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US 20110086407A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0086407 A1 Berka et al. (43) Pub. Date: Apr. 14, 2011 (54) BACILLUS LCHENFORMS Publication Classification CHROMOSOME (51) Int. Cl. (75) Inventors: Randy Berka, Davis, CA (US); CI2N 9/12 (2006.01) Michael Rey, Davis, CA (US); CI2N 9/90 (2006.01) Preethi Ramaiya, Walnut Creek, C07K I4/32 (2006.01) CA (US); Jens Tonne Andersen, CI2N 9/00 (2006.01) Naerum (DK); Michael Dolberg CI2N 9/56 (2006.01) Rasmussen, Vallensbaek (DK); CI2N 9/16 (2006.01) Peter Bjarke Olsen, Copenhagen O CI2N 9/10 (2006.01) CI2N 9/88 (2006.01) (DK) CI2N 9/78 (2006.01) (73) Assignees: Novozymes A/S, Bagsvaerd (DK); C07K I4/95 (2006.01) Novozymes, Inc., Davis, CA (US) (52) U.S. Cl. ......... 435/194; 435/233; 530/350: 435/183; (21) Appl. No.: 12/972,306 435/222; 435/196; 435/193; 435/232:435/227 (22) Filed: Dec. 17, 2010 Related U.S. Application Data (57) ABSTRACT The present invention relates to an isolated polynucleotide of (62) Division of application No. 12/322.974, filed on Feb. the complete chromosome of Bacillus licheniformis. The 9, 2009, now Pat. No. 7,863,032, which is a division of present invention also relates to isolated genes of the chro application No. 10/983,128, filedon Nov. 5, 2004, now mosome of Bacillus licheniformis which encode biologically Pat. No. 7,494,798. active Substances and to nucleic acid constructs, vectors, and (60) Provisional application No. 60/535.988, filed on Jan. host cells comprising the genes as well as methods for pro 9, 2004, provisional application No. 60/561,059, filed ducing biologically active substances encoded by the genes on Apr. 8, 2004, provisional application No. 60/572, and to methods of using the isolated genes of the complete 403, filed on May 18, 2004. chromosome of Bacillus licheniformis. US 2011/0086407 A1 Apr. 14, 2011 BACILLUS LCHENFORMS or water, and understanding of specialized systems used by CHROMOSOME microbial cells to live in natural environments. 0008 Bacillus licheniformis is a gram positive spore CROSS-REFERENCE TO RELATED forming bacterium that is widely distributed as a saprophytic APPLICATIONS organism in the environment. Unlike most other bacilli that 0001. This application is a divisional of U.S. application are predominantly aerobic, Bacillus licheniformis is a facul Ser. No. 12/322,974, filed Feb. 9, 2009, which is a divisional tative anaerobe which may allow it to grow in additional of U.S. application Ser. No. 10/983,128, filed Nov. 5, 2004, ecological niches. This species produces a diverse assortment now U.S. Pat. No. 7,494,798, which claims the benefit of U.S. of extracellular enzymes that are believed to contribute to the Provisional Application No. 60/535.988, filed Jan. 9, 2004, process of nutrient cycling in nature (Claus, D. and Berkeley, U.S. Provisional Application No. 60/561,059, filed Apr. 8, R. C. W., 1986. In Bergey's Manual of Systematic Bacteriol 2004, and U.S. Provisional Application No. 60/572,403, filed ogy, Vol. 2., eds. Sneath, P. H. A. et al., Williams and Wilkins May 18, 2004, which applications are incorporated herein by Co., Baltimore, Md., pp. 1105-1139). Certain Bacillus reference. licheniformis isolates are capable of denitrification, however, the relevance of this characteristic to environmental denitri REFERENCE TO ASEQUENCE LISTING fication may be small since the species generally persists in soil as endospores (Alexander, M., 1977, Introduction to Soil 0002 This application contains a Sequence Listing in Microbiology. John Wiley and Sons, Inc., New York). computer readable form, which is incorporated herein by 0009. There are numerous industrial and agricultural uses reference. for Bacillus licheniformis and its extracellular products. The species has been used for decades in the manufacture of BACKGROUND OF THE INVENTION industrial enzymes including several proteases, C.-amylase, 0003 1. Field of the Invention penicillinase, pentosanase, cycloglucosyltransferase, B-man 0004. The present invention relates to an isolated poly nanase, and several pectinolytic enzymes, owing largely to its nucleotide molecule comprising the complete chromosome ability to secrete sizeable amounts of degradative enzymes. of Bacillus licheniformis. The present invention also relates to Bacillus licheniformis is also used to produce peptide antibi features (genes) of the complete chromosomal DNA mol otics such as bacitracin and proticin, in addition to a number ecule of Bacillus licheniformis which encode biologically of specialty chemicals such as citric acid, inosine, inosinic active substances and to nucleic acid constructs, vectors, and acid, and poly-Y-glutamic acid. The proteases from Bacillus host cells comprising the features as well as methods for licheniformis are used in the detergent industry as well as for producing biologically active Substances encoded by the fea dehairing and batting of leather (Eveleigh, D. E., 1981, Sci tures and to methods of using the isolated features derived entific American 245, 155-178). Amylases from Bacillus from the complete chromosomal DNA molecule of Bacillus licheniformis are deployed for the hydrolysis of starch, desiz licheniformis. ing of textiles, and sizing of paper (Erickson, R. J., 1976. In 0005 2. Description of the Related Art Microbiology, ed. Schlesinger, D. (Am. Soc. Microbiol. 0006 Microbes, which make up most of the earth's biom Washington, D.C.), pp. 406-419.). Certain strains of Bacillus ass, have evolved for some 3.8 billion years. They are found licheniformis have shown efficacy to destroy fungal patho in virtually every environment, Surviving and thriving in gens affecting maize, grasses, and vegetable crops (U.S. Pat. extremes of heat, cold, radiation, pressure, salt, acidity, and No. 5,589.381; U.S. Pat. No. 5,665,354). As an endospore darkness. Often in these environments, no other forms of life forming bacterium, the ability of the organism to Survive are found and the only nutrients come from inorganic matter. under unfavorable environmental conditions may enhance its The diversity and range of their environmental adaptations potential as a natural control agent. indicate that microbes long ago 'solved’ many problems for 0010 Bacillus licheniformis can be differentiated from which scientists are still actively seeking Solutions. The value other bacillion the basis of metabolic and physiological tests in determining the complete genome sequence of microbes is (Logan, N. A. and Berkeley, R. C. W., 1981. In The Aerobic that it provides a detailed blueprint for the organism revealing Endospore-Forming Bacteria Classification and Identifica all of the biochemical pathways, Substrates, intermediates, tion, eds. Berkeley, R. C. W. and Goodfellow, M., Academic and end products as well as regulatory networks, and evolu Press, Inc., London, pp. 106-140; O'Donnell, A.G., Norris, J. tionary relationships to other microbes. A complete manifest R., Berkeley, R. C. W., Claus, D., Kanero, T., Logan, N.A., of proteins, both structural and catalytic, is encoded as a list of and Nozaki, R., 1980, Internat. J. Systematic Bacteriol. 30: features in the DNA molecule comprising the genome, as well 448-459). However, biochemical and phenotypic character as their likely cellular location. istics may be ambiguous among closely related species. Lapi 0007 Knowledge about the enormous range of microbial dus et al. (Lapidus, A., Galleron, N., Andersen, J. T., Jor capacities has broad and far-reaching implications for envi gensen, P. L. Ehrlich, S. D., and Sorokin, A., 2002, FEMS ronmental, energy, health, and industrial applications, such as Microbiol. Lett. 209: 23-30) recently constructed a physical cleanup of toxic-waste, production of novel therapeutic and map of the Bacillus licheniformis chromosome using a PCR preventive agents (drugs and vaccines), energy generation approach, and established a number of regions of co-linearity and development of renewable energy sources, production of where gene content and organization were ostensibly con chemical catalysts, reagents, and enzymes to improve effi served with the Bacillus subtilis chromosome. ciency of industrial processes, management of environmental 0011. It would be advantageous to the art to have available carbon, nitrogen and nutrient cycling, detection of disease the complete primary structure of the chromosomal DNA causing organisms and monitoring of the safety of food and molecule of the Bacillus licheniformis type strain ATCC water Supplies, use of genetically altered bacteria as living 14580. With the complete chromosome data in hand, it should sensors (biosensors) to detect harmful chemicals in soil, air, be possible to do comparative genomics and proteomics stud US 2011/0086407 A1 Apr. 14, 2011 ies that can lead to improved industrial strains as well as to a without an inducing Substrate, and labeled second nucleic better understanding of genome evolution among closely acid probes, isolated from the microbial strain cultured on related bacilli in the subtilis-licheniformis group. medium with the inducing Substrate, to an array of Bacillus 0012. It is an object of the present invention to provide an licheniformis genes selected from the group consisting of isolated polynucleotide with the sequence of the complete nucleotides SEQID NOS: 2-4198, complementary strands of chromosome of Bacillus licheniformis. SEQID NOS: 2-4198, or fragments of SEQID NOS: 2-4198, under conditions where the labeled nucleic acid probes SUMMARY OF THE INVENTION hybridize to complementary
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    INVASIVE PLANKTON Implications of and for ballast water management Dissertation Zur Erlangung der Würde des Doktors der Naturwissenschaften des Fachbereichs Biologie, der Fakultät für Mathematik, Informatik und Naturwissenschaften, der Universität Hamburg vorgelegt von Viola Liebich aus Berlin Hamburg, Dezember 2012 Title: INVASIVE PLANKTON - implications of and for ballast water management Contents: Chapter 1. Introduction: Invasive species and ballast water 4 Chapter 2. Understanding (marine) invasions through the application of a comprehensive stage-transition framework – review of invasion theory and terminology 15 Chapter 3. Re-growth of potential invasive phytoplankton following UV-based ballast water treatment 32 Chapter 4. Incubation experiments after ballast water treatment: focus on the forgotten fraction of organisms smaller than 10 µm 47 Chapter 5. Overall Discussion, Conclusion and Perspective 66 List of publications 78 Acknowledgement 79 Summary in English 80 Summary in German 82 Declaration on oath 85 Annex: Stehouwer PP, Liebich V, Peperzak L (2012) Flow cytometry, microscopy, and DNA analysis as complementary phytoplankton screening methods in ballast water treatment studies. Journal of Applied Phycology 86 2 Chapter 1. Introduction: Invasive species and ballast water Invasive species Invasive species are considered one of the biggest threats to our world’s oceans (in addition to marine pollution and overexploitation). The man-aided introduction of non- native organisms via a vector (transport element) into new areas and their successful establishment as invasive species pose risks to native biodiversity and habitats (Zaiko et al. 2011). Commonly recognized is the phenomenon of competitive exclusion of native populations by invasive species (Huxel 1999), with increasing probability due to climate change (Philippart et al.
  • Diphthamide Biosynthesis: Characterization and Mechanistic Studies of an Unconventional Radical Sam Enzyme Phdph2

    Diphthamide Biosynthesis: Characterization and Mechanistic Studies of an Unconventional Radical Sam Enzyme Phdph2

    DIPHTHAMIDE BIOSYNTHESIS: CHARACTERIZATION AND MECHANISTIC STUDIES OF AN UNCONVENTIONAL RADICAL SAM ENZYME PHDPH2 A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Xuling Zhu January 2011 © 2011 Xuling Zhu DIPHTHAMIDE BIOSYNTHESIS: CHARACTERIZATION AND MECHANISTIC STUDIES OF AN UNCONVENTIONAL RADICAL SAM ENZYME PHDPH2 Xuling Zhu, Ph. D. Cornell University 2011 Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). The proposed biosynthesis of diphthamide involves three steps. The first step is the formation of a C-C bond between the histidine residue and the 3-amino-3- carboxylpropyl group of S-adenosylmethionine (SAM), which is catalyzed by four enzymes Dph1-Dph4 in eukaryotic or only one enzyme Dph2 in archaea; the second step is the trimethylation of the amino group by Dph5; and the last step is an ATP depended amidation of the carboxyl group by an unknown enzyme. We have recently found that in an archaeal species Pyrococcus horikoshii (P. horikoshii), the first step uses an S-adenosyl-L-methionine (SAM)-dependent [4Fe– 4S] enzyme, PhDph2, to catalyze the formation of a C–C bond. Crystal structure shows that PhDph2 is a homodimer and each monomer contains three conserved cysteine residues that can bind a [4Fe–4S] cluster. In the reduced state, the [4Fe–4S] cluster can provide one electron to reductively cleave the bound SAM molecule. However, different from classical radical SAM enzymes, biochemical evidence suggests that a 3-amino-3-carboxypropyl radical is generated in PhDph2.