Toward functional genomics in bacteria: analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus.
As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes.
Rondon
M R
MR
Department of Plant Pathology, University of Wisconsin-Madison, 1630 Linden Drive, Russell Laboratories, Madison, WI 53706, USA.
Raffel
S J
SJ
Goodman
R M
RM
Handelsman
J
J
D001409:Bacillus cereus / Q000235:genetics*; D001431:Bacteriological Techniques; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics*; D004929:Esculin / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D005821:Genetic Techniques; D005822:Genetic Vectors; D006461:Hemolysis; D016296:Mutagenesis; D010957:Plasmids
D004269:DNA, Bacterial; D004929:Esculin