Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins

Jennings, Michael P.; Beacham, Ifor R.

doi:10.1128/jb.172.3.1491-1498.1990

Cited by 73 publications

(54 citation statements)

References 47 publications

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“…This effect was attributed to modification of a serine residue in position 120 of the recently corrected sequence [9]. However, the assignment was not unequivocal.…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

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A catalytic role for threonine‐12 of E. coli asparaginase II as established by site‐directed mutagenesis

et al. 1991

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A threonine-12 to alanine mutant of E. coli asparaginase II (EC 3.5.1.1) has less than 0.01% of the activity of wild-type enzyme. Both tertiary and quaternary structure of the enzyme are essentially unaffected by the mutation; thus the activity loss seems to be the result of a direct impairment of catalytic function. As aspartate is still bound by the mutant enzyme, Thr-12 appears not be involved in substrate binding.

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“…This effect was attributed to modification of a serine residue in position 120 of the recently corrected sequence [9]. However, the assignment was not unequivocal.…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

“…The first 16 residues were as expected [9], including a glycine residue in position 11 and an alanine in position 12.…”

Section: Wavelengthmentioning

confidence: 99%

A catalytic role for threonine‐12 of E. coli asparaginase II as established by site‐directed mutagenesis

et al. 1991

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“…6). Dual regulation by Fnr and CRP has been shown to exist in the ansB gene (16) (32). The CRP consensus (7) is shown, and bases that match are indicated by dots.…”

Section: Discussionmentioning

confidence: 99%

Anaerobic control of colicin E1 production

Eraso

Weinstock

1992

J Bacteriol

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Expression of the cea gene, which is carried by the ColEl plasmid and which encodes colicin El, was found to be greatly increased when the cells were grown anaerobically. By using cea-lacZ fusions to quantitate expression, aerobic levels were found to be only a few percent of the anaerobic levels. The anaerobic increase in expression was observed both in protein and in operon fusions, indicating that its regulation occurred at the level of transcription. It was also found to require a functionalfitr gene and to occur when the cea-lacZ fusion was present as a single copy in the bacterial chromosome instead of in the multicopy ColEl plasmid. Anaerobic expression was regulated by the SOS response and catabolite repression as is aerobic expression. The start site of the mRNA produced under anaerobic conditions was mapped by primer extension and found to be the same as the start for mRNA produced under aerobic conditions. These observations show that the cea gene is Regulation of the cea-kil operon is complex. Previous studies reported effects of the SOS system (9, 14), catabolite repression (10, 28, 41), the stringent response (23), and anaerobiosis (27,28). The repressor of the SOS response, LexA, is the repressor of the cea-kil operon (9, 11). When LexA is inactivated in the RecA-dependent response to DNA damage (22, 49), cea and kil are expressed. However, cea and kl expression shows a pronounced lag compared with expression of other SOS response genes (34, 56). Catabolite repression, mediated by binding of the cyclic Amp (CAMP) receptor protein (CRP)-cAMP complex upstream from the cea-kil promoter (32), stimulates transcription and affects the lag in induction (32,34). An effect of the stringent response has also been observed in vitro (23).Results of Nakazawa et al. (27,28) suggested that maximum production of colicin El occurred in minimal media when cells were grown anaerobically with glucose as a carbon source. We have reinvestigated this effect, using cea-lacZ protein and operon fusions to quantitate expression. Here we report that cea expression is much higher when cells are grown anaerobically than when they are grown aerobically. A 45-fold increase was observed in anaerobically growing cells. This stimulation of expression was due to transcriptional activation, since both protein and operon fusions showed the increase in expression. The anaerobic conditions started at the same site which had been previously reported for cea mRNA produced under aerobic conditions. MATERIALS AND METHODSMedia and chemicals. MCC medium was M63 salts supplemented with 0.4% glucose or glycerol, 0.4% vitamin-free Casamino Acids (Difco Laboratories, Detroit, Mich.), and 0.1 mg of thiamine per ml; 40 mM potassium fumarate was added as an electron acceptor for the anaerobic cultures. Other media were as described previously (42). Ampicillin and kanamycin were used at 150 and 25 ,ug/ml, respectively. 5-Bromo-4-chloro-3-indolyl-,-D-galactoside (X-Gal) was used to score the LacZ phenotype. Mitomycin C (MMC) was used at a final con...

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“…enterica analogue of the ansB gene of E. coli, which encodes a high-affinity L-asparaginase (Jennings & Beacham, 1990). An S. enterica cosmic library in the E. coli host DH1…”

Section: Cloning and Identification Of Anspmentioning

confidence: 99%

“…These systems have not been studied at the molecular level. In contrast, two enzymes for the subsequent degradation of L-asparagine have been described : a high-affinity, periplasmic L-asparaginase (asparaginase I1 ;Jennings & Beacham, 1990) and a low-affinity cytoplasmic Lasparaginase (asparaginase I; Jerlstrom et a/., 1889). The latter presumably functions to degrade L-asparagine when it has accumulated to an appropriate intracellular concentration.…”

Section: Introductionmentioning

confidence: 99%

Cloning and molecular analysis of the Salmonella enterica ansP gene, encoding an L-asparagine permease

Jennings

Anderson²,

Beacham³

1995

Microbiology

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A gene (ansP), which encodes an L-asparagine permease, has been isolated from a cosmid library of Salmonella enterica during screening for recombinant clones which encode L-asparaginase. Nucleotide sequence analysis reveals that the gene product is a polypeptide of 497 amino acid residues, containing 12 putative transmembrane segments. The calculated molecular mass is 54 kDa, although maxicell analysis by SDS-PAGE gave an apparent molecular mass of 37 kDa. Comparison of the deduced amino acid sequence with sequence databases showed significant homology with a family of basic and aromatic amino acid permeases. Strains containing the cloned ansP gene demonstrated a many-fold increase in L-asparagine uptake in comparison with control strains.

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Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins

Cited by 73 publications

References 47 publications

A catalytic role for threonine‐12 of E. coli asparaginase II as established by site‐directed mutagenesis

A catalytic role for threonine‐12 of E. coli asparaginase II as established by site‐directed mutagenesis

Anaerobic control of colicin E1 production

Cloning and molecular analysis of the Salmonella enterica ansP gene, encoding an L-asparagine permease

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