The ChrSA and HrrSA Two-Component Systems Are Required for Transcriptional Regulation of the hemA Promoter in Corynebacterium diphtheriae.

UNLABELLED: Corynebacterium diphtheriae utilizes heme and hemoglobin (Hb) as iron sources for growth in low-iron environments. In C. diphtheriae, the two-component signal transduction systems (TCSs) ChrSA and HrrSA are responsive to Hb levels and regulate the transcription of promoters for hmuO, hrtAB, and hemA ChrSA and HrrSA activate transcription at the hmuO promoter and repress transcription at hemA in an Hb-dependent manner. In this study, we show that HrrSA is the predominant repressor at hemA and that its activity results in transcriptional repression in the presence and absence of Hb, whereas repression of hemA by ChrSA is primarily responsive to Hb. DNA binding studies showed that both ChrA and HrrA bind to the hemA promoter region at virtually identical sequences. ChrA binding was enhanced by phosphorylation, while binding to DNA by HrrA was independent of its phosphorylation state. ChrA and HrrA are phosphorylated in vitro by the sensor kinase ChrS, whereas no kinase activity was observed with HrrS in vitro Phosphorylated ChrA was not observed in vivo, even in the presence of Hb, which is likely due to the instability of the phosphate moiety on ChrA. However, phosphorylation of HrrA was observed in vivo regardless of the presence of the Hb inducer, and genetic analysis indicates that ChrS is responsible for most of the phosphorylation of HrrA in vivo Phosphorylation studies strongly suggest that HrrS functions primarily as a phosphatase and has only minimal kinase activity. These findings collectively show a complex mechanism of regulation at the hemA promoter, where both two-component systems act in concert to optimize expression of heme biosynthetic enzymes. IMPORTANCE: Understanding the mechanism by which two-component signal transduction systems function to respond to environmental stimuli is critical to the study of bacterial pathogenesis. The current study expands on the previous analyses of the ChrSA and HrrSA TCSs in the human pathogen C. diphtheriae The findings here underscore the complex interactions between the ChrSA and HrrSA systems in the regulation of the hemA promoter and demonstrate how the two systems complement one another to refine and control transcription in the presence and absence of Hb.

Analysis of novel iron-regulated, surface-anchored hemin-binding proteins in Corynebacterium diphtheriae.

Corynebacterium diphtheriae utilizes hemin and hemoglobin (Hb) as iron sources during growth in iron-depleted environments, and recent studies have shown that the surface-exposed HtaA protein binds both hemin and Hb and also contributes to the utilization of hemin iron. Conserved (CR) domains within HtaA and in the associated hemin-binding protein, HtaB, are required for the ability to bind hemin and Hb. In this study, we identified and characterized two novel genetic loci in C. diphtheriae that encode factors that bind hemin and Hb. Both genetic systems contain two-gene operons that are transcriptionally regulated by DtxR and iron. The gene products of these operons are ChtA-ChtB and ChtC-CirA (previously DIP0522-DIP0523). The chtA and chtB genes are carried on a putative composite transposon associated with C. diphtheriae isolates that dominated the diphtheria outbreak in the former Soviet Union in the 1990s. ChtA and ChtC each contain a single N-terminal CR domain and exhibit significant sequence similarity to each other but only limited similarity with HtaA. The chtB and htaB gene products exhibited a high level of sequence similarity throughout their sequences, and both proteins contain a single CR domain. Whole-cell binding studies as well as protease analysis indicated that all four of the proteins encoded by these two operons are surface exposed, which is consistent with the presence of a transmembrane segment in their C-terminal regions. ChtA, ChtB, and ChtC are able to bind hemin and Hb, with ChtA showing the highest affinity. Site-directed mutagenesis showed that specific tyrosine residues within the ChtA CR domain were critical for hemin and Hb binding. Hemin iron utilization assays using various C. diphtheriae mutants indicate that deletion of the chtA-chtB region and the chtC gene has no affect on the ability of C. diphtheriae to use hemin or Hb as iron sources; however, a chtB htaB double mutant exhibits a significant decrease in hemin iron use, indicating a role in hemin transport for HtaB and ChtB.

The ChrA response regulator in Corynebacterium diphtheriae controls hemin-regulated gene expression through binding to the hmuO and hrtAB promoter regions.

Corynebacterium diphtheriae, the etiologic agent of diphtheria, utilizes heme and hemoglobin (Hb) as iron sources for growth. Heme-iron utilization involves HmuO, a heme oxygenase that degrades cytosolic heme, resulting in the release of heme-associated iron. Expression of the hmuO promoter is under dual regulation, in which transcription is repressed by DtxR and iron and activated by a heme source, such as hemin or Hb. Hemin-dependent activation is mediated primarily by the ChrAS two-component system, in which ChrS is a putative heme-responsive sensor kinase while ChrA is proposed to serve as a response regulator that activates transcription. It was recently shown that the ChrAS system similarly regulates the hrtAB genes, which encode an ABC transporter involved in the protection of C. diphtheriae from hemin toxicity. In this study, we characterized the phosphorelay mechanism in the ChrAS system and provide evidence for the direct regulation of the hmuO and hrtAB promoters by ChrA. A fluorescence staining method was used to show that ChrS undergoes autophosphorylation and that the phosphate moiety is subsequently transferred to ChrA. Promoter fusion studies identified regions upstream of the hmuO and hrtAB promoters that are critical for the heme-dependent regulation by ChrA. Electrophoretic mobility shift assays revealed that ChrA specifically binds at the hmuO and hrtAB promoter regions and that binding is phosphorylation dependent. A phosphorylation-defective mutant of ChrA [ChrA(D50A)] exhibited significantly diminished binding to the hmuO promoter region relative to that of wild-type ChrA. DNase I footprint analysis further defined the sequences in the hmuO and hrtAB promoters that are involved in ChrA binding, and this analysis revealed that the DtxR binding site at the hmuO promoter partially overlaps the binding site for ChrA. DNase I protection studies as well as promoter fusion analysis suggest that ChrA and DtxR compete for binding at the hmuO promoter. Collectively, these data demonstrate that the ChrA response regulator directly controls the expression of hmuO and the hrtAB genes and the binding activity of ChrA is dependent on phosphorylation by its cognate sensor kinase ChrS.

Dairy farm age and resistance to antimicrobial agents in Escherichia coli isolated from dairy topsoil.

Antimicrobial agent usage is common in animal agriculture for therapeutic and prophylactic purposes. Selective pressure exerted by these antimicrobials on soil bacteria could result in the selection of strains that are resistant due to chromosomal- or plasmid-derived genetic components. Multiple antimicrobial resistances in Escherichia coli and the direct relationship between antimicrobial agent use over time has been extensively studied, yet the relationship between the age of an animal agriculture environment such as a dairy farm and antibiotic resistance remains unclear. Therefore, we tested the hypothesis that antimicrobial-resistance profiles of E. coli isolated from dairy farm topsoil correlate with dairy farm age. E. coli isolated from eleven dairy farms of varying ages within Roosevelt County, NM were used for MIC determinations to chloramphenicol, nalidixic acid, penicillin, tetracycline, ampicillin, amoxicillin/clavulanic acid, gentamicin, trimethoprim/sulfamethoxazole, cefotaxime, and ciprofloxacin. The minimum inhibitory concentration values of four antibiotics ranged 0.75 to >256 µg/ml, 1 to >256 µg/ml, 12 to >256 µg/ml, and 0.75 to >256 µg/ml for chloramphenicol, nalidixic acid, penicillin, and tetracycline, respectively. The study did not show a direct relationship between antibiotic resistance and the age of dairy farms.

Expression of BfrH, a putative siderophore receptor of Bordetella bronchiseptica, is regulated by iron, Fur1, and the extracellular function sigma factor EcfI.

Iron (Fe) in soluble elemental form is found in the tissues and fluids of animals at concentrations insufficient for sustaining growth of bacteria. Consequently, to promote colonization and persistence, pathogenic bacteria evolved a myriad of scavenging mechanisms to acquire Fe from the host. Bordetella bronchiseptica, the etiologic agent of upper respiratory infections in a wide range of mammalian hosts, expresses a number of proteins for acquisition of Fe. Using proteomic and genomic approaches, three Fe-regulated genes were identified in the bordetellae: bfrH, a gene encoding a putative siderophore receptor; ecfI, a gene encoding a putative extracellular function (ECF) sigma factor; and ecfR, a gene encoding a putative EcfI modulator. All three genes are highly conserved in B. pertussis, B. parapertussis, and B. avium. Genetic analysis revealed that transcription of bfrH was coregulated by ecfI, ecfR, and fur1, one of two fur homologues carried by B. bronchiseptica. Overexpression of ecfI decoupled bfrH from Fe-dependent regulation. In contrast, expression of bfrH was significantly reduced in an ecfI deletion mutant. Deletion of ecfR, however, was correlated with a significant increase in expression of bfrH, due in part to a cis-acting nucleotide sequence within ecfR which likely reduces the frequency of readthrough transcription of bfrH from the Fe-dependent ecfIR promoter. Using a murine competition infection model, bfrH was shown to be required for optimal virulence of B. bronchiseptica. These experiments revealed ecfIR-bfrH as a locus encoding a new member of the growing family of Fe and ECF sigma factor-modulated regulons in the bordetellae.