The ecological dynamics underlying species invasions have been a major focus of research in macroorganisms for the last five decades. However, we still know little about the processes behind invasion by unicellular organisms. To expand our knowledge of microbial invasions, we studied the roles of propagule pressure, nutrient supply, and biotic resistance in the invasion success of a freshwater invasive alga, Prymnesium parvum, using microcosms containing natural freshwater microbial assemblages. Microcosms were subjected to a factorial design with two levels of nutrient-induced diversity and three levels of propagule pressure, and incubated for 7 d, during which P. parvum densities and microbial community composition were tracked. Successful invasion occurred in microcosms receiving high propagule pressure whereas nutrients or community diversity played no role in invasion success. Invaded communities experienced distinctive changes in composition compared with communities where the invasion was unsuccessful. Successfully invaded microbial communities had an increased abundance of fungi and ciliates, and decreased abundances of diatoms and cercozoans. Many of these changes mirrored the microbial community changes detected during a natural P. parvum bloom in the source system. This role of propagule pressure is particularly relevant for P. parvum in the reservoir-dominated southern United States because this species can form large, sustained blooms that can generate intense propagule pressures for downstream sites. Human impact and global climate change are currently causing widespread environmental changes in most southern US freshwater systems that may facilitate P. parvum establishment and, when coupled with strong propagule pressure, could put many more systems at risk for invasion.

The order and orientation (arrangement) of all 91 sequenced scaffolds in the 12 pseudomolecules of the recently published tomato (Solanum lycopersicum, 2n = 2x = 24) genome sequence were positioned based on marker order in a high-density linkage map. Here, we report the arrangement of these scaffolds determined by two independent physical methods, bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and optical mapping. By localizing BACs at the ends of scaffolds to spreads of tomato synaptonemal complexes (pachytene chromosomes), we showed that 45 scaffolds, representing one-third of the tomato genome, were arranged differently than predicted by the linkage map. These scaffolds occur mostly in pericentric heterochromatin where 77% of the tomato genome is located and where linkage mapping is less accurate due to reduced crossing over. Although useful for only part of the genome, optical mapping results were in complete agreement with scaffold arrangement by FISH but often disagreed with scaffold arrangement based on the linkage map. The scaffold arrangement based on FISH and optical mapping changes the positions of hundreds of markers in the linkage map, especially in heterochromatin. These results suggest that similar errors exist in pseudomolecules from other large genomes that have been assembled using only linkage maps to predict scaffold arrangement, and these errors can be corrected using FISH and/or optical mapping. Of note, BAC-FISH also permits estimates of the sizes of gaps between scaffolds, and unanchored BACs are often visualized by FISH in gaps between scaffolds and thus represent starting points for filling these gaps.

Chapter 32 The Staphylococcus aureus NCTC 8325 Genome Allison F. Gillaspy, Allison F. GillaspySearch for more papers by this authorVeronica Worrell, Veronica WorrellSearch for more papers by this authorJoshua Orvis, Joshua OrvisSearch for more papers by this authorBruce A. Roe, Bruce A. RoeSearch for more papers by this authorDavid W. Dyer, David W. DyerSearch for more papers by this authorJohn J. Iandolo, John J. IandoloSearch for more papers by this author Allison F. Gillaspy, Allison F. GillaspySearch for more papers by this authorVeronica Worrell, Veronica WorrellSearch for more papers by this authorJoshua Orvis, Joshua OrvisSearch for more papers by this authorBruce A. Roe, Bruce A. RoeSearch for more papers by this authorDavid W. Dyer, David W. DyerSearch for more papers by this authorJohn J. Iandolo, John J. IandoloSearch for more papers by this author Book Editor(s):Vincent A. Fischetti, Vincent A. FischettiSearch for more papers by this authorRichard P. Novick, Richard P. NovickSearch for more papers by this authorJoseph J. Ferretti, Joseph J. FerrettiSearch for more papers by this authorDaniel A. Portnoy, Daniel A. PortnoySearch for more papers by this authorJulian I. Rood, Julian I. RoodSearch for more papers by this author First published: 03 March 2006 https://doi.org/10.1128/9781555816513.ch32Citations: 1 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onEmailFacebookTwitterLinkedInRedditWechat Summary By focusing on the Staphylococcus aureus phage group III strain NCTC 8325, Pattee's laboratory utilized transduction and transformation to identify a series of linkage groups that were organized into a rough genetic map. Once physical techniques for genome mapping (restriction endonuclease digestion and pulsed field gel electrophoresis) became feasible, the researcher and his coworkers attempted to fit the genetic linkage data to the physical maps of the genome. The genome sequence and annotation are now complete and are the main focus of this chapter. In addition to strain NCTC 8325, the genome sequence and annotation for at least six other S. aureus strains (COL, N315, Mu50, MW2, MRSA252, MSSA476) have been completed in recent years. The complete circular genome map of NCTC 8325 shows the position of each predicted open reading frame (ORF) within the genome and the predicted functional role for each coding sequence. The current map for the NCTC 8325 genome shows the position of each of the 2,892 ORFs designated by color based on their predicted functional roles. Strain 8325 appears to contain components of at least nine potential insertion elements (IS). S. aureus NCTC 8325 appears to contain fewer of the previously identified genomic islands, pathogenicity islands, or transposons than other sequenced S. aureus strains. References Ajdic, D., W. M. McShan, R. E. McLaughlin, G. Savic, J. Chang, M. B. Carson, C. Primeaux, R. Tian, S. Kenton, H. Jia, S. Lin, Y. Qian, S. Li, H. Zhu, F. Najar, H. Lai, J. White, B. A. Roe, and J. J. Ferretti. 2002. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc. Natl. Acad. Sci. USA 99: 14434–14439. 10.1073/pnas.172501299 CASPubMedWeb of Science®Google Scholar Anagnostopoulos, C., P. J. Piggot, and J. A. Hoch. 1993. The genetic map of Bacillus subtilis , p. 425–462. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics. American Society for Microbiology, Washington, D.C. 10.1128/9781555818388.ch29 Google Scholar Baba, T., F. Takeuchi, M. Kuroda, H. Yuzawa, K. Aoki, A. Oguchi, Y. Nagai, N. Iwama, K. Asano, T. Naimi, H. Kuroda, L. Cui, K. Yamamoto, and K. Hiramatsu. 2002. Genome and virulence determinants of high virulence community-acquired MRSA. Lancet 359: 1819–1827. 10.1016/S0140-6736(02)08713-5 CASPubMedWeb of Science®Google Scholar Bachmann, B. J. 1983. Linkage map of Escherichia coli K-12, edition 7. Microbiol. Rev. 47: 180–230. 10.1128/MMBR.47.2.180-230.1983 CASPubMedWeb of Science®Google Scholar Barberis-Maino, L., B. Berger-Bachi, H. Weber, W. D. Beck, and F. H. Kayser. 1987. IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgaris . Gene 59: 107–113. 10.1016/0378-1119(87)90271-X CASPubMedWeb of Science®Google Scholar Beres, S. B., G. L. Sylva, K. D. Barbian, B. Lei, J. S. Hoff, N. D. Mammarella, M. Y. Liu, J. C. Smoot, S. F. Porcella, L. D. Parkins, D. S. Campbell, T. M. Smith, J. K. McCormick, D. Y. Leung, P. M. Schlievert, and J. M. Musser. 2002. Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence. Proc. Natl. Acad. Sci. USA 99: 10078–10083. 10.1073/pnas.152298499 CASPubMedWeb of Science®Google Scholar Betley, M. J., and J. J. Mekalanos. 1988. Nucleotide sequence of the type A staphylococcal enterotoxin gene. J. Bacteriol. 170: 34–41. 10.1128/jb.170.1.34-41.1988 CASPubMedWeb of Science®Google Scholar Blattner, F. R., G. Plunkett III, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277: 1453–1474. 10.1126/science.277.5331.1453 CASPubMedWeb of Science®Google Scholar Byrne, M. E., D. A. Rouch, and R. A. Skurray. 1989. Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001 . Gene 81: 361–367. 10.1016/0378-1119(89)90197-2 CASPubMedWeb of Science®Google Scholar Coleman, D. C., J. P. Arbuthnott, H. M. Pomeroy, and T. H. Birkbeck. 1986. Cloning and expression in Escherichia coli and Staphylococcus aureus of the beta-lysin determinant from Staphylococcus aureus: evidence that bacteriophage conversion of beta-lysin activity is caused by insertional inactivation of the beta-lysin determinant. Microb. Pathog. 1: 549–564. 10.1016/0882-4010(86)90040-9 CASPubMedWeb of Science®Google Scholar Coleman, D. C., D. J. Sullivan, R. J. Russell, J. P. Arbuthnott, B. F. Carey, and H. M. Pomeroy. 1989. Staphylococcus aureus bacteriophages mediating the simultaneous lysogenic conversion of beta-lysin, staphylokinase and enterotoxin A: molecular mechanism of triple conversion. J. Gen. Microbiol. 135: 1679–1697. CASPubMedWeb of Science®Google Scholar Derbise, A., K. G. Dyke, and N. el Solh. 1994. Isolation and characterization of IS1181, an insertion sequence from Staphylococcus aureus . Plasmid 31: 251–264. 10.1006/plas.1994.1027 CASPubMedWeb of Science®Google Scholar Ferretti, J. J., W. M. McShan, D. Ajdic, D. J. Savic, G. Savic, K. Lyon, C. Primeaux, S. Sezate, A. N. Suvorov, S. Kenton, H. S. Lai, S. P. Lin, Y. Qian, H. G. Jia, F. Z. Najar, Q. Ren, H. Zhu, L. Song, J. White, X. Yuan, S. W. Clifton, B. A. Roe, and R. McLaughlin. 2001. Complete genome sequence of an M1 strain of Streptococcus pyogenes . Proc. Natl. Acad. Sci. USA 98: 4658–4663. 10.1073/pnas.071559398 CASPubMedWeb of Science®Google Scholar Fitzgerald, J. R., S. R. Monday, T. J. Foster, G. A. Bohach, P. J. Hartigan, W. J. Meaney, and C. J. Smyth. 2001. Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens. J. Bacteriol. 183: 63–70. 10.1128/JB.183.1.63-70.2001 CASPubMedWeb of Science®Google Scholar Gillespie, M. T., B. R. Lyon, and R. A. Skurray. 1988. Structural and evolutionary relationships of beta-lactamase transposons from Staphylococcus aureus . J. Gen. Microbiol. 134: 2857–2866. CASPubMedWeb of Science®Google Scholar Holden, M. T., E. J. Feil, J. A. Lindsay, S. J. Peacock, N. P. Day, M. C. Enright, T. J. Foster, C. E. Moore, L. Hurst, R. Atkin, A. Barron, N. Bason, S. D. Bentley, C. Chillingworth, T. Chillingworth, C. Churcher, L. Clark, C. Corton, A. Cronin, J. Doggett, L. Dowd, T. Feltwell, Z. Hance, B. Harris, H. Hauser, S. Holroyd, K. Jagels, K. D. James, N. Lennard, A. Line, R. Mayes, S. Moule, K. Mungall, D. Ormond, M. A. Quail, E. Rabbinowitsch, K. Rutherford, M. Sanders, S. Sharp, M. Simmonds, K. Stevens, S. Whitehead, B. G. Barrell, B. G. Spratt, and J. Parkhill. 2004. Complete genomes of two clinical Staphylococcus aureus strains: evidence for the rapid evolution of virulence and drug resistance. Proc. Natl. Acad. Sci. USA 101: 9786–9791. 10.1073/pnas.0402521101 CASPubMedWeb of Science®Google Scholar Iandolo, J. J., J. P. Bannantine, and G. C. Stewart. 1997. Genetic and physical map of the chromosome of Staphylococcus aureus , p. 39–54. In G. L. Archer and K. Crosley (ed.), Staphylococci and Staphylococcal Diseases. Churchill-Livingstone, New York, N.Y. Google Scholar Iandolo, J. J., V. Worrell, K. H. Groicher, Y. Qian, R. Tian, S. Kenton, A. Dorman, H. Ji, S. Lin, P. Loh, S. Qi, H. Zhu, and B. A. Roe. 2002. Comparative analysis of the genomes of the temperate bacteriophages phi 11, phi 12 and phi 13 of Staphylococcus aureus 8325. Gene 289: 109–118. 10.1016/S0378-1119(02)00481-X CASPubMedWeb of Science®Google Scholar Kondo, I., and K. Fujise. 1977. Serotype B staphylococcal bacteriophage singly converting staphylokinase. Infect. Immun. 18: 266–272. 10.1128/IAI.18.2.266-272.1977 CASPubMedWeb of Science®Google Scholar Kuroda, M., T. Ohta, I. Uchiyama, T. Baba, H. Yuzawa, I. Kobayashi, L. Cui, A. Oguchi, K. Aoki, Y. Nagai, J. Lian, T. Ito, M. Kanamori, H. Matsumaru, A. Maruyama, H. Murakami, A. Hosoyama, Y. Mizutani-Ui, N. K. Takahashi, T. Sawano, R. Inoue, C. Kaito, K. Sekimizu, H. Hirakawa, S. Kuhara, S. Goto, J. Yabuzaki, M. Kanehisa, A. Yamashita, K. Oshima, K. Furuya, C. Yoshino, T. Shiba, M. Hattori, N. Ogasawara, H. Hayashi, and K. Hiramatsu. 2001. Whole genome sequencing of meticillin-resistant Staphylococcus aureus . Lancet 357: 1225–1240. 10.1016/S0140-6736(00)04403-2 CASPubMedWeb of Science®Google Scholar Lee, C. Y. 1995. Association of staphylococcal type-1 capsule-encoding genes with a discrete genetic element. Gene 167: 115–119. 10.1016/0378-1119(95)00684-2 CASPubMedWeb of Science®Google Scholar Lee, C. Y., and J. J. Iandolo. 1986. Lysogenic conversion of staphylococcal lipase is caused by insertion of the bacteriophage L54a genome into the lipase structural gene. J. Bacteriol. 166: 385–391. 10.1128/jb.166.2.385-391.1986 CASPubMedWeb of Science®Google Scholar Lindsay, J. A., and M. T. Holden. 2004. Staphylococcus aureus: superbug, super genome? Trends Microbiol. 12: 378–385. 10.1016/j.tim.2004.06.004 CASPubMedWeb of Science®Google Scholar Lindsay, J. A., A. Ruzin, H. F. Ross, N. Kurepina, and R. P. Novick. 1998. The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus . Mol. Microbiol. 29: 527–543. 10.1046/j.1365-2958.1998.00947.x CASPubMedWeb of Science®Google Scholar Lyon, B. R., J. W. May, and R. A. Skurray. 1984. Tn4001: a gentamicin and kanamycin resistance transposon in Staphylococcus aureus . Mol. Gen. Genet. 193: 554–556. 10.1007/BF00382099 CASPubMedWeb of Science®Google Scholar Murphy, E., S. Phillips, I. Edelman, and R. P. Novick. 1981. Tn554: isolation and characterization of plasmid insertions. Plasmid 5: 292–305. 10.1016/0147-619X(81)90006-8 CASPubMedWeb of Science®Google Scholar Novick, R. P., P. Schlievert, and A. Ruzin. 2001. Pathogenicity and resistance islands of staphylococci. Microbes Infect. 3: 585–594. 10.1016/S1286-4579(01)01414-9 CASPubMedWeb of Science®Google Scholar Pattee, P. A. 1993. The genetic map of Staphylococcus aureus , p. 489–496. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics. American Society for Microbiology, Washington, D.C. Google Scholar Rouch, D. A., L. J. Messerotti, L. S. Loo, C. A. Jackson, and R. A. Skurray. 1989. Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257 . Mol. Microbiol. 3: 161–175. 10.1111/j.1365-2958.1989.tb01805.x CASPubMedWeb of Science®Google Scholar Rowland, S. J., and K. G. Dyke. 1989. Characterization of the staphylococcal beta-lactamase transposon Tn552 . EMBO J. 8: 2761–2773. 10.1002/j.1460-2075.1989.tb08418.x CASPubMedWeb of Science®Google Scholar Ruzin, A., J. Lindsay, and R. P. Novick. 2001. Molecular genetics of SaPI1—a mobile pathogenicity island in Staphylococcus aureus . Mol. Microbiol. 41: 365–377. 10.1046/j.1365-2958.2001.02488.x CASPubMedWeb of Science®Google Scholar Townsend, D. E., S. Bolton, N. Ashdown, D. I. Annear, and W. B. Grubb. 1986. Conjugative, staphylococcal plasmids carrying hitch-hiking transposons similar to Tn554: intra- and interspecies dissemination of erythromycin resistance. Aust. J. Exp. Biol. Med. Sci. 64: 367–379. 10.1038/icb.1986.39 CASPubMedWeb of Science®Google Scholar Ubeda, C., M. A. Tormo, C. Cucarella, P. Trotonda, T. J. Foster, I. Lasa, and J. R. Penades. 2003. Sip, an integrase protein with excision, circularization and integration activities, defines a new family of mobile Staphylococcus aureus pathogenicity islands. Mol. Microbiol. 49: 193–210. 10.1046/j.1365-2958.2003.03577.x CASPubMedWeb of Science®Google Scholar van der Vijver, J. C., M. van Es-Boon, and M. F. Michel. 1972. Lysogenic conversion in Staphylococcus aureus to leucocidin production. J. Virol. 10: 318–319. 10.1128/JVI.10.2.318-319.1972 PubMedWeb of Science®Google Scholar Zhang, Y. Q., S. X. Ren, H. L. Li, Y. X. Wang, G. Fu, J. Yang, Z. Q. Qin, Y. G. Miao, W. Y. Wang, R. S. Chen, Y. Shen, Z. Chen, Z. H. Yuan, G. P. Zhao, D. Qu, A. Danchin, and Y. M. Wen. 2003. Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC12228). Mol. Microbiol. 49: 1577–1593. 10.1046/j.1365-2958.2003.03671.x CASPubMedWeb of Science®Google Scholar Citing Literature Gram‐Positive Pathogens, Second Edition ReferencesRelatedInformation

ABSTRACT Arhodomonas sp. strain Seminole was isolated from a crude oil-impacted brine soil and shown to degrade benzene, toluene, phenol, 4-hydroxybenzoic acid (4-HBA), protocatechuic acid (PCA), and phenylacetic acid (PAA) as the sole sources of carbon at high salinity. Seminole is a member of the genus Arhodomonas in the class Gammaproteobacteria , sharing 96% 16S rRNA gene sequence similarity with Arhodomonas aquaeolei HA-1. Analysis of the genome predicted a number of catabolic genes for the metabolism of benzene, toluene, 4-HBA, and PAA. The predicted pathways were corroborated by identification of enzymes present in the cytosolic proteomes of cells grown on aromatic compounds using liquid chromatography-mass spectrometry. Genome analysis predicted a cluster of 19 genes necessary for the breakdown of benzene or toluene to acetyl coenzyme A (acetyl-CoA) and pyruvate. Of these, 12 enzymes were identified in the proteome of toluene-grown cells compared to lactate-grown cells. Genomic analysis predicted 11 genes required for 4-HBA degradation to form the tricarboxylic acid (TCA) cycle intermediates. Of these, proteomic analysis of 4-HBA-grown cells identified 6 key enzymes involved in the 4-HBA degradation pathway. Similarly, 15 genes needed for the degradation of PAA to the TCA cycle intermediates were predicted. Of these, 9 enzymes of the PAA degradation pathway were identified only in PAA-grown cells and not in lactate-grown cells. Overall, we were able to reconstruct catabolic steps for the breakdown of a variety of aromatic compounds in an extreme halophile, strain Seminole. Such knowledge is important for understanding the role of Arhodomonas spp. in the natural attenuation of hydrocarbon-impacted hypersaline environments.

The reniform nematode (RN), Rotylenchulus reniformis, is an agriculturally important pest with a broad host range that results in a large economic impact in tropical, subtropical and in warm temperate zones. In an initial effort to understand the transcriptome and gene expression in RN, we present EST results that reveal numerous putative parasitism-related genes some of which play roles in plant cell wall modification. The characterized contigs included 8362 (40.6%) matches to unique proteins. Coding contigs predicted were 10,656 (51.7%) or 3079 (14.9%), that was similar to those identified in Brugia malayi and Caenorhabditis elegans as reference organisms respectively. Specific transcripts studied in more detail include putative plant parasitism genes, prominent among them were several plant cell wall modification genes. Contigs matching 14 parasitism genes found in sedentary endoparasitic nematodes included expansins, hexosaminidase, glycosyl hydrolases family, 14-3-3 protein, xylanases, glutathione peroxidase, pectate lyase, β-1,4-endoglucanase, major sperm protein, aminopeptidase, c-type lectin, chitin synthase, FMR famide-like peptide, and calreticulin. These genes function in suppression of host defenses and development of feeding sites.

High-throughput pyrosequencing of SSU rDNA genes was used to obtain monthly snapshots of eukaryotic and bacterial diversity and community structure at two locations in Lake Texoma, a low salinity lake in the south central United States, over 1 year. The lake experienced two disturbance events (i) a localized bloom of Prymnesium parvum restricted to one of the locations that lasted from January to April, and (ii) a large (17 cm), global rain event in the beginning of May, overlaid onto seasonal environmental change. Eukaryotic species richness as well as both eukaryotic and bacterial community similarity exhibited seasonal patterns, including distinct responses to the rain event. The P. parvum bloom created a natural experiment in which to directly explore the effects of an Ecosystem Disruptive Algal Bloom (EDAB) on the microbial community separated from seasonal changes. Microbial species richness was unaffected by the bloom, however, the eukaryotic community structure (evenness) and the patterns of both eukaryotic and bacterial community similarity at bloom and non-bloom sites were statistically distinct during the 4 months of the bloom. These results indicate that physical and biological disturbances as well as seasonal environmental forces contribute to the structure of both the eukaryotic and bacterial communities.

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

Although new and emerging next-generation sequencing (NGS) technologies have reduced sequencing costs significantly, much work remains to implement them for de novo sequencing of complex and highly repetitive genomes such as the tetraploid genome of Upland cotton (Gossypium hirsutum L.). Herein we report the results from implementing a novel, hybrid Sanger/454-based BAC-pool sequencing strategy using minimum tiling path (MTP) BACs from Ctg-3301 and Ctg-465, two large genomic segments in A12 and D12 homoeologous chromosomes (Ctg). To enable generation of longer contig sequences in assembly, we implemented a hybrid assembly method to process ~35x data from 454 technology and 2.8-3x data from Sanger method. Hybrid assemblies offered higher sequence coverage and better sequence assemblies. Homology studies revealed the presence of retrotransposon regions like Copia and Gypsy elements in these contigs and also helped in identifying new genomic SSRs. Unigenes were anchored to the sequences in Ctg-3301 and Ctg-465 to support the physical map. Gene density, gene structure and protein sequence information derived from protein prediction programs were used to obtain the functional annotation of these genes. Comparative analysis of both contigs with Arabidopsis genome exhibited synteny and microcollinearity with a conserved gene order in both genomes. This study provides insight about use of MTP-based BAC-pool sequencing approach for sequencing complex polyploid genomes with limited constraints in generating better sequence assemblies to build reference scaffold sequences. Combining the utilities of MTP-based BAC-pool sequencing with current longer and short read NGS technologies in multiplexed format would provide a new direction to cost-effectively and precisely sequence complex plant genomes.

We used a comparative genomics approach to investigate the evolution of a complex nucleotide-binding (NB)-leucine-rich repeat (LRR) gene cluster found in soybean (Glycine max) and common bean (Phaseolus vulgaris) that is associated with several disease resistance (R) genes of known function, including Rpg1b (for Resistance to Pseudomonas glycinea1b), an R gene effective against specific races of bacterial blight. Analysis of domains revealed that the amino-terminal coiled-coil (CC) domain, central nucleotide-binding domain (NB-ARC [for APAF1, Resistance genes, and CED4]), and carboxyl-terminal LRR domain have undergone distinct evolutionary paths. Sequence exchanges within the NB-ARC domain were rare. In contrast, interparalogue exchanges involving the CC and LRR domains were common, consistent with both of these regions coevolving with pathogens. Residues under positive selection were overrepresented within the predicted solvent-exposed face of the LRR domain, although several also were detected within the CC and NB-ARC domains. Superimposition of these latter residues onto predicted tertiary structures revealed that the majority are located on the surface, suggestive of a role in interactions with other domains or proteins. Following polyploidy in the Glycine lineage, NB-LRR genes have been preferentially lost from one of the duplicated chromosomes (homeologues found in soybean), and there has been partitioning of NB-LRR clades between the two homeologues. The single orthologous region in common bean contains approximately the same number of paralogues as found in the two soybean homeologues combined. We conclude that while polyploidization in Glycine has not driven a stable increase in family size for NB-LRR genes, it has generated two recombinationally isolated clusters, one of which appears to be in the process of decay.

ABSTRACT Pyrosequencing analysis of 16S rRNA genes was used to examine impacts of elevated CO2 (eCO2) on soil microbial communities from 12 replicates each from ambient CO2 (aCO2) and eCO2 settings. The results suggest that the soil microbial community composition and structure significantly altered under conditions of eCO2, which was closely associated with soil and plant properties.