An analysis of “revertants” of a deletion mutant in the C gene of the l-arabinose gene complex in Escherichia coli B/r: Isolation of initiator constitutive mutants (Ic)

Englesberg, Ellis; Sheppard, David E.; Squires, Catherine L.; Meronk, Frank

doi:10.1016/0022-2836(69)90268-x

Cited by 74 publications

(64 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mutants have been isolated that express the arabinose operon in the absence of araC protein (24,25 (26,27 a requirement for the complete set of proteins necessitates that the binding of the proteins to DNA be cooperative. Such a regulatory mechanism is versatile and easily accommodates situations in which large numbers of inducers must all be present in order for a gene to be induced.…”

Section: Resultsmentioning

confidence: 99%

The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.

Ogden¹,

Haggerty²,

Stoner³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

168

123

View full text Add to dashboard Cite

The locations of DNA binding by the proteins involved with positive and negative regulation of transcription initiation of the L-arabinose operon in Escherichia coli have been determined by the DNase I protection method. Two cyclic AMP receptor protein sites were found, at positions -78 to -107 and -121 to -146, an araCprotein-arabinose binding site was found at position -40 to -78, and an araC protein-fucose binding site was found at position -106 to -144. These locations, combined with in vivo data on induction of the two divergently oriented arabinose promoters, suggest the following regulatory mechanism: induction of the araBAD operon occurs when cyclic AMP receptor protein, araC protein, and RNA polymerase are all present and able to bind to DNA. Negative regulation is accomplished by the repressing form of araC protein binding to a site in the regulatory region such that it simultaneously blocks access of cyclic AMP receptor protein to two sites on the DNA, one site of which serves each of the two promoters. Thus, from a single operator site, the negative regulator represses the two outwardly oriented ara promoters. This regulatory mechanism explains the known positive and negative regulatory properties of the ara promoters.Studies on the L-arabinose operon of Escherichia coli have established the following important facts on regulation of the divergently oriented ara promoters PC and PBAD (see Fig. 1).The activity of both promoters is stimulated by the cyclic AMP (cAMP) receptor protein (CRP) in the presence of cyclic AMP (1-4). The promoter PBAD is positively regulated by araC protein in the presence of arabinose-i.e., the protein is required for activity of the promoter (1, 2, 5). Under noninducing conditions, the araC protein instead acts negatively to repress both the PC and PBAD promoters (1-3, 6). At least part of the DNA site necessary for repression of PBAD lies upstream from all of the sites necessary for induction of PBAD, as shown by the existence of deletions entering the ara regulatory region from the Pc side that abolish repression of PBAD without affecting induction of PBAD (6, 7).The requisite components are now available for in vitro studies of the mechanism of regulation of the ara operon. The regulatory region DNA has been isolated, and its nucleotide sequence has been determined (8,9) and found to contain elements similar to the RNA polymerase-binding sites seen in other E. coli promoters at about 10 and 35 bases before the start sites of transcription (8). The sequence also contains several stretches that resemble the CRP-binding site in the gal operon (10). Also available are the proteins involved in the regulation of the ara operon: araC protein (11), CRP (12), and RNA polymerase (13).In the work reported here, we have utilized the DNA se- MATERIALS AND METHODS DNA fragments for sequence determination and protection were obtained from plasmid pMB9ara440 (8) by EcoRI endonuclease digestion and polyethylene glycol precipitation (16). CRP was purified as described (12), and ara...

show abstract

Section: Resultsmentioning

confidence: 99%

The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.

Ogden¹,

Haggerty²,

Stoner³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

168

123

View full text Add to dashboard Cite

show abstract

“…The revertants contained the original deletion plus a second mutation mapping to the promoter region. They have a cis-dominant constitutive phenotype characteristic of such transcriptional mutants (12). By analogy, constitutive CsgA-dependent gene expression might bypass the csgA mutation.…”

Section: Discussionmentioning

confidence: 99%

Developmental bypass suppression of Myxococcus xanthus csgA mutations

Rhie

Shimkets

1989

J Bacteriol

View full text Add to dashboard Cite

The csgA mutations of Myxococcus xanthus (formerly known as spoC) inhibit sporulation as well as rippling, which involves ridges of cells moving in waves. Sporulating revertants of CsgA cells were isolated by direct selection, since spores are much more resistant to heat and ultrasonic treatment than are vegetative cells. The revertants fell into seven groups on the basis of phenotype and the chromosomal location of the suppressor alleles. Group 1 contained one allele that was a back mutation of the original csgA mutation. Group 2 contained two linked alleles that were unlinked to the csgA locus and restored fruiting-body formation, sporulation, and rippling. Group 3 revertants regained the ability to sporulate in fruiting bodies but not the ability to ripple. Revertants in groups 4 to 7 were able to sporulate but unable to form fruiting bodies or ripples. The suppressors were all found to be bypass suppressors even though they were not selected as such in most cases. The csgA mutation prevented expression of several developmentally regulated promoters, each fused to a lacZ reporter gene and assayed by 3-galactosidase production. In four of five suppressor groups (groups 4 to 7), expression of each of these csgA-dependent fusions was restored, which suggests that bypass suppression restores developmental gene expression near the point at which expression is disrupted in CsgA mutants. Bypass suppression did not restore production of C factor, and morphological manifestations of development such as rippling and fruiting-body formation were usually abnormal. One interpretation of these results is that C factor has multiple functions and few suppressors can compensate for all of them.

show abstract

“…Plasmid pAH5 contains part of araA, all of araB, the araI(Con) allele, and the (araC0)766 deletion. The araI(Con) mutation allows a low level of expression of the araBAD promoter in the absence of AraC activation (7,9). An Aratransformant was grown in TYE-tetracycline broth for 24 h and plated on MAT medium to obtain isolated colonies.…”

Section: Methodsmentioning

confidence: 99%

Novel activation of araC expression and a DNA site required for araC autoregulation in Escherichia coli B/r

Cass

Wilcox

1988

J Bacteriol

View full text Add to dashboard Cite

Mutations in the araC gene have been isolated which alter both the activator and autoregulatory functions of AraC protein (L. G. Cass and G. Wilcox, J. Bacteriol. 166:892-900, 1986). In this study, the effect of each araC mutation on autoregulation was characterized in vivo and in vitro in the presence of L-arabinose. The effect of L-arabinose in some of these araC mutants revealed a novel activation of araC expression which was not observed in the araC+ cell. Experiments were therefore focused on understanding the mechanism of this novel activation. We describe a systematic analysis of the effect of mutations within the known regulatory binding sites for araBAD and araC transcription on araC expression. Our results suggest that the novel activation of araC expression requires the AraC activator-binding site, aral, and the cyclic AMP receptor protein-cyclic AMP complex-binding site. We also found that in the absence of L-arabinosej the aral site was required for maximal autoregulation by the wild-type AraC protein.The AraC protein functions as both a positive and negative regulator of gene expression in Escherichia coli (8,17). Transcription of the araBAD operon is repressed by AraC protein in the absence of L-arabinose and is activated by AraC protein in the presence of L-arabinose (8, 17). In the presence or absence of L-arabinose, the araC gene is autogenously regulated (2,12,19). Maximal expression of both the araBAD operon and the araC gene requires the presence of the cyclic AMP (cAMP) receptor protein-cAMP complex (CRP-cAMP) (8, 17).The araBAD operon and araC gene promoters are separated by 147 base pairs (bp) (33) and are transcribed in opposite directions (34). The DNA sequence of this ara regulatory region is known (10, 30). Genetic and biochemical analyses have defined the binding sites for the regulatory proteins AraC and CRP and for RNA polymerase (6, 19, 28) (Fig. 1). Based on these studies, models for the regulation of the araCBAD gene complex have been proposed, aspects of which are summarized as follows. (Fig. 1).The Escherichia coli araC gene encodes a 292-amino-acid polypeptide, as predicted by DNA sequence analysis (23). Mutations in the araC gene have been isolated which affect both the activator and autoregulatory functions of AraC * Corresponding author. t Present address: Department of Biological Sciences, University of California, Santa Barbara, CA 93106. protein (5). The effect of each araC mutation on autoregulation was characterized in vivo and in vitro in the absence of L-arabinose. Previous studies have shown that in the wildtype cell, induction of araC expression by L-arabinose does not occur (2). In the present study, we have reexamined the effect of these araC mutations on autoregulation in the presence of L-arabinose. The effect of L-arabinose in some of these araC mutants revealed a novel activation of araC expression which is not observed in the araC+ cell. Therefore, experiments were focused on understanding the mechanism of this novel activation. We describe a systematic...

show abstract

An analysis of “revertants” of a deletion mutant in the C gene of the l-arabinose gene complex in Escherichia coli B/r: Isolation of initiator constitutive mutants (Ic)

Cited by 74 publications

References 11 publications

The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.

The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.

Developmental bypass suppression of Myxococcus xanthus csgA mutations

Novel activation of araC expression and a DNA site required for araC autoregulation in Escherichia coli B/r

Contact Info

Product

Resources

About