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Identification and characterization of the Chlamydia trachomatis L2 S-adenosylmethionine transporter.

Binet R, Fernandez RE, Fisher DJ, Maurelli AT - MBio (2011)

Bottom Line: Moreover, CTL843 conferred a growth advantage to a Δpfs E. coli mutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843.The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle of Chlamydia and identifies an excellent chemotherapeutic target.The transporter, CTL843, allows Chlamydia trachomatis L2 to steal S-adenosylmethionine (SAM) from the eukaryotic host cytosol and to likely remove the toxic S-adenosylhomocysteine (SAH) formed when SAM loses its methyl group, acting as a SAM/SAH transporter (SAMHT).

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA. rachel.binet@fda.hhs.gov

ABSTRACT

Unlabelled: Methylation is essential to the physiology of all cells, including the obligate intracellular bacterium Chlamydia. Nevertheless, the methylation cycle is under strong reductive evolutionary pressure in Chlamydia. Only Parachlamydia acanthamoebae and Waddlia chondrophila genome sequences harbor homologs to metK, encoding the S-adenosylmethionine (SAM) synthetase required for synthesis of SAM, and to sahH, which encodes the S-adenosylhomocysteine (SAH) hydrolase required for detoxification of SAH formed after the transfer of the methyl group from SAM to the methylation substrate. Transformation of a conditional-lethal ΔmetK mutant of Escherichia coli with a genomic library of Chlamydia trachomatis L2 identified CTL843 as a putative SAM transporter based on its ability to allow the mutant to survive metK deficiency only in the presence of extracellular SAM. CTL843 belongs to the drug/metabolite superfamily of transporters and allowed E. coli to transport S-adenosyl-L-[methyl-(14)C]methionine with an apparent K(m) of 5.9 µM and a V(max) of 32 pmol min(-1) mg(-1). Moreover, CTL843 conferred a growth advantage to a Δpfs E. coli mutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843. We propose that CTL843 acts as a SAM/SAH transporter (SAMHT) serving a dual function by allowing Chlamydia to acquire SAM from the host cell and excrete the toxic by-product SAH. The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle of Chlamydia and identifies an excellent chemotherapeutic target.

Importance: Obligate intracellular parasites like Chlamydia have followed a reductive evolutionary path that has made them almost totally dependent on their host cell for nutrients. In this work, we identify a unique transporter of a metabolite essential for all methylation reactions that potentially bypasses the need for two enzymatic reactions in Chlamydia. The transporter, CTL843, allows Chlamydia trachomatis L2 to steal S-adenosylmethionine (SAM) from the eukaryotic host cytosol and to likely remove the toxic S-adenosylhomocysteine (SAH) formed when SAM loses its methyl group, acting as a SAM/SAH transporter (SAMHT). In addition to reflecting the adaptation of Chlamydia to an obligate intracellular lifestyle, the specific and central roles of SAMHT in Chlamydia metabolism provide a target for the development of therapeutic agents for the treatment of chlamydial infections.

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Growth of E. coli ∆metK mutant in the presence or absence of extracellular SAM. ATM778 (∆metK) transformed with the indicated plasmids was grown at 37°C in LB supplemented with Ap, Cm, 0.2% glucose (solid lines), or 0.2% arabinose (dotted lines), in the absence (A) or presence (B) of 1 mM SAM. Absorbance (OD600) was measured in a Bioscreen growth curve analyzer and plotted against time (hours). Error bars represent the standard deviations from four replicates. Symbols: ○, pUC-empty vector; □, pREF77-ctl843; ▵, pRAK368-ctl843; ▿, pRAK368-ctl843 with IPTG.
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f1: Growth of E. coli ∆metK mutant in the presence or absence of extracellular SAM. ATM778 (∆metK) transformed with the indicated plasmids was grown at 37°C in LB supplemented with Ap, Cm, 0.2% glucose (solid lines), or 0.2% arabinose (dotted lines), in the absence (A) or presence (B) of 1 mM SAM. Absorbance (OD600) was measured in a Bioscreen growth curve analyzer and plotted against time (hours). Error bars represent the standard deviations from four replicates. Symbols: ○, pUC-empty vector; □, pREF77-ctl843; ▵, pRAK368-ctl843; ▿, pRAK368-ctl843 with IPTG.

Mentions: metK is essential in E. coli, and E. coli cells are impermeable to extracellular SAM (14–16). To construct a conditional-lethal ΔmetK mutant of E. coli, the rescuing copy of metK needs to be tightly controlled. Although metK deletion was first obtained in ATM770 (Table 1), this strain was still able to grow in the absence of IPTG (isopropyl-β-d-thiogalactopyranoside), indicating that the lactose promoter controlling metK in pREF71 was still leaky in the absence of inducer (data not shown), as previously encountered with the ara promoter (16). On the other hand, by placement of a copy of the metK gene containing an alternate GUG start codon (to reduce translational efficiency) under the control of the arabinose-inducible, glucose-repressible ara promoter, the growth and/or survival of the ΔmetK ATM778 mutant was made dependent on the presence of arabinose (Fig. 1A). We screened a C. trachomatis serovar L2 genomic DNA library in ATM778 in the presence of glucose and identified several colonies on medium supplemented with 1 mM SAM, while no colonies were detected under the same conditions in the absence of extracellular SAM. Two of 12 independent colonies characterized displayed consistent SAM-dependent growth in the presence of glucose (i.e., repression of E. coli metK expression) (Fig. 1B). Rescuing plasmids were isolated and analyzed by restriction mapping and sequence analyses. All library clones revealed the same 2,528-bp insert harboring C. trachomatis L2 CTL843 and 123 bp of upstream sequence in the same orientation as the lactose promoter of pUC, followed by CTL842 with 7 bp of upstream sequence, in reverse orientation. Subcloning of this ORF into pBluescript (i.e., pRAK368, Table 1) confirmed that CTL843 conferred a SAM-dependent growth phenotype to ATM778 in the presence of glucose and IPTG (Fig. 1B), suggesting that CTL843 functions as a transporter for SAM.


Identification and characterization of the Chlamydia trachomatis L2 S-adenosylmethionine transporter.

Binet R, Fernandez RE, Fisher DJ, Maurelli AT - MBio (2011)

Growth of E. coli ∆metK mutant in the presence or absence of extracellular SAM. ATM778 (∆metK) transformed with the indicated plasmids was grown at 37°C in LB supplemented with Ap, Cm, 0.2% glucose (solid lines), or 0.2% arabinose (dotted lines), in the absence (A) or presence (B) of 1 mM SAM. Absorbance (OD600) was measured in a Bioscreen growth curve analyzer and plotted against time (hours). Error bars represent the standard deviations from four replicates. Symbols: ○, pUC-empty vector; □, pREF77-ctl843; ▵, pRAK368-ctl843; ▿, pRAK368-ctl843 with IPTG.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104491&req=5

f1: Growth of E. coli ∆metK mutant in the presence or absence of extracellular SAM. ATM778 (∆metK) transformed with the indicated plasmids was grown at 37°C in LB supplemented with Ap, Cm, 0.2% glucose (solid lines), or 0.2% arabinose (dotted lines), in the absence (A) or presence (B) of 1 mM SAM. Absorbance (OD600) was measured in a Bioscreen growth curve analyzer and plotted against time (hours). Error bars represent the standard deviations from four replicates. Symbols: ○, pUC-empty vector; □, pREF77-ctl843; ▵, pRAK368-ctl843; ▿, pRAK368-ctl843 with IPTG.
Mentions: metK is essential in E. coli, and E. coli cells are impermeable to extracellular SAM (14–16). To construct a conditional-lethal ΔmetK mutant of E. coli, the rescuing copy of metK needs to be tightly controlled. Although metK deletion was first obtained in ATM770 (Table 1), this strain was still able to grow in the absence of IPTG (isopropyl-β-d-thiogalactopyranoside), indicating that the lactose promoter controlling metK in pREF71 was still leaky in the absence of inducer (data not shown), as previously encountered with the ara promoter (16). On the other hand, by placement of a copy of the metK gene containing an alternate GUG start codon (to reduce translational efficiency) under the control of the arabinose-inducible, glucose-repressible ara promoter, the growth and/or survival of the ΔmetK ATM778 mutant was made dependent on the presence of arabinose (Fig. 1A). We screened a C. trachomatis serovar L2 genomic DNA library in ATM778 in the presence of glucose and identified several colonies on medium supplemented with 1 mM SAM, while no colonies were detected under the same conditions in the absence of extracellular SAM. Two of 12 independent colonies characterized displayed consistent SAM-dependent growth in the presence of glucose (i.e., repression of E. coli metK expression) (Fig. 1B). Rescuing plasmids were isolated and analyzed by restriction mapping and sequence analyses. All library clones revealed the same 2,528-bp insert harboring C. trachomatis L2 CTL843 and 123 bp of upstream sequence in the same orientation as the lactose promoter of pUC, followed by CTL842 with 7 bp of upstream sequence, in reverse orientation. Subcloning of this ORF into pBluescript (i.e., pRAK368, Table 1) confirmed that CTL843 conferred a SAM-dependent growth phenotype to ATM778 in the presence of glucose and IPTG (Fig. 1B), suggesting that CTL843 functions as a transporter for SAM.

Bottom Line: Moreover, CTL843 conferred a growth advantage to a Δpfs E. coli mutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843.The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle of Chlamydia and identifies an excellent chemotherapeutic target.The transporter, CTL843, allows Chlamydia trachomatis L2 to steal S-adenosylmethionine (SAM) from the eukaryotic host cytosol and to likely remove the toxic S-adenosylhomocysteine (SAH) formed when SAM loses its methyl group, acting as a SAM/SAH transporter (SAMHT).

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA. rachel.binet@fda.hhs.gov

ABSTRACT

Unlabelled: Methylation is essential to the physiology of all cells, including the obligate intracellular bacterium Chlamydia. Nevertheless, the methylation cycle is under strong reductive evolutionary pressure in Chlamydia. Only Parachlamydia acanthamoebae and Waddlia chondrophila genome sequences harbor homologs to metK, encoding the S-adenosylmethionine (SAM) synthetase required for synthesis of SAM, and to sahH, which encodes the S-adenosylhomocysteine (SAH) hydrolase required for detoxification of SAH formed after the transfer of the methyl group from SAM to the methylation substrate. Transformation of a conditional-lethal ΔmetK mutant of Escherichia coli with a genomic library of Chlamydia trachomatis L2 identified CTL843 as a putative SAM transporter based on its ability to allow the mutant to survive metK deficiency only in the presence of extracellular SAM. CTL843 belongs to the drug/metabolite superfamily of transporters and allowed E. coli to transport S-adenosyl-L-[methyl-(14)C]methionine with an apparent K(m) of 5.9 µM and a V(max) of 32 pmol min(-1) mg(-1). Moreover, CTL843 conferred a growth advantage to a Δpfs E. coli mutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843. We propose that CTL843 acts as a SAM/SAH transporter (SAMHT) serving a dual function by allowing Chlamydia to acquire SAM from the host cell and excrete the toxic by-product SAH. The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle of Chlamydia and identifies an excellent chemotherapeutic target.

Importance: Obligate intracellular parasites like Chlamydia have followed a reductive evolutionary path that has made them almost totally dependent on their host cell for nutrients. In this work, we identify a unique transporter of a metabolite essential for all methylation reactions that potentially bypasses the need for two enzymatic reactions in Chlamydia. The transporter, CTL843, allows Chlamydia trachomatis L2 to steal S-adenosylmethionine (SAM) from the eukaryotic host cytosol and to likely remove the toxic S-adenosylhomocysteine (SAH) formed when SAM loses its methyl group, acting as a SAM/SAH transporter (SAMHT). In addition to reflecting the adaptation of Chlamydia to an obligate intracellular lifestyle, the specific and central roles of SAMHT in Chlamydia metabolism provide a target for the development of therapeutic agents for the treatment of chlamydial infections.

Show MeSH
Related in: MedlinePlus