The ArgP protein stimulates the Klebsiella pneumoniae gdhA promoter in a lysine-sensitive manner

Abstract

The lysine-sensitive factor that binds to the upstream region of the Klebsiella pneumoniae gdhA promoter and stimulates gdhA transcription during growth in minimal medium has been proposed to be the K. pneumoniae ArgP protein (M. R. Nandineni, R. S. Laishram, and J. Gowrishankar, J. Bacteriol. 186:6391-6399, 2004). A knockout mutation of the K. pneumoniae argP gene was generated and used to assess the roles of exogenous lysine and argP in the regulation of the gdhA promoter. Disruption of argP reduced the strength and the lysine-dependent regulation of the gdhA promoter. Electrophoretic mobility shift assays using crude extracts prepared from wild-type and argP-defective strains indicted the presence of an argP-dependent factor whose ability to bind the gdhA promoter was lysine sensitive. DNase I footprinting studies using purified K. pneumoniae ArgP protein indicated that ArgP bound the region that lies approximately 50 to 100 base pairs upstream of the gdhA transcription start site in a manner that was sensitive to the presence of lysine. Substitutions within the region bound by ArgP affected the binding of ArgP to the gdhA promoter region in vitro and the argP-dependent stimulation of the gdhA promoter in vivo. These observations suggest that elevated intracellular levels of lysine reduce the affinity of ArgP for its binding site at the gdhA promoter, preventing ArgP from binding to and stimulating transcription from the promoter in vivo.

FIG. 1.

Plasmids used for allelic replacement on the K. pneumoniae W70 chromosome. pKAS46-based plasmids pCB1578 (A) and pCB1590 (B) carry the pKAS46 backbone (gray arcs) and DNA fragments which were either amplified from the K. pneumoniae W70 chromosome (open arcs with arrowheads) or excised from plasmids (black arcs), where the arrowheads at the ends of arcs indicate the polarity of the open reading frames. The pKAS46 component of these plasmids carries the pir-dependent oriR6K replicon, npt, specifying Kan^r; bla, specifying Amp^r; and wild-type E. coli rpsL⁺, conferring Str^s. (A) The DNA fragments amplified from the K. pneumoniae chromosome carry the carboxyl terminally truncated rbsA open reading frame (ΔrbsA75) and the amino terminally truncated rbsK open reading frame (ΔrbsK3). The DNA fragments cloned from pRS415 and pBC KS(+) carry transcriptional terminators (T₁/T₂, large black arc) and the MCR (MCR) with the indicated restriction sites that are unique on pCB1578 (small black arc), respectively. (B) The DNA fragment amplified from the K. pneumoniae W70 chromosome carries the divergently transcribed rpiA (rpiA^Kpn) and argP (argP^Kpn) genes. The argP gene was disrupted by the insertion of a DNA fragment that carries cat, specifying Cam^r, from pBC KS(+) (black arc) into the argP open reading frame, generating the argP28Ωcat allele.

FIG. 2.

Electrophoretic mobility shift assay for gdhAp-binding activities. End-labeled DNA (0.1 fmol) from EcoRI-HindIII-digested pCB725 carrying gdhAp was incubated with sonication buffer (lane 1), extracts prepared in sonication buffer (lanes 2 to 8), storage buffer (lane 9), or purified ArgP-His in storage buffer (lanes 10 to 12). The extracts were prepared from cultures of either argP28 strain KC6540 (lane 2) or wild-type strain KC5998 (lanes 3 to 8), grown in minimal medium without (−) (lanes 2 and 5 to 8) or with (+) (lanes 3 and 4) a 550 μM l-lysine HCl supplement. Purified ArgP-His (1.8 pmol monomer/μl) was diluted 81-fold (lane 12) or 243-fold (lanes 10 and 11), and 1-μl portions were added to the DNA-binding solutions. For lanes 2 to 12, the DNA-binding solutions contained the indicated concentrations of l-lysine HCl. The samples were loaded onto a polyacrylamide gel, and the free and shifted DNA fragments were separated by electrophoresis. The positions of the end-labeled DNA were visualized by autoradiography of the dried gels. The labeled arrows at the left indicate the extent of migration of the free and bound DNA targets through the gel.

FIG. 3.

DNase I footprinting of ArgP bound to the gdhA promoter region. Portions (0.15 pmol) of end-labeled pCB725 DNA were used undigested (lane 1) or digested with DNase I in the absence (lane 2) or presence of either 0.75 pmol (lane 3), 1.5 pmol (lane 4), or 3.0 pmol (lanes 5 and 6) of purified ArgP-His monomer. In lane 6, the DNase I digestion solution contained 700 μM l-lysine HCl prior to the addition of ArgP-His. The same end-labeled DNA was subjected to partial chemical degradation, generating an A+G sequence ladder (lane A+G). The regions of ArgP-dependent DNase I protection and hypersensitivity are indicated by the boxes superimposed on lanes 3 to 5 and a horizontal arrow to the right of the A+G lane, respectively. The numbers at the right of the A+G lane correspond to nucleotides positions relative to the gdhA transcription start site, as indicated in Fig. 4.

FIG. 4.

DNA sequence homologies of ArgP-regulated promoters and summary of substitutions and footprinting data for K. pneumoniae gdhAp. The sequences of K. pneumoniae gdhAp (Kpn gdhAp) from −105 to −46, E. coli gdhAp (Eco gdhAp) from −108 to −49, and E. coli argOp (Eco argOp) from −79 to −21, relative to their respective transcription start sites, are aligned to show the regions of DNA sequence homology. The vertical lines between the sequences indicate nucleotides that are identical in all three sequences. The boxed T and A residues within the DNA sequences represent those of the LTTR consensus binding motif indicated below the sequences. For the K. pneumoniae gdhAp sequence, the approximate extents of the ArgP RBS and ABS (long horizontal brackets) and the ArgP-dependent regions of DNase I protection (solid horizontal bars) are indicated above the sequence. The vertical arrow indicates the approximate position of ArgP-induced DNase I hypersensitivity. The positions of the substitutions A(−96)T, T(−65)A, and GTC(−54 to −52)TAT are indicated above the sequence. For the E. coli argOp sequence, one gap was introduced to improve its homology with the gdhAp sequences.

FIG. 5.

Electrophoretic mobility shift assay for the influences of substitutions within the ArgP binding site on ArgP binding. End-labeled DNA from EcoRI-HindIII-digested pCB725 carrying the wild-type (WT) gdhA promoter (lanes 3 to 6 and 11 to 12) or comparable pRJ800 derivatives carrying modified gdhA promoters (lanes 1 to 2, 7 to 10, and 13 to 14) were incubated with sonication buffer (lanes 1, 3, 5, 7, and 9) or KC5998 extract (lanes 2, 4, 6, and 8 and 10 to 14) prepared as described in Fig. 2. For lanes 12 and 14, the DNA-binding solutions were supplemented with 55 μM l-lysine HCl. After the extract was added to the DNA-binding solution, the samples were subjected to electrophoresis as described in the legend to Fig. 2. The substitutions carried by the modified gdhA promoters are shown below the appropriate lanes at the bottom of the figure and above the K. pneumoniae gdhAp sequence in Fig. 4. The labeled arrows indicate the extents of migration of the free and bound target fragments through the gel.