Complex regulation of urease formation from the two promoters of the ure operon of Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae can use urea as the sole source of nitrogen, thanks to a urease encoded by the ureDABCEFG operon. Expression of this operon is independent of urea and is regulated by the supply of nitrogen in the growth medium. When cells were growth rate limited for nitrogen, the specific activity of urease was about 70 times higher than that in cells grown under conditions of excess nitrogen. Much of this nitrogen regulation of urease formation depended on the nitrogen regulatory system acting through the nitrogen assimilation control protein, NAC. In a strain deleted for the nac gene, nitrogen limitation resulted in only a 7-fold increase in the specific activity of urease, in contrast to the 70-fold increase seen in that of the wild type. The ure operon was transcribed from two promoters. The proximal promoter (P1) had an absolute requirement for NAC; little or no transcription was seen in the absence of NAC. The distal promoter (P2) was independent of NAC, but its activity increased about threefold when the growth rate of the cells was limited by the nitrogen source. Transcriptional regulation of P1 and P2 accounted for most of the changes in urease activity seen under various nitrogen conditions. However, when transcription of ureDABCEFG was less than 20% of its maximum, the amount of active urease formed per transcript of ure decreased almost linearly with decreasing transcription. This may reflect a defect in the assembly of active urease and accounted for as much as a threefold activity difference under the conditions tested here. Thus, the ure operon was transcribed from a NAC-independent promoter (P2) and the most strongly NAC-dependent promoter known (P1). Most of the regulation of urease formation was transcriptional, but when ure transcription was low, assembly of active urease also was defective.

FIG. 1.

Sequence of the ureD promoter region. Bent arrowheads indicate the start of transcription from the two promoters (P2 and P1, respectively) in the region. Underlined regions indicate the presumed −35 and −10 regions corresponding to P2. The boxed nucleotides indicate the NAC binding site. The double-underlined nucleotides are the beginning of the ureD coding region. The vertical bars with numbers indicate the endpoints of the deletions and fusions used. Primer 200 and Primer 240 indicate the positions of the 5′ end of the 29-mer and 20-mer primers, respectively, used in primer extension experiments.

FIG. 2.

The ureD-lacZ transcriptional fusions used. Solid lines indicate DNA sequences present, and the numbers on the lines indicate the last nucleotide remaining, using the numbering of Fig. 1. P1 and P2 indicate the approximate positions of the two promoters, and SD indicates the approximate position of the Shine-Dalgarno sequence in front of the ureD coding sequence. Full refers to the 262-bp fragment illustrated in Fig. 1. The negative numbers indicate constructs that contain K. pneumoniae DNA upstream of the sequence shown in Fig. 1. lacZ indicates the promoterless lacZ sequence in plasmid pRS415 (19).

FIG. 3.

Primer extension of mRNA from the ureD promoter region. Primers extending back to bp 200 and 240 were used to determine the start of transcription. The same primers were used to generate a DNA sequencing ladder. Arrows indicate the start of transcription, and the DNA sequence (complementary to the sequencing ladder but matching the sequence shown in Fig. 1) is indicated. GATC indicates the DNA sequencing ladder. RT indicates the experimental lane with reverse transcriptase present during the reaction. Primer indicates a control reaction with no reverse transcriptase. Note that Primer 200 gave an artifactual signal, somewhat shorter than the actual runoff transcript generated by reverse transcriptase. (A) Primer extension of mRNA isolated from strain DH5α carrying a plasmid with the complete ureDp region (designated Full in Fig. 2) and a plasmid expressing K. pneumoniae nac constitutively. (B) Primer extension of mRNA from DH5α carrying only a plasmid with ureDp₂ (designated P2 in Fig. 2).

FIG. 4.

Amount of urease activity formed per transcript as a function of transcription amount. Three strains differing only in their nac allele were used, KC2653 (Nac⁺), KC6122 (Nac⁻), and KC6146 (Nac^const) (which was grown both with and without IPTG present), and each carried a wild-type ure operon and a ure-lacZ fusion (designated Full in Fig. 2) inserted at the rbs locus. The strains were grown in glucose minimal medium with five different nitrogen sources, resulting in a variety of levels of ure transcription. The specific activity of the urease formed by the cells was determined by direct assay of urease, and the amount of transcription from the ure promoter was determined indirectly by measuring β-galactosidase from the ure-lac fusion. The ratio of urease activity to β-galactosidase activity (urease activity per transcript) was plotted against β-galactosidase activity (amount of transcription). The raw data for the 20 different conditions are given in Table S1 in the supplemental material. All data were the result of three or more assays of independent cultures with standard errors of the means of less than 10% in all cases.