Impact of Mutating the Key Residues of a Bifunctional 5,10-Methylenetetrahydrofolate Dehydrogenase-Cyclohydrolase from <i>Escherichia coli</i> on Its Activities

Methylenetetrahydrofolate dehydrogenase-cyclohydrolase (FolD) catalyzes interconversion of 5,10-methylene-tetrahydrofolate and 10-formyl-tetrahydrofolate in the one-carbon metabolic pathway. In some organisms, the essential requirement of 10-formyl-tetrahydrofolate may also be fulfilled by formyltetrahydrofolate synthetase (Fhs). Recently, we developed an <i>Escherichia coli</i> strain in which the <i>folD</i> gene was deleted in the presence of <i>Clostridium perfringens fhs</i> (<i>E. coli</i> Δ<i>folD</i>/p-<i>fhs</i>) and used it to purify FolD mutants (free from the host-encoded FolD) and determine their biological activities. Mutations in the key residues of <i>E. coli</i> FolD, as identified from three-dimensional structures (D121A, Q98K, K54S, Y50S, and R191E), and a genetic screen (G122D and C58Y) were generated, and the mutant proteins were purified to determine their kinetic constants. Except for the R191E and K54S mutants, others were highly compromised in terms of both dehydrogenase and cyclohydrolase activities. While the R191E mutant showed high cyclohydrolase activity, it retained only a residual dehydrogenase activity. On the other hand, the K54S mutant lacked the cyclohydrolase activity but possessed high dehydrogenase activity. The D121A and G122D (in a loop between two helices) mutants were highly compromised in terms of both dehydrogenase and cyclohydrolase activities. <i>In vivo</i> and <i>in vitro</i> characterization of wild-type and mutant (R191E, G122D, D121A, Q98K, C58Y, K54S, and Y50S) FolD together with three-dimensional modeling has allowed us to develop a better understanding of the mechanism for substrate binding and catalysis by <i>E. coli</i> FolD

A Genetic Analysis of the Functional Interactions within Mycobacterium tuberculosis Single-Stranded DNA Binding Protein

Single-stranded DNA binding proteins (SSBs) are vital in all organisms. SSBs of Escherichia coli (EcoSSB) and Mycobacterium tuberculosis (MtuSSB) are homotetrameric. The N-terminal domains (NTD) of these SSBs (responsible for their tetramerization and DNA binding) are structurally well defined. However, their C-terminal domains (CTD) possess undefined structures. EcoSSB NTD consists of beta 1-beta 1'-beta 2-beta 3-alpha-beta 4-beta 45(1)-beta 45(2)-beta 5 secondary structure elements. MtuSSB NTD includes an additional beta-strand (beta 6) forming a novel hook-like structure. Recently, we observed that MtuSSB complemented an E. coli Delta ssb strain. However, a chimeric SSB (m beta 4-beta 5), wherein only the terminal part of NTD (beta 4-beta 5 region possessing L-45 loop) of EcoSSB was substituted with that from MtuSSB, failed to function in E. coli in spite of its normal DNA binding and oligomerization properties. Here, we designed new chimeras by transplanting selected regions of MtuSSB into EcoSSB to understand the functional significance of the various secondary structure elements within SSB. All chimeric SSBs formed homotetramers and showed normal DNA binding. The m beta 4-beta 6 construct obtained by substitution of the region downstream of beta 5 in m beta 4-beta 5 SSB with the corresponding region (beta 6) of MtuSSB complemented the E. coli strain indicating a functional interaction between the L-45 loop and the beta 6 strand of MtuSSB

Physiological role of FolD (methylenetetrahydrofolate dehydrogenase), FchA (methenyltetrahydrofolate cyclohydrolase) and Fhs (formyltetrahydrofolate synthetase) from Clostridium perfringens in a heterologous model of Escherichia coli

Most organisms possess bifunctional FolD 5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] to generate NADPH and 10-formyltetrandrofolate (10-CHO-THF) required in various metabolic steps. In addition, some organisms including Clostridium perfringens possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF. Here, we show that unlike the bifunctional FolD of Escherichia coli (Eco FolD), and contrary to its annotated bifunctional nature, C. perfringens FolD (Cpe FoID) is a monofunctional 5,10-CH2-THF dehydrogenase. The dehydrogenase activity of Cpe FoID is about five times more efficient than that of Eco FolD. The 5,10-methenyltetrahydrofolate (5,10-CH+-THF) cyclohydrolase activity in C. perfringens is provided by another protein, FchA (5,10-CH+-THF cyclohydrolase), whose cyclohydrolase activity is similar to 10 times more efficient than that of Eco FolD. Kinetic parameters for Cpe Fhs were also determined for utilization of all of its substrates. Both Cpe FoID and Cpe FchA are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of Cpe FoID and Cpe FchA is also necessary to rescue an E coli folD deletion strain (harbouring Cpe Fhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD, FchA and Fhs) is required to maintain folate homeostasis in the cell

One-Carbon Metabolic Pathway Rewiring in Escherichia coli Reveals an Evolutionary Advantage of 10-Formyltetrahydrofolate Synthetase (Fhs) in Survival under Hypoxia

In cells, N-10-formyltetrahydrofolate (N-10-fTHF) is required for formylation of eubacterial/organellar initiator tRNA and purine nucleotide biosynthesis. Biosynthesis of N-10-fTHF is catalyzed by 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) and/or 10-formyltetrahydrofolate synthetase (Fhs). All eubacteria possess FolD, but some possess both FolD and Fhs. However, the reasons for possessing Fhs in addition to FolD have remained unclear. We used Escherichia coli, which naturally lacks fhs, as our model. We show that in E. coli, the essential function of folD could be replaced by Clostridium perfringens fhs when it was provided on a medium-copy-number plasmid or integrated as a single-copy gene in the chromosome. The fhs-supported folD deletion (Delta folD) strains grow well in a complex medium. However, these strains require purines and glycine as supplements for growth in M9 minimal medium. The in vivo levels of N-10-fTHF in the Delta folD strain (supported by plasmid-borne fhs) were limiting despite the high capacity of the available Fhs to synthesize N-10-fTHF in vitro. Auxotrophy for purines could be alleviated by supplementing formate to the medium, and that for glycine was alleviated by engineering THF import into the cells. The Delta folD strain (harboring fhs on the chromosome) showed a high NADP(+)-to-NADPH ratio and hypersensitivity to trimethoprim. The presence of fhs in E. coli was disadvantageous for its aerobic growth. However, under hypoxia, E. coli strains harboring fhs outcompeted those lacking it. The computational analysis revealed a predominant natural occurrence of fhs in anaerobic and facultative anaerobic bacteria

Species-Specific Interactions of Arr with RpIK Mediate Stringent Response in Bacteria

D Bacteria respond to stressful growth conditions through a conserved phenomenon of stringent response mediated by synthesis of stress alarmones ppGpp and pppGpp referred to as (p)ppGpp]. (p)ppGpp synthesis is known to occur by ribosome-associated RelA. In addition, a dual-function protein, SpoT (with both synthetase and hydrolase activities), maintains (p)ppGpp homeostasis. The presence of (p)ppGpp is also known to contribute to antibiotic resistance in bacteria. Mycobacterium smegmatis possesses Arr, which inactivates rifampin by its ADP ribosylation. Arr has been shown to be upregulated in response to stress. However, the roles Arr might play during growth have remained unclear. We show that Arr confers growth fitness advantage to M. smegmatis even in the absence of rifampin. Arr deficiency in M. smegmatis resulted in deficiency of biofilm formation. Further, we show that while Arr does not interact with the wild-type Escherichia coli ribosomes, it interacts with them when the E. coli ribosomal protein L11 (a stringent response regulator) is replaced with its homolog from M. smegmatis. The Arr interaction with E. coli ribosomes occurs even when the N-terminal 33 amino acids of its L11 protein were replaced with the corresponding sequence of M. smegmatis L11 (Msm-EcoL11 chimeric protein). Interestingly, Arr interaction with the E. coli ribosomes harboring M. smegmatis L11 or Msm-EcoL11 results in the synthesis of ppGpp in vivo. Our study shows a novel role of antibiotic resistance gene arr in stress response. IMPORTANCE Mycobacterium smegmatis, like many other bacteria, possesses an ADP-ribosyltransferase, Arr, which confers resistance to the first-line antituberculosis drug, rifampin, by its ADP ribosylation. In this report, we show that in addition to its known property of conferring resistance to rifampin, Arr confers growth fitness advantage to M. smegmatis even when there is no rifampin in the growth medium. We then show that Arr establishes species-specific interactions with ribosomes through the N-terminal sequence of ribosomal protein L11 (a stringent response regulator) and results in ppGpp (stress alarmone) synthesis. Deficiency of Arr in M. smegmatis results in deficiency of biofilm formation. Arr protein is physiologically important both in conferring antibiotic resistance as well as in mediating stringent response

Comparison of <i>Eco</i>SSB and <i>Mtu</i>SSB.

<p>(A) <i>Eco</i>SSB and <i>Mtu</i>SSB sequences were aligned with ClustalW program. Identical amino acid residues (*), very similar amino acid residues (:) and similar amino acid residues (.) are indicated. Secondary structural elements (α helix and β strands) are shown as per <i>Eco</i>SSB nomenclature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094669#pone.0094669-Raghunathan1" target="_blank">[17]</a>. Acidic amino acids in <i>Mtu</i>SSB L₄₅ region are highlighted in ‘red’. (B) DNA binding domains of <i>Eco</i>SSB and <i>Mtu</i>SSB tertiary structures were superposed using Pymole. Various secondary structural elements mentioned in Fig. 1A are indicated. L₄₅ loop in both the SSBs (connecting β45₁ and β45₂) are also indicated.</p

List of strains, plasmids and DNA oligomers.

<p>List of strains, plasmids and DNA oligomers.</p

Complementation assays with various SSB constructs.

<p>(A) <i>E. coli</i> TG1 strains harboring pBAD constructs of SSBs (as shown) were grown to mid log phase in 2–3 ml cultures. Aliquots (1 ml) were either not supplemented (−) or supplemented (+) with 0.02% arabinose, and grown further for 3 h. Cells were harvested and processed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094669#pone.0094669-Bharti1" target="_blank">[22]</a>. Cell-free extracts (∼10 μg total protein) were resolved on SDS-PAGE (15%). (B) Transformants of <i>E. coli</i> RDP 317 harboring chimeric SSBs obtained in the presence of IPTG were suspended in LB and streaked on LB-agar (Kan, Amp) containing IPTG or arabinose (0.002–0.2%) and incubated at 37°C for ∼12 h. Sectors: 1, pBAD vector; 2, pBAD<i>Eco</i>SSB; 3, pBAD<i>Mtu</i>SSB; 4, pBADmβ4-β5; 5, pBADmβ4-β5(acidic); 6, pBADmβ4-β6; 7, pBADmβ1-α; 8, pBADmβ6; 9, pBADmβ6-CTD;10, pBADmCTD.</p

^*Plating efficiencies of various SSBs.

<p>*Plating efficiencies were determined by taking ratios of number of transformants obtained with various SSB constructs in <i>E. coli</i> RDP317-1/pHYD<i>Eco</i>SSB by plating equal volumes from the same transformation mixes on Kan, Amp and 0.02% arabinose plates <i>vs</i> Kan, Amp and IPTG plates. The values have been tabulated from five independent experiments (with three replicates each). Averages with S.D. values are shown.</p