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. 2020 Mar 11;202(7):e00770-19.
doi: 10.1128/JB.00770-19. Print 2020 Mar 11.

Roles of TroA and TroR in Metalloregulated Growth and Gene Expression in Treponema denticola

Prakaimuk Saraithong #  1 , M Paula Goetting-Minesky #  1 , Peter M Durbin  1 , Spencer W Olson  1 , Frank C Gherardini  2 , J Christopher Fenno  3
Affiliations

Roles of TroA and TroR in Metalloregulated Growth and Gene Expression in Treponema denticola

Prakaimuk Saraithong et al. J Bacteriol. .

Abstract

The availability of divalent metal cations required as cofactors for microbial metabolism is severely limited in the host environment. Bacteria have evolved highly regulated uptake systems to maintain essential metal homeostasis to meet cellular demands while preventing toxicity. The Tro operon (troABCDR), present in all sequenced Treponema spp., is a member of a highly conserved family of ATP-binding cassette transporters involved in metal cation uptake whose expression is controlled by TroR, a DtxR-like cation-responsive regulatory protein. Transcription of troA responds to divalent manganese and iron (T. denticola) or manganese and zinc (T. pallidum), and metal-dependent TroR binding to the troA promoter represses troA transcription. We report here the construction and complementation of defined T. denticola ΔtroR and ΔtroA strains to characterize (i) the role of TroA in metal-dependent T. denticola growth and (ii) the role of TroR in T. denticola gene expression. We show that TroA expression is required for T. denticola growth under iron- and manganese-limited conditions. Furthermore, TroR is required for the transcriptional regulation of troA in response to iron or manganese, and deletion of troR results in significant differential expression of more than 800 T. denticola genes in addition to troA These results suggest that (i) TroA-mediated cation uptake is important in metal homeostasis in vitro and may be important for Treponema survival in the host environment and (ii) the absence of TroR results in significant dysregulation of nearly one-third of the T. denticola genome. These effects may be direct (as with troA) or indirect due to dysregulation of metal homeostasis.IMPORTANCETreponema denticola is one of numerous host-associated spirochetes, a group including commensals, pathobionts, and at least one frank pathogen. While most T. denticola research concerns its role in periodontitis, its relative tractability for growth and genetic manipulation make it a useful model for studying Treponema physiology, metabolism, and host-microbe interactions. Metal micronutrient acquisition and homeostasis are highly regulated both in microbial cells and by host innate defense mechanisms that severely limit metal cation bioavailability. Here, we characterized the T. denticolatroABCDR operon, the role of TroA-mediated iron and manganese uptake in growth, and the effects of TroR on global gene expression. This study contributes to our understanding of the mechanisms involved in cellular metal homeostasis required for survival in the host environment.

Keywords: metalloregulation; spirochetes; transcription.

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Figures

FIG 1
FIG 1
RT-PCR analysis of the tro locus. (A) RT-PCR using oligonucleotide primer sets to amplify junctions of troA-troB, troB-troC, troC-troD, and troD-troR . Lanes showing troD-R PCR products were spliced from a different region of the same agarose gel due to the number of lanes required. (B) RT-PCR using oligonucleotide primer sets specific for TDE1227-troA. T. denticola PCR templates: cDNA (lanes 1), genomic DNA (lanes 2), and RNA (lanes 3). Template cDNAs were made with random hexamer primers (A) or a troR “reverse” primer (B). (C) Quantitative RT-PCR (RT-qPCR) following 2 days of growth in NOS medium. The transcript levels of individual genes in the tro operon are shown relative to troA. The oligonucleotide primer sets are listed in Table S2.
FIG 2
FIG 2
RT-qPCR analysis of troA and troR transcription in response to cation starvation and supplementation with Mn2+ or Fe2+. T. denticola 35405 was grown for 2 days in NOS medium under standard conditions (405N), chelated conditions (405C), chelated conditions plus Mn2+ at 20 μM (405C+Mn), or chelated conditions plus Fe2+ at 20 μM (405C+Fe). Statistically significant (P < 0.05) differences between gene expressions under different growth conditions are indicated by asterisks.
FIG 3
FIG 3
Expression of TroA during growth. Samples of T. denticola 35405 were taken on days 1 to 4 during growth in NOS medium and subjected to Western immunoblotting. The upper panel was probed with anti-TroA antibodies. The lower panel was probed with anti-FlaA antibodies. Lanes 1 to 4 represent samples taken on the indicated day.
FIG 4
FIG 4
Mutagenesis and complementation of the T. denticola tro operon. The construction of isogenic T. denticola strains mutated in troA or troR (designated ΔtroA and ΔtroR strains, respectively) and the ΔtroA::troA and ΔtroR::troR complemented mutant strains (designated C-ΔtroA and C-ΔtroR, respectively) are illustrated below the map. Each of the four mutant strains was constructed independently. Each antibiotic cassette (aphA2 encoding Kmr and ermB encoding Emr) is under transcriptional control of a ermB promoter (P). The sequence of the tro promoter/operator (P/O) region containing the promoter and putative TroR binding site in T. denticola 35405 is shown above the map.
FIG 5
FIG 5
T. denticola ΔtroA mutant requires cation supplementation. T. denticola parent strain 35405 (“405”) and isogenic ΔtroR and ΔtroA were grown in NOS medium or NOS medium supplemented with Fe2+, Mn2+, or both Fe2+ and Mn2+ .
FIG 6
FIG 6
Expression of the T. denticola tro locus in wild-type, mutant, and complemented mutant strains. (A) RT-qPCR of each of the genes in the tro operon in T. denticola ΔtroA and C-ΔtroA strains relative to its expression level in T. denticola 35405, which is set at 1.0 (dashed line). (B) TroA protein expression after 2 days of growth. Immunoblots were probed with antibodies raised against TroA (upper) or FlaA (lower). (C) RT-qPCR of each of the genes in the tro operon in T. denticola ΔtroR and C-ΔtroR strains relative to its expression level in T. denticola 35405, which is set at 1.0 (dashed line). (D) TroR protein expression after 2 days growth. Immunoblots were probed with antibodies raised against TroR (upper) or FlaA (lower).
FIG 7
FIG 7
Comparison of RNA-seq and RT-qPCR data. Comparison of transcription levels (fold change, base 10) between T. denticola parent strain (WT) and ΔtroR mutant in 12 differentially expressed genes as determined by the two methods. The Pearson correlation coefficient between RNA-seq and RT-qPCR was 0.997. TDE0439, TDE0610, TDE0479, TDE0040, TDE1017, TDE2337, TDE0765, and TDE0389 were among the top 20 upregulated genes in RNA-seq in T. denticola ΔtroR, while TDE2061 and TDE2063 were downregulated. As expected, TDE1226 (troA) was also upregulated in both RNA-seq and RT-qPCR.
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References

    1. Hood MI, Skaar EP. 2012. Nutritional immunity: transition metals at the pathogen-host interface. Nat Rev Microbiol 10:525–537. doi:10.1038/nrmicro2836. - DOI - PMC - PubMed
    1. Nelson N. 1999. Metal ion transporters and homeostasis. EMBO J 18:4361–4371. doi:10.1093/emboj/18.16.4361. - DOI - PMC - PubMed
    1. Boyd J, Oza MN, Murphy JR. 1990. Molecular cloning and DNA sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae. Proc Natl Acad Sci U S A 87:5968–5972. doi:10.1073/pnas.87.15.5968. - DOI - PMC - PubMed
    1. Que Q, Helmann JD. 2000. Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Mol Microbiol 35:1454–1468. doi:10.1046/j.1365-2958.2000.01811.x. - DOI - PubMed
    1. Leyn SA, Rodionov DA. 2015. Comparative genomics of DtxR family regulons for metal homeostasis in Archaea. J Bacteriol 197:451–458. doi:10.1128/JB.02386-14. - DOI - PMC - PubMed

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