Positive regulation of Leptospira interrogans kdp expression by KdpE as Demonstrated with a novel β-galactosidase reporter in Leptospira biflexa

Abstract

Leptospirosis is a potentially deadly zoonotic disease that afflicts humans and animals. Leptospira interrogans, the predominant agent of leptospirosis, encounters diverse conditions as it proceeds through its life cycle, which includes stages inside and outside the host. Unfortunately, the number of genetic tools available for examining the regulation of gene expression in L. interrogans is limited. Consequently, little is known about the genetic circuits that control gene expression in Leptospira. To better understand the regulation of leptospiral gene expression, the L. interrogans kdp locus, encoding homologs of the P-type ATPase KdpABC potassium transporter with their KdpD sensors and KdpE response regulators, was selected for analysis. We showed that a kdpE mutation in L. interrogans prevented the increase in kdpABC mRNA levels observed in the wild-type L. interrogans strain when external potassium levels were low. To confirm that KdpE was a positive regulator of kdpABC transcription, we developed a novel approach for constructing chromosomal genetic fusions to the endogenous bgaL (β-galactosidase) gene of the nonpathogen Leptospira biflexa. We demonstrated positive regulation of a kdpA'-bgaL fusion in L. biflexa by the L. interrogans KdpE response regulator. A control lipL32'-bgaL fusion was not regulated by KdpE. These results demonstrate the utility of genetic fusions to the bgaL gene of L. biflexa for examining leptospiral gene regulation.

Fig 1

Location of the Himar1 insertion in the predicted structure of L. interrogans KdpE. The alignment of the amino acid sequence of the carboxy terminus of L. interrogans KdpE with that of the winged helix-turn-helix of E. coli OmpR was aided by an earlier published alignment of E. coli KdpE and OmpR (26). The secondary structure of OmpR is depicted with cylinders (α helix) and open arrows (β strand). The location of the Himar1 insertion in the L. interrogans M45 strain is marked by an arrow below the KdpE sequence.

Fig 2

Effect of the kdpE mutation on kdp transcript levels in low-potassium medium. Transcript levels were determined by quantitative reverse transcriptase (qRT) PCR as described in Materials and Methods. Signals from the kdpABC mRNA were normalized to flaA2 transcript levels. Representative results from three independent experiments are shown. The error bars denote the standard deviation. *, P < 0.01; **, P < 0.001; ***, P < 0.0001 (2-way ANOVA).

Fig 3

Cotranscription of adjacent genes in kdpABC. Total RNA was extracted from L. interrogans grown under low-potassium conditions. Primer pairs flanking the intergenic regions of kdpABC were used for reverse transcriptase PCR. The primer pairs that included the forward oligonucleotide targeting the kdp promoter region served as a negative control.

Fig 4

Integration of bgaL fusions into the chromosome of L. biflexa. (A) Sequence features of pRAT562. Restriction sites used for cloning are shown. The first 293 of the 661 codons of L. biflexa bgaL are present in pRAT562. (B) Scheme for integration of the kdpA promoter and translation initiation signals upstream of the bgaL coding region on the chromosome of L. biflexa. The kdpA promoter and 5′ untranslated region cloned into pRAT562 are depicted by the stippled segment. The bgaL sequences are shaded in light gray. Homologous recombination between the plasmid and chromosome occurred within the first 879 bp of bgaL. The integrated sequence was detected by PCR with a forward primer that annealed within the plasmid vector sequence and a reverse primer that annealed within the bgaL sequence downstream of the cloned segment. The nucleotide sequences of the NdeI recognition site and flanking regions are shown. The Shine-Dalgarno sequence and start codon are underlined. Lowercase, kdpA sequence; italics, NdeI recognition sequence; uppercase, second and third bgaL codons. (C) PCR products were digested with NdeI and subjected to electrophoresis in a 1.0% agarose gel. The expected sizes of the smaller fragments are indicated in parentheses. (D) β-Galactosidase activities measured from various chromosomal bgaL fusions. Each bar represents the mean β-galactosidase expression from three independent cultures. The error bars denote the standard deviations.

Fig 5

Positive regulation of the kdpA′-bgaL fusion by KdpE. (A) The L. biflexa kdpA′-bgaL strain was transformed with pSLe94 (vector) or a KdpE expression plasmid. (B) Same as panel A, except the L. biflexa strain harbored the lipL32′-bgaL fusion. Each bar represents the mean β-galactosidase expression from three independent cultures. The error bars denote the standard deviations. *, P < 0.05 (1-way ANOVA and Dunnett's multiple-comparison posttest).