Molybdenum-sensitive transcriptional regulation of the chlD locus of Escherichia coli

Abstract

The chiD gene in Escherichja coli is required for the incorporation and utilization of molybdenum when the cells are grown with low concentrations of molybdate. We constructed chiD-lac operon fusions and measured expression of the fusion, Mo cofactor, and nitrate reductase activities under a variety of growth conditions. The chlD-lac fusion was highly expressed when cells were grown with less than 10 nm molybdate. Increasing concentrations of molybdate caused loss of activity, with less than 5% of the activity remaining at 500 nM molybdate; when tungstate replaced molybdate, it bad an identical affect on chiD expression. Expression of chiD-lac was increased in cells grown with nitrate. Strains with chiD-lac plus an additional mutation in a chl or nar gene were constructed to test whether the regulation of chiD-lac required the concerted action of gene products involved with Mo cofactor or nitrate reductase synthesis. Mutations in narL prevented the increase in activity in response' to nitrate; mutations in chM, narC, or narI resulted in partial constitutive expression of the chiD-lac fusion: the fusion was regulated by molybdate, but it no longer required the presence of nitrate for maximal activity. Mutations in chMA, chE, or chiG which affect Mo cofactor metabolism, did not affect the expression of chiD-lac.

Characterization of the ModE DNA‐binding sites in the control regions of modABCD and moaABCDE of Escherichia coli

“…We then compared expression from both plasmids when grown in a modC strain in the presence and absence of molybdate. The modC mutation abolishes the high-affinity molybdateuptake system resulting in derepression of modA-lacZ expression due to molybdate limitation (Miller et al, 1987;Rech et al, 1995). To minimize the titration effects resulting from multiple copies of the modABCD regulatory elements, we lowered plasmid copy number by introducing a pcnB allele into the test strain.…”

“…Sequence analysis demonstrated a high degree of homology in both the amino acid sequence of the gene products, and in the organization of these genes into discrete operons. Still more striking is the finding that in the bacteria examined to date, these mod operons appear to be regulated in a similar manner (Miller et al, 1987;Mouncey et al, 1995;Walkenhorst et al, 1995;Grunden et al, 1996;Kutsche et al, 1996).…”

Activation in vitro of respiratory nitrate reductase of Escherichia coli K12 grown in the presence of tungstate

“…The component required for the activation, other than protein FA, is absent in chlA and chlE strains which are deficient in cofactor content [15]. It is present in both bound and free forms as is the cofactor [S] and it is absent from cells grown with tungstate as is the molybdenum cofactor .8 cm) column.…”

“…This biochemical complementation requires the participation of inactive nitrate reductase as well as the active products of the chlA and chlB genes, all of which are present in the combined soluble fractions [16,18,191. Protein FA is the presumed product of the chlB locus [18] and it is thought to be involved in the modification of the molybdenum cofactor during molybdoenzyme biosynthesis [15]. Protein FA brings about the activation of nitrate reductase when it is incubated with the soluble fraction from chlB mutants [18].…”

“…Mutations in any one of these loci, with the exception of chZC (narC) which determines the structure of the enzyme [13], lead to the pleiotropic loss of all molybdoenzyme activity. This suggests that the products of these genes are required for the synthesis of the molybdenum cofactor [11,[13][14][15] which is the common component of molybdoenzymes. The chlA, chlB, chlE and chlG mutants possess inactive forms of nitrate reductase which are present in the soluble cell fraction [16].…”