Expression of the L-arabinose regulon in Escherichia coli B/r requires, among other things, cyclic adenosine-3',5'-monophosphate (cAMP) and the cAMP receptor protein (CRP). Mutants deficient in adenyl cyclase (cya-), the enzyme which synthesizes cAMP, or CRP (crp-) are unable to utilize a variety of carbohydrates, including L-arabinose. Ara+ revertants of a cya-crp-strain were isolated on 0.2% minimal L-arabinose plates, conditions which require the entire ara regulon to be activated in the absence of cAMP and CRP. Evidence from genetic and physiological studies is consistent with placing these mutations in the araC regulatory gene. Deletion mapping with one mutant localized the site within either araO or araC, and complementation tests indicated the mutants acted trans to confer the ability to utilize L-arabinose in a cyacrp-genetic background. Since genetic analysis supports the conclusion that the mutant sites are in the araC regulatory gene, the mutants were designated araCi, indicating a mutation in the regulatory gene affecting the cAMP-CRP requirement. Physiological analysis of one mutant, araC'1, illustrates the transacting nature of the mutation. In a cya-crpgenetic background, araC1l promoted synthesis of both isomerase, a product of the araBAD operon, and permease, a product of the araE operon. Isomerase and permease levels in araCi1 cya+ crp+ were hyperinducible, and the sensitivity of each to cAMP was altered. Two models are presented that show the possible mutational lesion in the araCi strains.
Five of the genes required for phosphorylative catabolism of glucose in Pseudomonas aeruginosa were ordered on two different chromosomal fragments. Analysis of a previously isolated 6.0-kb EcoRI fragment containing three structural genes showed that the genes were present on a 4.6-kb fragment in the order glucose-binding protein (gltB)-glucokinase (glk)-6-phosphogluconate dehydratase (edd). Two genes, glucose-6-phosphate dehydrogenase (zwf) and 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), shown by transductional analysis to be linked to gltB and edd, were cloned on a separate 11-kb BamHI chromosomal DNA fragment and then subcloned and ordered on a 7-kb fragment. The 6.0-kb EcoRI fragment had been shown to complement a regulatory mutation, hexR, which caused noninducibility of four glucose catabolic enzymes. In this study, hexR was mapped coincident with edd. A second regulatory function, hexC, was cloned within a 0.6-kb fragment contiguous to the edd gene but containing none of the structural genes. The phenotypic effect of the hexC locus, when present on a multicopy plasmid, was elevated expression of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase activities in the absence of inducer.Glucose catabolism in Pseudomonas aeruginosa proceeds by either an oxidative or a phosphorylative pathway ( Fig. 1; reviewed in reference 24). In the direct phosphorylative pathway, glucose is transported into the cell by a periplasmic glucose-binding protein (GLTB)-dependent active transport system. Intracellular glucose is phosphorylated by glucokinase (GLK) and converted to 6-phosphogluconate (6PG) by glucose-6-phosphate dehydrogenase (ZWF). The 6PG from this pathway and from the oxidative pathway ( Fig. 1) is further metabolized to glyceraldehyde-3-phosphate and pyruvate by the Entner-Doudoroff enzymes 6PG dehydratase (EDD) and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EDA). GLK, ZWF, EDD, and EDA are strictly co-inducible, and 6PG is thought to serve as the physiological inducer (2,8,19). The glucose transport functions are separately regulated (20).All of the genes known to be required for direct phosphorylative catabolism of glucose to pyruvate and glyceraldehyde-3-phosphate are clustered in the 39-min region of the Pseudomonas aeruginosa chromosome (7,8,36 MATERIALS AND METHODS Bacterial strains and plasmids. All bacterial strains used in this study were derived from prototrophic P. aeruginosa PAO (16) and have been described previously. The edd lesions in strains PFB57 (edd-8 hexR1) and PFB2 (edd4 hexR2) have been described previously (2,8). Both of these mutant strains also were noninducible for the other glucose catabolic enzymes ZWF, GLK, and EDA (2, 8). We have designated these regulatory mutations hexRi and hexR2.Other strains used in this study were PFB9 (edd-1) and PFB52 (edd-2) (2), PFB362 (gltBi) (7), PRP444 (glk-1) (8), PFB98 (zwf-1) (36), PFB103 (zwf-2) (37), and PAO1838 (eda-9001 met-9020) (29).Plasmids in this study w...
Raising the concentration of phenylalanine and other amino acids in MEM leads to the inhibition of growth and in some cases to death of A9. Balb 3T3 , SV40 Balb 3T3 (SVT2), CHO, and WI38. All cells tested exhibited some similar senstivities to certain of the amino acids. but there were some unique differences. Phenylalanine-resistant mutants (Pher) of A9 were isolated that had modified phenylalanine-transport properties. These mutants can be isolated by a single-step selection procedure. A Lineweaver-Burk plot of initial rates of phenylalanine uptake by A9 and mutants showed a biphasic curve suggesting two transport systems. The Pher mutants had altered properties of both systems. It is suggested that the selection of clones resistant to high concentration of several of the natural amino acid may be used as a general method for the isolation of mutants affecting the various amino acid transport systems in mammalian cells.
A series of deletions beginning in the leu operon and continuing into the araC gene and also into the ara controlling site region were analyzed in reciprocal merodiploids, e.g., F' A2Cc67/B24delta719, F' B24delta719/A2Cc67, for their effects on catabolite deactivation (CD). The results of these experiments are consistent with placing the catabolite gene activator-cyclic AMP sensitive site in the controlling site region between araB and araO. With a deletion mutant, delta1109, that places araBAD under leu control when transcription begins at leuP, the araBAD operon is immune to CD even though araCGA, araP and araI are intact and functional. To focus attention on the fine structure and related functions of this region we propose that the three proteins that function therein have separate sites of action: araI (initiator-site for activator), araP (promoter-site for RNA polymerase) and ara(CGA) (catabolite gene activator-site for CGA-cAMP). None of the eighteen initiator constitutive mutants (Ic) tested have any significant effect on catabolite derepression or on the maximal level of expression of the operon supporting the view that the araI site may be distinct from araP and ARA(CGA). A series of constitutive mutants in the araC gene (Cc) also have no pronounced effect on catabolite deactivation.
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