In the """"""""baby machine"""""""" culture system, cells are immobilized on a surface such that newborn cells are shed continuously from the culture. When an immobilized cell divides, one of the new progeny cells remains attached and the other is released. This culture system has been used to decipher many of the cell cycle/DNA replication-segregation/cell division properties of E. coli B/r. Its usefulness has been limited, however, by the number of newborn cells released and the inability to use the many E. coli K12 derivatives. Recognizing that both problems required solutions to make real headway in solving regulatory aspects of the cell cycle, the baby machine has been modified to function with most microorganism, and the cell yield has been increased 5 to 20-fold. Accordingly, a variety of K12 derivatives will be used to determine molecular mechanisms involved in the control of chromosome replication/segregation and cell division. Among the projects to be undertaken are: 1) Continued modifications of the baby machine, primarily to develop covalent attachment of the cells, in order to develop its full potential for cell cycle/cell aging studies with a variety of species; 2) Analyze cell cycle parameters of K12 derivatives and identify the interactions among the components of the circuit controlling the timing of initiation of chromosome replication; 3) Test theories on control of the timing and toporegulation of cell division with K12 derivatives; 4) Determine the replication patterns of low copy F and PI plasmids in the cell. cycle; 5) Identify the determinants of nonrandom chromosome segregation; 6) Examine OriC-envelope interactions in relation to initiation of replication and segregation; 7) Determine the involvement of superhelicity in minichromosome stability and partitioning; 8) Assay cell cycle-specific transcription of genes. involved In initiation of chromosome replication; 9) Identify the cell cycle-dependent appearance of modulators of chromosome replication in an in vitro minichromosome replication system. The ability to perform physiologically sound cell cycle studies using strain K12 with improved yields should help resolve many unanswered questions on the control and coordination between chromosome replication/segregation and cell division in E. coli.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Florida Institute of Technology
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Bogan, J A; Grimwade, J E; Thornton, M et al. (2001) P1 and NR1 plasmid replication during the cell cycle of Escherichia coli. Plasmid 45:200-8
Gomez-Eichelmann, M C; Helmstetter, C E (1999) Transcription level of operon ftsYEX and activity of promoter P1 of rpoH during the cell cycle in Escherichia coli. J Basic Microbiol 39:237-42
Bogan, J A; Helmstetter, C E (1997) DNA sequestration and transcription in the oriC region of Escherichia coli. Mol Microbiol 26:889-96
Helmstetter, C E; Thornton, M; Zhou, P et al. (1997) Replication and segregation of a miniF plasmid during the division cycle of Escherichia coli. J Bacteriol 179:1393-9
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Helmstetter, C E (1997) Gravity and the orientation of cell division. Proc Natl Acad Sci U S A 94:10195-8
Bogan, J A; Helmstetter, C E (1996) mioC transcription, initiation of replication, and the eclipse in Escherichia coli. J Bacteriol 178:3201-6
Cassler, M R; Grimwade, J E; Leonard, A C (1995) Cell cycle-specific changes in nucleoprotein complexes at a chromosomal replication origin. EMBO J 14:5833-41
Zhou, P; Helmstetter, C E (1994) Relationship between ftsZ gene expression and chromosome replication in Escherichia coli. J Bacteriol 176:6100-6
Theisen, P W; Grimwade, J E; Leonard, A C et al. (1993) Correlation of gene transcription with the time of initiation of chromosome replication in Escherichia coli. Mol Microbiol 10:575-84

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