Treponema pallidum 3-phosphoglycerate mutase is a heat-labile enzyme that may limit the maximum growth temperature for the spirochete.

In the causative agent of syphilis, Treponema pallidum, the gene encoding 3-phosphoglycerate mutase, gpm, is part of a six-gene operon (tro operon) that is regulated by the Mn-dependent repressor TroR. Since substrate-level phosphorylation via the Embden-Meyerhof pathway is the principal way to generate ATP in T. pallidum and Gpm is a key enzyme in this pathway, Mn could exert a regulatory effect on central metabolism in this bacterium. To study this, T. pallidum gpm was cloned, Gpm was purified from Escherichia coli, and antiserum against the recombinant protein was raised. Immunoblots indicated that Gpm was expressed in freshly extracted infective T. pallidum. Enzyme assays indicated that Gpm did not require Mn(2+) while 2,3-diphosphoglycerate (DPG) was required for maximum activity. Consistent with these observations, Mn did not copurify with Gpm. The purified Gpm was stable for more than 4 h at 25 degrees C, retained only 50% activity after incubation for 20 min at 34 degrees C or 10 min at 37 degrees C, and was completely inactive after 10 min at 42 degrees C. The temperature effect was attenuated when 1 mM DPG was added to the assay mixture. The recombinant Gpm from pSLB2 complemented E. coli strain PL225 (gpm) and restored growth on minimal glucose medium in a temperature-dependent manner. Increasing the temperature of cultures of E. coli PL225 harboring pSLB2 from 34 to 42 degrees C resulted in a 7- to 11-h period in which no growth occurred (compared to wild-type E. coli). These data suggest that biochemical properties of Gpm could be one contributing factor to the heat sensitivity of T. pallidum.

Lack of a role for iron in the Lyme disease pathogen.

A fundamental tenet of microbial pathogenesis is that bacterial pathogens must overcome host iron limitation to establish a successful infection. Surprisingly, the Lyme disease pathogen Borrelia burgdorferi has bypassed this host defense by eliminating the need for iron. B. burgdorferi grew normally and did not alter gene expression in the presence of iron chelators. Furthermore, typical bacterial iron-containing proteins were not detected in cell lysates, nor were the genes encoding such proteins identified in the genome sequence. The intracellular concentration of iron in B. burgdorferi was estimated to be less than 10 atoms per cell, well below a physiologically relevant concentration.

Characterization of a manganese-dependent regulatory protein, TroR, from Treponema pallidum.

Genome sequence analysis of Treponema pallidum, the causative agent of syphilis, suggests that this bacterium has a limited iron requirement with few, if any, proteins that require iron. Instead, T. pallidum may use manganese-dependent enzymes for metabolic pathways. This strategy apparently alleviates the necessity of T. pallidum to acquire iron from the host, thus overcoming iron limitation, which is a primary host defense. Interestingly, a putative metal-dependent regulatory protein, TroR, which has homology with the diphtheria toxin regulatory protein, DtxR, from Corynebacterium diphtheriae was identified from T. pallidum. We describe here the characterization of TroR, a regulatory protein. Mobility-shift DNA binding and DNase I footprint assays indicated that purified TroR bound to a 22-nt region of dyad symmetry that overlaps the -10 region of the promoter of the tro operon, which contains the genes for a putative metal transport system, the glycolytic enzyme phosphoglycerate mutase, and TroR. Unlike other metal-dependent regulatory proteins like diphtheria toxin regulatory protein and the ferric ion uptake regulator, Fur, which can be activated by divalent metals such as Fe(2+), Mn(2+), Co(2+), Ni(2+), and Zn(2+), TroR is activated only by Mn(2+). The TroR-Mn(2+) complex binds its target sequence and blocks transcription of the troPO/lacZ fusion, suggesting that TroR acts as a metal-dependent repressor in vivo. In addition, TroR exists as a dimer in both its inactive (metal free) and active states as indicated by chemical crosslinking experiments. Based on these data, we propose that TroR represents a unique regulatory system for controlling gene expression in T. pallidum in response to Mn(2+).

Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa.

We have cloned a 3.6-kb genomic DNA fragment from Pseudomonas aeruginosa harboring the rpoA, rplQ, katA, and bfrA genes. These loci are predicted to encode, respectively, (i) the alpha subunit of RNA polymerase; (ii) the L17 ribosomal protein; (iii) the major catalase, KatA; and (iv) one of two iron storage proteins called bacterioferritin A (BfrA; cytochrome b1 or b557). Our goal was to determine the contributions of KatA and BfrA to the resistance of P. aeruginosa to hydrogen peroxide (H2O2). When provided on a multicopy plasmid, the P. aeruginosa katA gene complemented a catalase-deficient strain of Escherichia coli. The katA gene was found to contain two translational start codons encoding a heteromultimer of approximately 160 to 170 kDa and having an apparent Km for H2O2 of 44.7 mM. Isogenic katA and bfrA mutants were hypersusceptible to H2O2, while a katA bfrA double mutant demonstrated the greatest sensitivity. The katA and katA bfrA mutants possessed no detectable catalase activity. Interestingly, a bfrA mutant expressed only approximately 47% the KatA activity of wild-type organisms, despite possessing wild-type katA transcription and translation. Plasmids harboring bfrA genes encoding BfrA altered at critical amino acids essential for ferroxidase activity could not restore wild-type catalase activity in the bfrA mutant. RNase protection assays revealed that katA and bfrA are on different transcripts, the levels of which are increased by both iron and H2O2. Mass spectrometry analysis of whole cells revealed no significant difference in total cellular iron levels in the bfrA, katA, and katA bfrA mutants relative to wild-type bacteria. Our results suggest that P. aeruginosa BfrA may be required as one source of iron for the heme prosthetic group of KatA and thus for protection against H2O2.