. influenzae strains. The finite supragenome model predicts that these data include greater than 85% of all non-rare genes (where rare genes are defined as those present in less than 10% of sequenced strains). The veracity of the array was tested by comparing the whole genome sequences of eight strains with their hybridization data obtained using the supragenome array. The array predictions were correct and reproducible for ~ 98% of the gene content of all of the sequenced strains. This technology was then applied to an investigation of the gene content of 193 geographically and clinically diverse H. influenzae clinical strains. These strains came from multiple locations from five different continents and Papua New Guinea and include isolates from: the middle ears of persons with otitis media and otorrhea; lung aspirates and sputum samples from pneumonia and COPD patients, blood specimens from patients with sepsis; cerebrospinal fluid from patients with meningitis, as well as from pharyngeal specimens from healthy persons. Conclusions These analyses provided the most comprehensive and detailed genomic/phylogenetic look at this species to date, and identified a subset of highly divergent strains that form a separate lineage within the species. This array provides a cost-effective and high-throughput tool to determine the gene content of any H. influenzae isolate or lineage. Furthermore, the method for probe selection can be applied to any species, given a group of available whole genome sequences.

ABSTRACT Gardnerella vaginalis is associated with a spectrum of clinical conditions, suggesting high degrees of genetic heterogeneity among stains. Seventeen G. vaginalis isolates were subjected to a battery of comparative genomic analyses to determine their level of relatedness. For each measure, the degree of difference among the G. vaginalis strains was the highest observed among 23 pathogenic bacterial species for which at least eight genomes are available. Genome sizes ranged from 1.491 to 1.716 Mb; GC contents ranged from 41.18% to 43.40%; and the core genome, consisting of only 746 genes, makes up only 51.6% of each strain's genome on average and accounts for only 27% of the species supragenome. Neighbor-grouping analyses, using both distributed gene possession data and core gene allelic data, each identified two major sets of strains, each of which is composed of two groups. Each of the four groups has its own characteristic genome size, GC ratio, and greatly expanded core gene content, making the genomic diversity of each group within the range for other bacterial species. To test whether these 4 groups corresponded to genetically isolated clades, we inferred the phylogeny of each distributed gene that was present in at least two strains and absent in at least two strains; this analysis identified frequent homologous recombination within groups but not between groups or sets. G. vaginalis appears to include four nonrecombining groups/clades of organisms with distinct gene pools and genomic properties, which may confer distinct ecological properties. Consequently, it may be appropriate to treat these four groups as separate species.

Two multidrug resistant strains of Streptococcus pneumoniae - SV35-T23 (capsular type 23F) and SV36-T3 (capsular type 3) were recovered from the nasopharynx of two adult patients during an outbreak of pneumococcal disease in a New York hospital in 1996. Both strains belonged to the pandemic lineage PMEN1 but they differed strikingly in virulence when tested in the mouse model of IP infection: as few as 1000 CFU of SV36 killed all mice within 24 hours after inoculation while SV35-T23 was avirulent. Whole genome sequencing (WGS) of the two isolates was performed (i) to test if these two isolates belonging to the same clonal type and recovered from an identical epidemiological scenario only differed in their capsular genes? and (ii) to test if the vast difference in virulence between the strains was mostly - or exclusively - due to the type III capsule. WGS demonstrated extensive differences between the two isolates including over 2500 single nucleotide polymorphisms in core genes and also differences in 36 genetic determinants: 25 of which were unique to SV35-T23 and 11 unique to strain SV36-T3. Nineteen of these differences were capsular genes and 9 bacteriocin genes. Using genetic transformation in the laboratory, the capsular region of SV35-T23 was replaced by the type 3 capsular genes from SV36-T3 to generate the recombinant SV35-T3* which was as virulent as the parental strain SV36-T3* in the murine model and the type 3 capsule was the major virulence factor in the chinchilla model as well. On the other hand, a careful comparison of strains SV36-T3 and the laboratory constructed SV35-T3* in the chinchilla model suggested that some additional determinants present in SV36 but not in the laboratory recombinant may also contribute to the progression of middle ear disease. The nature of this determinants remains to be identified.

Background: Staphylococcus aureus is associated with a spectrum of symbiotic relationships with its human host from carriage to sepsis and is frequently associated with nosocomial and community-acquired infections, thus the differential gene content among strains is of interest.Results: We sequenced three clinical strains and combined these data with 13 publically available human isolates and one bovine strain for comparative genomic analyses. All genomes were annotated using RAST, and then their gene similarities and differences were delineated. Gene clustering yielded 3,155 orthologous gene clusters, of which 2,266 were core, 755 were distributed, and 134 were unique. Individual genomes contained between 2,524 and 2,648 genes. Gene-content comparisons among all possible S. aureus strain pairs (n = 136) revealed a mean difference of 296 genes and a maximum difference of 476 genes. We developed a revised version of our finite supragenome model to estimate the size of the S. aureus supragenome (3,221 genes, with 2,245 core genes), and compared it with those of Haemophilus influenzae and Streptococcus pneumoniae. There was excellent agreement between RAST's annotations and our CDS clustering procedure providing for high fidelity metabolomic subsystem analyses to extend our comparative genomic characterization of these strains.Conclusions: Using a multi-species comparative supragenomic analysis enabled by an improved version of our finite supragenome model we provide data and an interpretation explaining the relatively larger core genome of S. aureus compared to other opportunistic nasopharyngeal pathogens. In addition, we provide independent validation for the efficiency and effectiveness of our orthologous gene clustering algorithm.

Bacillus pseudofirmus OF4 is an extreme but facultative alkaliphile that grows non-fermentatively in a pH range from 7.5 to above 11.4 and can withstand large sudden increases in external pH. It is a model organism for studies of bioenergetics at high pH, at which energy demands are higher than at neutral pH because both cytoplasmic pH homeostasis and ATP synthesis require more energy. The alkaliphile also tolerates a cytoplasmic pH > 9.0 at external pH values at which the pH homeostasis capacity is exceeded, and manages other stresses that are exacerbated at alkaline pH, e.g. sodium, oxidative and cell wall stresses. The genome of B. pseudofirmus OF4 includes two plasmids that are lost from some mutants without viability loss. The plasmids may provide a reservoir of mobile elements that promote adaptive chromosomal rearrangements under particular environmental conditions. The genome also reveals a more acidic pI profile for proteins exposed on the outer surface than found in neutralophiles. A large array of transporters and regulatory genes are predicted to protect the alkaliphile from its overlapping stresses. In addition, unanticipated metabolic versatility was observed, which could ensure requisite energy for alkaliphily under diverse conditions.

We report on the comparative genomics and characterization of the virulence phenotypes of four S. pneumoniae strains that belong to the multidrug resistant clone PMEN1 (Spain(23F) ST81). Strains SV35-T23 and SV36-T3 were recovered in 1996 from the nasopharynx of patients at an AIDS hospice in New York. Strain SV36-T3 expressed capsule type 3 which is unusual for this clone and represents the product of an in vivo capsular switch event. A third PMEN1 isolate - PN4595-T23 - was recovered in 1996 from the nasopharynx of a child attending day care in Portugal, and a fourth strain - ATCC700669 - was originally isolated from a patient with pneumococcal disease in Spain in 1984. We compared the genomes among four PMEN1 strains and 47 previously sequenced pneumococcal isolates for gene possession differences and allelic variations within core genes. In contrast to the 47 strains - representing a variety of clonal types - the four PMEN1 strains grouped closely together, demonstrating high genomic conservation within this lineage relative to the rest of the species. In the four PMEN1 strains allelic and gene possession differences were clustered into 18 genomic regions including the capsule, the blp bacteriocins, erythromycin resistance, the MM1-2008 prophage and multiple cell wall anchored proteins. In spite of their genomic similarity, the high resolution chinchilla model was able to detect variations in virulence properties of the PMEN1 strains highlighting how small genic or allelic variation can lead to significant changes in pathogenicity and making this set of strains ideal for the identification of novel virulence determinants.

Benjamin Janto,1 Azad Ahmed,1 Masahiro Ito,2 Jun Liu,3 David B. Hicks,3 Sarah Pagni,3 Oliver J. Fackelmayer,3 Terry-Ann Smith,3 Joshua Earl,1 Liam D.H. Elbourne,4 Karl Hassan,4 Ian T. Paulsen,4 Anne-Brit Kolstø,5 Nicolas J. Tourasse,6 Garth D. Ehrlich,1 Robert Boissy,1 D. Mack Ivey,7 Gang Li,8 Yanfen Xue,8 Yanhe Ma,8 Fen Z. Hu1* and Terry A. Krulwich3* Allegheny General Hospital, Allegheny-Singer Research Institute, Center for Genomic Sciences and Department of Microbiology and Immunology, Drexel University College of Medicine, Allegheny Campus, Pittsburgh, PA 15212, USA. Graduate School of Life Sciences, Toyo University, Ora-gun, Gunma 374-0193, Japan. Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney 2109, Australia. Laboratory for Microbial Dynamics and Department of Pharmaceutical Biosciences, University of Oslo, Blindern 0316 Oslo, Norway. Institut de Biologie Physico-Chimique, Université Pierre et Marie Curie, 75005 Paris, France. Department of Biological Sciences, University of Arkansas, Fayetteville, AR 7270, USA. Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.

In many macroorganisms, the ultimate source of potent biologically active natural products has remained elusive due to an inability to identify and culture the producing symbiotic microorganisms. As a model system for developing a meta-omic approach to identify and characterize natural product pathways from invertebrate-derived microbial consortia, we chose to investigate the ET-743 (Yondelis) biosynthetic pathway. This molecule is an approved anticancer agent obtained in low abundance (10(-4)-10(-5) % w/w) from the tunicate Ecteinascidia turbinata and is generated in suitable quantities for clinical use by a lengthy semisynthetic process. On the basis of structural similarities to three bacterial secondary metabolites, we hypothesized that ET-743 is the product of a marine bacterial symbiont. Using metagenomic sequencing of total DNA from the tunicate/microbial consortium, we targeted and assembled a 35 kb contig containing 25 genes that comprise the core of the NRPS biosynthetic pathway for this valuable anticancer agent. Rigorous sequence analysis based on codon usage of two large unlinked contigs suggests that Candidatus Endoecteinascidia frumentensis produces the ET-743 metabolite. Subsequent metaproteomic analysis confirmed expression of three key biosynthetic proteins. Moreover, the predicted activity of an enzyme for assembly of the tetrahydroisoquinoline core of ET-743 was verified in vitro. This work provides a foundation for direct production of the drug and new analogues through metabolic engineering. We expect that the interdisciplinary approach described is applicable to diverse host-symbiont systems that generate valuable natural products for drug discovery and development.

ABSTRACT Cellulosilyticum ruminicola H1 is a newly described bacterium isolated from yak ( Bos grunniens ) rumen and is characterized by its ability to grow on a variety of hemicelluloses and degrade cellulosic materials. In this study, we performed the whole-genome sequencing of C. ruminicola H1 and observed a comprehensive set of genes encoding the enzymes essential for hydrolyzing plant cell wall. The corresponding enzymatic activities were also determined in strain H1; these included endoglucanases, cellobiohydrolases, xylanases, mannanase, pectinases, and feruloyl esterases and acetyl esterases to break the interbridge cross-link, as well as the enzymes that degrade the glycosidic bonds. This bacterium appears to produce polymer hydrolases that act on both soluble and crystal celluloses. Approximately half of the cellulytic activities, including cellobiohydrolase (50%), feruloyl esterase (45%), and one third of xylanase (31%) and endoglucanase (36%) activities were bound to cellulosic fibers. However, only a minority of mannase (6.78%) and pectinase (1.76%) activities were fiber associated. Strain H1 seems to degrade the plant-derived polysaccharides by producing individual fibrolytic enzymes, whereas the majority of polysaccharide hydrolases contain carbohydrate-binding module. Cellulosome or cellulosomelike protein complex was never isolated from this bacterium. Thus, the fibrolytic enzyme production of strain H1 may represent a different strategy in cellulase organization used by most of other ruminal microbes, but it applies the fungal mode of cellulose production.

Although there is tremendous interest in understanding the evolutionary roles of horizontal gene transfer (HGT) processes that occur during chronic polyclonal infections, to date there have been few studies that directly address this topic. We have characterized multiple HGT events that most likely occurred during polyclonal infection among nasopharyngeal strains of Streptococcus pneumoniae recovered from a child suffering from chronic upper respiratory and middle-ear infections. Whole genome sequencing and comparative genomics were performed on six isolates collected during symptomatic episodes over a period of seven months. From these comparisons we determined that five of the isolates were genetically highly similar and likely represented a dominant lineage. We analyzed all genic and allelic differences among all six isolates and found that all differences tended to occur within contiguous genomic blocks, suggestive of strain evolution by homologous recombination. From these analyses we identified three strains (two of which were recovered on two different occasions) that appear to have been derived sequentially, one from the next, each by multiple recombination events. We also identified a fourth strain that contains many of the genomic segments that differentiate the three highly related strains from one another, and have hypothesized that this fourth strain may have served as a donor multiple times in the evolution of the dominant strain line. The variations among the parent, daughter, and grand-daughter recombinant strains collectively cover greater than seven percent of the genome and are grouped into 23 chromosomal clusters. While capturing in vivo HGT, these data support the distributed genome hypothesis and suggest that a single competence event in pneumococci can result in the replacement of DNA at multiple non-adjacent loci.

Objectives: We sought to construct and partially characterize complementary DNA (cDNA) libraries prepared from the middle ear mucosa (MEM) of chinchillas to better understand pathogenic aspects of infection and inflammation, particularly with respect to leukotriene biogenesis and response. Methods: Chinchilla MEM was harvested from controls and after middle ear inoculation with nontypeable Haemophilus influenzae. RNA was extracted to generate cDNA libraries. Randomly selected clones were subjected to sequence analysis to characterize the libraries and to provide DNA sequence for phylogenetic analyses. Reverse transcription—polymerase chain reaction of the RNA pools was used to generate cDNA sequences corresponding to genes associated with leukotriene biosynthesis and metabolism. Results: Sequence analysis of 921 randomly selected clones from the uninfected MEM cDNA library produced approximately 250,000 nucleotides of almost entirely novel sequence data. Searches of the GenBank database with the Basic Local Alignment Search Tool provided for identification of 515 unique genes expressed in the MEM and not previously described in chinchillas. In almost all cases, the chinchilla cDNA sequences displayed much greater homology to human or other primate genes than with rodent species. Genes associated with leukotriene metabolism were present in both normal and infected MEM. Conclusions: Based on both phylogenetic comparisons and gene expression similarities with humans, chinchilla MEM appears to be an excellent model for the study of middle ear inflammation and infection. The higher degree of sequence similarity between chinchillas and humans compared to chinchillas and rodents was unexpected. The cDNA libraries from normal and infected chinchilla MEM will serve as useful molecular tools in the study of otitis media and should yield important information with respect to middle ear pathogenesis.

Dupuytren's contracture (DC) is the most common inherited connective tissue disease of humans and is hypothesized to be associated with aberrant wound healing of the palmar fascia. Fibroblasts and myofibroblasts are believed to play an important role in the genesis of DC and the fibroproliferation and contraction that are hallmarks of this disease. This study compares the gene expression profiles of fibroblasts isolated from DC patients and controls in an attempt to identify key genes whose regulation might be significantly altered in fibroblasts found within the palmar fascia of Dupuytren's patients. Total RNA isolated from diseased palmar fascia (DC) and normal palmar fascia (obtained during carpal tunnel release; 6 samples per group) was subjected to quantitative analyses using two different microarray platforms (GE Code Link™ and Illumina™) to identify and validate differentially expressed genes. The data obtained was analyzed using The Significance Analysis of Microarrays (SAM) software through which we identified 69 and 40 differentially regulated gene transcripts using the CodeLink™ and Illumina™ platforms, respectively. The CodeLink™ platform identified 18 upregulated and 51 downregulated genes. Using the Illumina™ platform, 40 genes were identified as downregulated, eleven of which were identified by both platforms. Quantitative RT-PCR confirmed the downregulation of three high-interest candidate genes which are all components of the extracellular matrix: proteoglycan 4 (PRG4), fibulin-1 (FBLN-1) transcript variant D, and type XV collagen alpha 1 chain. Overall, our study has identified a variety of candidate genes that may be involved in the pathophysiology of Dupuytren's contracture and may ultimately serve as attractive molecular targets for alternative therapies.