Defined mutations of acrA or acrB (formerly acrE) genes increased the susceptibility of Escherichia coli to a range of small inhibitor molecules. Deletion of acrAB increased susceptibility to cephalothin and cephaloridine, but the permeability of these beta-lactams across the outer membrane was not increased. This finding is inconsistent with the earlier hypothesis that acrAB mutations increase drug susceptibility by increasing the permeability of the outer membrane, and supports our model that acrAB codes for a multi-drug efflux pump. The natural environment of an enteric bacterium such as E. coli is enriched in bile salts and fatty acids. An acrAB deletion mutant was found to be hypersusceptible to bile salts and to decanoate. In addition, acrAB expression was elevated by growth in 5 mM decanoate. These results suggest that one major physiological function of AcrAB is to protect E. coli against these and other hydrophobic inhibitors. Transcription of acrAB is increased by other stress conditions including 4% ethanol, 0.5 M NaCl, and stationary phase in Luria-Bertani medium. Finally, acrAB expression was shown to be increased in mar (multiple-antibiotic-resistant) mutants.

The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test (4,24

No abstract

The relative rotation between RNA polymerase and DNA during transcription elongation can lead to supercoiling of the DNA template. However, the variables that influence the efficiency of supercoiling by RNA polymerase in vivo are poorly understood, despite the importance of supercoiling for DNA metabolism. We describe a model system to measure the rate ofsupercoiling by transcription and to estimate the rates of topoisomerase turnover in Eseherichia coil. Transcription in a strain lacking topoisomerase I can lead to optimal supercoiling, wherein nearly one positive and one negative superturn are produced for each 10.4 base pairs transcribed. This rapid efficient supercoiling is observed during transcription of membrane-associated gene products, encoded by tet (the gene for tetracycline resistance) and phoA (the gene for E. coil alkaline phosphatase), when the genes are oppositely oriented.Replacement oftet by cat, the gene from Tn9 encoding resistance to chloramphenicol, whose gene product is soluble in the cytosol, reduces the efficiency of supercoiling by RNA polymerase. In a wild-type topoisomerase background, both gyrase and topoisomerase I are kinetically competent to relieve superturns produced by transcription. These results suggest that the level of DNA supercoiling in vivo is probably determined by topoisomerase activity, not by transcription.Transcription can lead to localized supercoiling of DNA because the topology of the elongation complex requires a relative rotation between RNA polymerase and DNA (1, 2). Experimental evidence for this model was originally based on two observations concerning the topology ofplasmid pBR322 in Escherichia coli: (i) inhibition ofDNA gyrase results in the formation of positively supercoiled plasmids (3); and (ii) transcription of tet in AtopA strains leads to the accumulation of a heterogeneous population of pBR322 topoisomers, some of which are hypernegatively supercoiled (4). These seminal observations led Liu and Wang (2) to propose that the required relative rotation of RNA polymerase about DNA (1) generates positive supercoils downstream from and negative supercoils upstream from the elongating polymerase. Many studies have verified the essential features of this model both in vitro and in bacteria and yeast (5-10). An important conclusion from these studies is that topoisomerases function as swivels to relieve torsional stress during transcription (11).Since transcription is a ubiquitous process in cells, a fundamental biological question is the extent to which transcription determines the level ofDNA supercoiling in vivo (5, 12, 13). The mechanistic factors which influence the efficiency and extent of localized supercoiling during transcription, however, are not well understood. First, the forces which may anchor RNA polymerase in vivo and thus necessitate the rotation of DNA through the transcription ternary complex have not been clearly identified. Frictional drag on polymerase with its nascent RNA and associated ribonucleoprotein complexes in the visc...

Isolated chloroplasts form labeled sugar phosphates in the light in the presence of C14O, and suitable cofactors (1,4, 5,15,30). They contain, therefore, all the enzymes needed for the uptake and reduction of CO., and the transformation of the resultant compounds (22). It is not known, however, whether the photosynthetic formation of sugars (or sugar phosphates) in the leaf cell is confined to the chloroplast or proceeds in chloroplast and cytoplasm alike. In the latter case a specific function of the chloroplast vould consist in the light dependent formation of ATP and TPNH3. Enzymes of photosynthetic dark reactions would then be distributed throughout the plasm. In the former case at least some of these enzymes could be expected to be located exclusively in the chloroplasts. The It was important to apply further purification steps in addition to the normal procedure to obtain pure preparations (21). The chloroplast isolation was performed mostly at 00 and only in few cases at room temperature. In the former case, very little chlorophyll was extracted (or destroyed) by the organic solvent (0-5 %). In the latter case, more chlorophyll was lost (up to 25 %). The results of chlorophyll determinations of the fractions were corrected for the loss of chlorophyll due to extraction as follows: The total chlorophyll content of all the fractions was compared with that of the starting miiaterial. From this the percent loss of chlorophyll due to extraction into petroleum ether/carbon tetrachloride and due to destruction could be calculated. Values were then corrected assuming proportionate losses in all fractions.Fractions Used for Enzyme Determinations. Enzymes were determined in the chloroplast fraction, in unfractionated tissue and in chloroplast-depleted tissue, which is the material remaining after the removal of the chloroplast fraction. Since yields of chloroplasts were in the order of 15 to 50 % in our isolation procedure, chloroplast-depleted tissue contained between 50