Myxococcus xanthus genome sequences and uses thereof

Details Myxococcus xanthus genome sequences and uses thereof Myxococcus xanthus genome sequences and uses thereof

2001-07-10

2004-12-21

Marschel, Ardin H. (Department: 1631)

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

Organic compounds

Carbohydrates or derivatives

C435S252300, C800S013000

Reexamination Certificate

active

06833447

ABSTRACT:

INCORPORATION OF SEQUENCE LISTING
Two copies of the sequence listing (Copy 1 and Copy 2) and a computer readable form of the sequence listing, all on CD-ROMs, each containing the file named Pa
—
00359.rpt which is 39,705,377 bytes (measured in MS-WINDOWS) and was created on Jun. 12, 2001 are herein incorporated by reference.
INCORPORATION OF TABLES 1, 2 AND 3
Two copies of Table 1 on CD-ROMs, each containing 998,830 bytes (measured in MS-WINDOWS) and all having the file name pa
—
00359.txt Table all created on Jun. 12, 2001, are herein incorporated by reference.
FIELD OF THE INVENTION
Included in the disclosure are nucleic acid molecules representing the, genome of the bacterium
Myxococcus xanthus
and, in particular, to nucleic acid molecules having nucleic acid sequences corresponding to DNA replication elements, genes, promoters, and other regulatory elements found in the
M. xanthus
genome. Also disclosed are homologous nucleic acid molecules, complementary nucleic acid molecules, polypeptides expressed by
M. xanthus
gene sequences, constructs comprising
M. xanthus
promoters, regulatory elements and/or genes, transformed cells and organisms comprising
M. xanihus
promoters, regulatory elements and/or genes, primers useful for replicating all or portions of
M. xanthus
genes or other
M. xanthus
nucleic acid molecules, computer readable media comprising sets of
M. xanthus
nucleic acid sequences, polypeptides and oligonucleotides, collections of
M. xanthus
nucleic acid molecules and methods of using such molecules and sequences including the use of collections of nucleic acid molecules in gene identification and gene expression analysis, development of a stoichiometric metabolic model, and preparation of constructs.
BACKGROUND OF THE INVENTION
Myxococcus xanthus
is a Gram-negative, rod-shaped bacterium with gliding motility that is classified within the delta subgroup of bacteria. It is a member of a group of microorganisms, commonly called myxobacteria, that generally survive by degrading organic material and other organisms in the soil. Of particular interest within the myxobacteria is the social behavior among cells. Myxobacteria form social interactions that facilitate feeding and, when nutrients become scarce, sporulation. They are the only bacteria that practice both types of social behavior, and the mechanism of communication among cells has been the subject of much research (see
Myxobacteria II.
1993. Martin Dworkin and Dale Kaiser (ed.), American Society for Microbiology, Washington, D.C.).
M. xanthus
has been particularly well studied, and is the member of the myxobacteria with the firmest genetic and physical map on which to build a genome project. An ordered YAC library and physical map of the
M. xanthus
genome have been constructed (He et al.,
Proc Natl Acad Sci USA.
91:9584-9587 (1994); Kuspa et al.,
Proc Natl Acad Sci U S
91:8917-8921 (1989)). The circular genome has been estimated to be around 9.5 Mbp (Shimkets, “The Myxobacterial Genome,” in
Myxobacteria II. American Society for Microbiology
, Dworkin and Kaiser (eds.), Washington, D.C., pp. 85-107 (1993)), which is quite large for a bacterial genome. It also has a very high G+C content (around 70%: Kaiser et al.,
Ann. Rev. Microbiol.
33:595-639 (1979)) which makes sequencing and assembly of the genome a significant technical challenge.
Ecology and Life Cycle of
M. xanthus
Myxobacteria are predatory organisms that can attack and degrade many other types of bacteria. Whole colonies of myxobacteria generally migrate together (swarm), and the combined production of extracellular enzymes allows more efficient solubilization of nutrients. Motility is accomplished by gliding, but the mechanism of gliding motility is not understood, either for myxobacteria or other types of gliding bacteria. The cells continue to feed communally until nutrients have been exhausted. Once nutrients become limiting, myxobacteria initiate a complex developmental process that leads to the production of fruiting bodies containing myxospores. Myxospores are resistant to heat, desiccation and other environmental insults, and serve as the resting phase for myxobacteria. The myxospores remain dormant until nutrients are again available, at which point they germinate to produce a new swarm of motile cells.
The sporulation process requires aggregation of many cells to an area where the fruiting body will eventually form. Both aggregation and fruiting body formation require a complex set of cell-to-cell communication networks, and a series of genetic switches within individual cells. The genetic cascade leads to differentiation of certain cells within the fruiting body, thereby producing myxospores.
The cells initially form a small, translucent mound. A portion of the cells within the mound begin to develop into myxospores, and the fruiting body eventually becomes about 0.1 mM high and dark as the thick spore walls are formed. The spores allow
M. xanthus
to survive harsh conditions for a long period of time, thus allowing the cells to be safely transported to a new location, perhaps by wind or within the gut of an animal.
Genetic analyses have identified a series of Myxococcus regulatory mutants that are defective in fruiting body formation. These mutants terminate at various points along the developmental pathway, and have defined four different chemical signaling factors, designated A, B, C, and D, that are required for normal sporulation (Kroos et al.,
Genes and Development
1:840-854 (1987); Losick et al.,
Scientific American.
276:68-73 (1997); Lee et al.,
J. Bacteriol.
178:977-984 (1996); Munoz et al.,
Microbiologia Madrid.
11:429-438 (1995); Kim,
Trends in Genetics.
7:361-365 (1991)). Factors A and C are the best studied. A-factor is required for aggregation of the cells. It is actually a combination of factors, including a heat stable component that appears to be a complex mixture of amino acids (Kuspa et al.,
J. Bacteriol.
174:3319-3326 (1992)) and a heat labile portion that includes a mixture of peptidases that presumably generate amino acids (Plamann et al.,
J. Bacteriol.
174:3311-3318 (1992)). A-factor is diffusible, and therefore does not require direct cell-to-cell contact for signal transmission. In contrast, C-Factor is normally found tightly associated with the cell surface of the signal producer, and transmission requires close contact between the signal producer and the recipient. Thus, C-signaling requires cellular motion and the close physical contact of the swarming cells in an aggregate. Both signal types provide the necessary format for the required message; A-factor to attract distant cells to a focus, and C-factor to maintain communication within the developing fruiting body. Each of the signals leads to a cascade of genetic switches that continues the cell differentiation process.
Many of the downstream regulatory and effecter genes have now been identified in
M. xanthus
using genetic and biochemical approaches, and it is the speed and efficiency with which bacteria allow analysis of the complex networks and metabolic pathways that provides a primary utility of the genome sequence.
The nucleic acid molecules and sequences disclosed herein represent a substantial portion of the
M. xanthus
genome. These molecules and sequences may be used to identify novel genes, for example genes involved in antibiotic production, and sequences in regulatory regions of the
Myxococcus
genes provided herein. The
M. xanthus
molecules and sequences also permit identification of genetic sequences from other organisms, including plants, mammals such as humans, bacteria, other filamentous fungi and non-filamentous fungi such as a yeast, e.g. by comparison of such sequences with
M. xanthus
sequences. The availability of a substantially complete set of genes or partial genes of the
M. xanthus
genome permits the definition of primers for fabricating representative nucleic acid molecules of the genome which can be used on microarrays to facilitate transcription profile studies. Such stu

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