Specific growth rate dependent gene expression changes of Escherichia coli K12 MG1655 were studied by microarray and real-time PCR analyses. The bacteria were cultivated on glucose limited minimal medium using the accelerostat method (A-stat) where starting from steady state conditions (chemostat culture) dilution rate is constantly increased. At specific growth rate (mu) 0.47h(-1), E. coli had focused its metabolism to glucose utilization by down-regulation of alternative substrate transporters expression compared to mu=0.3h(-1). It was found that acetic acid accumulation began at mu=0.34+/-0.01h(-1) and two acetate synthesis pathways - phosphotransacetylase-acetate kinase (pta-ackA) and pyruvate oxidase (poxB) - contributed to the synthesis at the beginning of overflow metabolism, i.e. onset of acetate excretion. On the other hand, poxB, pta and ackA expression patterns suggest that pyruvate oxidase may be the only enzyme synthesizing acetate at mu=0.47h(-1). Loss of glucose and acetate co-utilization represented by down-regulation of acs-yjcH-actP operon between specific growth rates 0.3-0.42h(-1) and acetic acid accumulation from mu=0.34+/-0.01h(-1) allows one to surmise that the acetate utilization operon expression might play an important role in overflow metabolism.