Characterization of the Pasteurella multocida hgbA gene encoding a hemoglobin-binding protein

Abstract

Reverse transcriptase PCR analyses have demonstrated that open reading frames (ORFs) PM0298, PM0299, and PM0300 of the animal pathogen Pasteurella multocida constitute a single transcriptional unit. By cloning and overexpression studies in Escherichia coli cells, the product of ORF PM0300 was shown to bind hemoglobin in vitro; this ORF was therefore designated hgbA. In vitro and in vivo quantitative assays demonstrated that the P. multocida hgbA mutant bound hemoglobin to the same extent as the wild-type strain, although the adsorption kinetics was slightly slower for the hgbA cells. In agreement with this, the virulence of P. multocida hgbA cells was not affected, suggesting that other functional hemoglobin receptor proteins must be present in this organism. On the other hand, P. multocida mutants defective in PM0298 and PM0299 could be isolated only when a plasmid containing an intact copy of the gene was present in the cells, suggesting that these genes are essential for the viability of this bacterial pathogen. By adapting the recombinase-based expression technology in vivo to P. multocida, we also demonstrated that the transcriptional PM0298-PM0299-hgbA unit is iron regulated and that its expression is triggered in the first 2 h following infection in a mouse model. Furthermore, hybridization experiments showed that the hgbA gene is widespread in P. multocida strains regardless of their serotype or the animal from which they were isolated.

FIG. 1.

(A) Genetic organization of the P. multocida chromosomal region containing the PM0298-PM0299-PM0300 (hgbA) transcriptional unit. The translational start and stop codons for the PM0298, PM0299, and PM0300 ORFs are indicated by boldface type, by boldface type and underlining, and by italics, respectively. The positions of primers (Table 2) used to show the existence of a single mRNA by RT-PCR, as well as in the several constructions used in this study, are indicated. The numbers in parentheses indicate the positions with reference to the ORF PM0298 translational start point. The location of ORF PM0297 immediately upstream of PM0298 is also shown. The solid box downstream of ORF PM0300 (hgbA) represents the putative rho-independent transcriptional terminator of the operon. (B) RT-PCR analysis of the PM0298-PM0299-PM0300 (hgbA) transcript in P. multocida cells performed with PM0298intup and PhgbArp as the upper and lower primers, respectively, in the presence of RNA (lane 3). As a positive control, the PCR fragment obtained when P. multocida chromosomal DNA was used as the template (lane 4) was also examined. The negative controls were a preparation containing RNA template but no RT (lane 2) and a preparation lacking both RNA and DNA (lane 5). Lane 1 contained BstEII-digested λ DNA employed as the molecular size marker.

FIG. 1.

(A) Genetic organization of the P. multocida chromosomal region containing the PM0298-PM0299-PM0300 (hgbA) transcriptional unit. The translational start and stop codons for the PM0298, PM0299, and PM0300 ORFs are indicated by boldface type, by boldface type and underlining, and by italics, respectively. The positions of primers (Table 2) used to show the existence of a single mRNA by RT-PCR, as well as in the several constructions used in this study, are indicated. The numbers in parentheses indicate the positions with reference to the ORF PM0298 translational start point. The location of ORF PM0297 immediately upstream of PM0298 is also shown. The solid box downstream of ORF PM0300 (hgbA) represents the putative rho-independent transcriptional terminator of the operon. (B) RT-PCR analysis of the PM0298-PM0299-PM0300 (hgbA) transcript in P. multocida cells performed with PM0298intup and PhgbArp as the upper and lower primers, respectively, in the presence of RNA (lane 3). As a positive control, the PCR fragment obtained when P. multocida chromosomal DNA was used as the template (lane 4) was also examined. The negative controls were a preparation containing RNA template but no RT (lane 2) and a preparation lacking both RNA and DNA (lane 5). Lane 1 contained BstEII-digested λ DNA employed as the molecular size marker.

FIG. 2.

Solid-phase assay for binding of bovine hemoglobin to sonicated and intact E. coli BL21(λDE3)/pUA963 cells (lanes 1 and 2, respectively). Sonicated E. coli BL21(λDE3)/pET22b cells in the presence and absence of IPTG were the negative controls (lanes 3 and 4, respectively).

FIG. 3.

(A) PCR analysis of P. multocida mutants. Chromosomal DNA from hgbA (lane 3) and PM0298 (lane 6) mutants were subjected to PCR analysis with the Aad oligonucleotide (Table 2) as the upper primer and the HgbArp and HgbAintrp oligonucleotides (Fig. 1A), respectively, as the lower primers. PCRs performed with chromosomal DNA from the wild-type strain (lanes 2 and 5) and these primer pairs and PCRs performed with preparations lacking DNA template (lanes 4 and 7) were the negative controls. Lane 1 contained HindIII-digested λ DNA as the molecular size marker. (B) RT-PCR study of the P. multocida hgbA mutant. RNA from hgbA (lane 3) and wild-type (lane 4) cells were subjected to RT-PCR analysis with the RTHgbAup and RTHgbArp oligonucleotides (Table 2). The control for RNA integrity was an RT-PCR amplification performed with RNA from hgbA cells and oligonucleotides RecAup and RecAdw belonging to the internal sequence of the P. multocida recA gene (lane 2). PCR performed with chromosomal DNA from the wild-type strain and oligonucleotides RTHgbAup and RTHgbArp (lane 5) and RT-PCR amplification of preparations containing the same primers but lacking both DNA and RNA templates (lane 6) were the product size and negative controls, respectively. Lane 1 contained BstEII-digested λ DNA as the molecular size marker.

FIG. 4.

(A) Solid-phase assay for in vitro binding of bovine hemoglobin to whole P. multocida wild-type (lane 1) or hgbA mutant (lane 2) cells. (B) In vivo quantitative analysis of hemoglobin adsorption to P. multocida wild-type (▪) and hgbA (▴) cells. The behavior of E. coli cells (•) was used as a negative control. All values are the means of three experiments (each performed in triplicate), and the standard error of any value was never greater than 10%.

FIG. 5.

Schematic representation of specific single recombination between the pUA961 plasmid and the P. multocida chromosome through the 370-bp internal fragment of the PM0298-PM0299-hgbA transcriptional unit cloned upstream of the tnpR gene-encoding region.

FIG. 6.

(A) Kinetics of transcription of the PM0298-PM0299-hgbA operon in mice inoculated intraperitoneally with the P. multocida PM1077 strain, measured as the proportion of Cm^s bacteria recovered from the peritoneal cavity (♦). The absence of Cm^s bacteria in cultures of the P. multocida PM1077 strain growing in BPW medium without chloramphenicol demonstrates the genetic stability of the construction (▪). Three animals were used for each point, and each Cm^s percentage is the average of three determinations for each of the inoculated mice. In all cases, the standard error of any value was never greater than 10%. (B) Kinetics of transcription of the PM0298-PM0299-hgbA operon in the P. multocida PM1077 strain measured in vitro as the proportion of Cm^s bacteria recovered from BPW medium (•) or BPW medium supplemented with either DPD (50 μg/ml) (♦), DPD and FeSO₄ (1 mM) (▴), or DPD and hemoglobin (40 mM) (▪). All values are the means of at least three experiments (each performed in triplicate), and the standard error of any value was never greater than 10%.

FIG. 7.

Presence of the hgbA gene in P. multocida strains: dot blot hybridization of chromosomal DNA from 34 P. multocida strains obtained from different animal sources and belonging to several serotypes with a digoxigenin-labeled 989-bp internal fragment of the hgbA gene. The DNA position corresponding to each of the strains is indicated above the nitrocellulose membrane. Equal amounts of chromosomal DNA (1 μg) of all strains were applied to the nitrocellulose membrane, and hybridization was performed under high-stringency conditions.