Nucleic acids and proteins from Cenarchaeum symbiosum

Details Nucleic acids and proteins from Cenarchaeum symbiosum Nucleic acids and proteins from Cenarchaeum symbiosum

1999-09-29

2003-10-14

Marschel, Ardin H. (Department: 1631)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C530S350000

Reexamination Certificate

active

06632937

ABSTRACT:

BACKGROUND OF THE INVENTION
The identification and characterization of organisms which inhabit a diverse range of ecosystems leads to a greater understanding of the operation of such ecosystems. In addition, because the physiology of such organisms is adapted to function in the particular habitat which the organism inhabits, the enzymes which carry out the organism's physiological processes may possess characteristics which provide advantages when they are utilized in therapeutic procedures, industrial applications, or research applications. Furthermore, by determining the sequences of these organisms' genes, insight into their biochemical pathways and processes may be gained without the necessity of culturing the organisms in the laboratory, thereby enabling the physiological characterization of organisms which are recalcitrant to growth in the laboratory.
Molecular phylogenetic surveys have recently revealed an ecologically widespread Crenarchaeal group that inhabits cold and temperate terrestrial and marine environments. To date these organisms have resisted isolation in pure culture, so their phenotypic and genotypic characteristics remain largely unknown. In order to characterize the physiology of these archaea, to develop methodological approaches for characterizing uncultivated microorganisms and identifying their presence in a sample, and to identify enzymes produced by these archae which may be useful in therapeutic, industrial, or laboratory applications, genomic analyses of the non-thermophilic crenarchaeote
Cenarchaeum symbiosum
was undertaken.
Non-thermophilic Crenarchaeota are one of the more abundant, widespread and frequently recovered prokaryotic groups revealed by molecular phylogenetic approaches. These microorganisms were originally detected in high abundance in temperate ocean waters and polar seas. (DeLong, E. F. 1992. Archaea in coastal marine environments.
Proc. Natl. Acad. Sci.
89, 5685-5689; DeLong, E. F et al. 1994. High abundance of Archaea in Antarctic marine picoplankton.
Nature
371, 695-697; Fuhrman, J. A., et al. Davis. 1992. Novel major archaebacterial group from marine plankton.
Nature
356, 148-149; Massana, R., et al. 1997. Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel.
Appl. Env. Microb.
63, 50-56; McInerney, J. O. et al. 1995. Recovery and phylogenetic analysis of novel archaeal rRNA sequences from a deep-sea deposit feeder.
Appl. Env. Microb.
61, 1646-1648; Preston, C. M. et al. 1996. A psychrophilic crenarchaeon inhabits a marine sponge:
Cenarchaeum symbiosum
gen.
nov.,
sp.
nov. Proc. Natl. Acad. Sci.
USA 93, 6241-6246) Representatives have now been reported in terrestrial environments and freshwater lake sediments, indicating a widespread distribution. (Bintrim, S. B. et al. 1997. Molecular phylogeny of Archaea from soil.
Proc. Natl. Acad Sci.
USA 94, 277-282; Jurgens, G. et al. 1997. Novel group within the kingdom Crenarchaeota from boreal forest soil.
Appl. Env. Mircob.
63, 803-80515, Kudo, Y. et al. 1997. Peculiar archaea found in Japanese paddy soils.
Biosc. Biotech. Biochem.
61, 917-920; Ueda, et al. 1995. Molecular phylogenetic analysis of a soil microbial community.
Eur. J. Soil Sci.
46, 415-421; Hershberger, K. L. et al. 1996. Wide diversity of Crenarchaeota.
Nature
384, 420; MacGregor, B. J. 1997. Crenarchaeota in Lake Michigan sediment.
Appl. Env. Microb.
63, 1178-1181 et al.; Schleper, C.et al. 1997. Recovery of crenarchaeotal ribosomal DNA sequences from freshwater-lake sediments.
Appl. Env. Microb.
63, 321-323) The ecological distribution of these organisms was initially surprising, since their closest cultivated relatives are all thermophilic or hyperthermophilic. No representative of this new archaeal group has yet been obtained in pure culture, so the phenotypic and metabolic properties of these organisms, as well as their impact on the environment and global nutrient cycling, remain unknown. Since growth temperature and habitat characteristics vary so widely between non-thermophilic and the hyperthermophilic Creanarchaeota, these groups are likely to differ greatly with respect to their specific physiology and metabolism.
To gain a better perspective on the genetic and physiological characteristics of non-thermophilic crenarchaeotes, a genomic study of
Cenarchaeum symbiosum
was begun. This archaeon lives in specific association with the marine sponge
Axinella mexicana
off the coast of California, allowing access to relatively large amounts of biomass from this species. (Preston, C. M. et al. 1996. A psychrophilic crenarchaeon inhabits a marine sponge:
Cenarchaeum symbiosum
gen.
nov.,
sp.
nov. Proc. Natl. Acad. Sci.
USA 93, 6241-6246) The approach taken herein differs in several respects from now standard genomic characterization of cultivated organisms, and also from comparable studies of uncultivated obligate parasites or symbionts.
C. symbiosum
has not been completely physically separated from the tissues of its metazoan host. Therefore, its genetic material needs to be identified within the context of complex genomic libraries that contain significant amounts of eucaryotic DNA, as well as DNA derived from members of Bacteria.
Molecular phylogenetic surveys of mixed microbial populations have revealed the existence of many new lineages undetected by classical microbiological approaches. (DeLong, E. F. 1997. Marine microbial diversity: the tip of the iceberg.
Tibtech
15, 2-9.; Pace, N. R. 1997. A molecular view of microbial diversity and the biosphere.
Science
276, 734-740) Furthermore, quantitative rRNA hybridization experiments demonstrate that some of these novel prokaryotic groups represent major components of natural microbial communities. These molecular phylogenetic approaches have altered current views of microbial diversity and ecology, and have demonstrated that traditional cultivation techniques may recover only a small, skewed fraction of naturally occurring microbes. However, phylogenetic identification using single gene sequences provides a limited perspective on other biological properties, particularly for novel lineages only distantly related to cultivated and characterized organisms. Consequently, additional approaches are necessary to better characterize ecologically abundant and potentially biotechnologically useful microorganisms, many of which resist cultivation attempts.
SUMMARY OF THE INVENTION
One embodiment of the present invention is an isolated, purified, or enriched nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, the sequences complementary to SEQ ID NO: 1 and SEQ ID NO: 2, fragments comprising at least 10 consecutive nucleotides of SEQ ID NO: 1 and SEQ ID NO: 2, and fragments comprising at least 10 consecutive nucleotides of the sequences complementary to SEQ ID NO: 1 and SEQ ID NO: 2. One aspect of the present invention is an isolated, purified, or enriched nucleic acid capable of hybridizing to the nucleic acid of this embodiment under conditions of high stringency. Another aspect of the present invention is an isolated, purified, or enriched nucleic acid capable of hybridizing to the nucleic acid of this embodiment under conditions of moderate stringency. Another aspect of the present invention is an isolated, purified, or enriched nucleic acid capable of hybridizing to the nucleic acid of this embodiment under conditions of low stringency. Another aspect of the present invention is an isolated, purified, or enriched nucleic acid having at least 70% homology to the nucleic acid of this embodiment as determined by analysis with BLASTN version 2.0 with the default parameters. Another aspect of the present invention is an isolated, purified, or enriched nucleic acid having at least 99% homology to the nucleic acid of this embodiment as determined by analysis with BLASTN version 2.0 with the default parameters.
Another embodiment of the present invention is an isolated, purified, or enriched nucleic acid compris

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