Properties of HflX, an enigmatic protein from Escherichia coli

Abstract

The Escherichia coli gene hflX was first identified as part of the hflA operon, mutations in which led to an increased frequency of lysogenization upon infection of the bacterium by the temperate coliphage lambda. Independent mutational studies have also indicated that the HflX protein has a role in transposition. Based on the sequence of its gene, HflX is predicted to be a GTP-binding protein, very likely a GTPase. We report here purification and characterization of the HflX protein. We also specifically examined its suggested functional roles mentioned above. Our results show that HflX is a monomeric protein with a high (30% to 40%) content of helices. It exhibits GTPase as well as ATPase activities, but it has no role in lambda lysogeny or in transposition.

FIG. 1.

(A) Purification of His₆-HflX. The 12.5% SDS-PAGE gel contained uninduced (lane 1) and induced (lane 2) cells, the pellet (lane 3) and supernatant (lane 4) obtained after sonication and high-speed centrifugation, the pellet (lane 5) and supernatant (lane 6) obtained after ultracentrifugation, the flowthrough (lane 7) and wash (lane 8) fractions from the Ni²⁺ affinity column, and purified protein bands (lanes 9 and 10, indicated by arrowhead) obtained by elution using 500 mM imidazole, along with molecular weight markers (lane 11). The numbers on the right indicate molecular masses (in kDa). (B) Recombinant proteins after removal of the tag by thrombin cleavage. The 12.5% SDS-PAGE gel contained His₆-HflX (lane 1) and GST-HflX (lane 3) and their thrombin cleavage products (lanes 2 and 4, respectively). Lane 5 contained molecular mass markers. The numbers on the right indicate molecular masses (in kDa).

FIG. 2.

(A) Gel filtration analysis of His₆-HflX: plot of R_f ([V_e − V₀]/[V_t − V₀], where V_e, V₀, and V_t are the elution volume, void volume, and total column volume respectively)] versus the logarithm of molecular weight (MW) for the four standard proteins BSA, E. coli cyclic AMP receptor protein, soybean trypsin inhibitor, and lysozyme. The position of His₆-HflX (□), which eluted as a 54-kDa protein, is indicated. (B) Size distribution of His₆-HflX as obtained from dynamic light scattering. The average diameters obtained for His₆-HflX (solid line) and BSA (dashed line) by an analysis of scattering data at various concentrations (5 μM to 25 μM) are shown.

FIG. 3.

Far-UV CD spectrum of HflX. The spectrum was recorded at 25°C with 4 μM of protein.

FIG. 4.

GTP and ATP binding assays. Lane 1, molecular weight markers; lane 2, His₆-HflX; lanes 3 to 6, His₆-HflX incubated with [α-³²P]ATP (lanes 3 and 4) or [α-³²P]GTP (lanes 5 and 6). Samples in lanes 4 and 6 were subjected to UV cross-linking. Lanes 1 to 2 were stained with Coomassie blue, and lanes 3 to 6 were autoradiographed.

FIG. 5.

GTPase and ATPase activities of HflX. The time kinetics of liberation of phosphate from [γ-³²P]ATP (○) and from [γ-³²P]GTP (•) by HflX are shown.

FIG. 6.

GST pull-down assay for the interaction between GST-HflX and His₆-HflK or His₆-HflC. Lane 1, crude extract of cells overexpressing His₆-HflK and His₆-HflC (indicated by arrowheads); lane 2, glutathione Sepharose beads bound with GST-HflX were mixed with cell lysate overexpressing His₆-HflKC and incubated at the ambient temperature for 1 h; lane 3, preparation after the mixture described above was washed with buffer P. The samples were run on a 12.5% SDS-PAGE gel.

FIG. 7.

Effect of HflX and HflKC on transposition, examined by the papillation assay. Wild-type (DH10B) E. coli cells (wt) and the mutants in which either the hflX gene (ΔX) or the hflKC genes (ΔKC) were removed by in-frame deletion (DH10BΔX or DH10BΔKC) were separately transformed with plasmids containing IS903, Tn10, and Tn552. Each of these plasmids bears a transposon with a cryptic lacZ reporter. The number of blue papillae was counted for each colony. The average number of papillae per colony is indicated below each panel.