Chimeras of Escherichia coli and Mycobacterium tuberculosis Single-Stranded DNA Binding Proteins: Characterization and Function in Escherichia coli

Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (mβ1, mβ1′β2, mβ1–β5, mβ1–β6 and mβ4–β5) by transplanting β1, β1′β2, β1–β5, β1–β6 and β4–β5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, mβ1′β2ESWR SSB was generated by mutating the MtuSSB specific ‘PRIY’ sequence in the β2 strand of mβ1′β2 SSB to EcoSSB specific ‘ESWR’ sequence. Biochemical characterization revealed that except for mβ1 SSB, all chimeras and a control construct lacking the C-terminal domain (ΔC SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, mβ1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that mβ1–β6, MtuSSB, mβ1′β2 and mβ1–β5 SSBs complemented E. coli Δssb in a dose dependent manner. Complementation by the mβ1–β5 SSB was poor. In contrast, mβ1′β2ESWR SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function

Simultaneous presence of fhs and purT genes is disadvantageous for the fitness of Escherichia coli growth

In bacteria, alternate mechanisms are known to synthesize N-10-formyltetrahydrofolate (N10-formyl-THF) and formyl glycinamide ribotide (fGAR), which are important in purine biosynthesis. In one of the mechanisms, a direct transfer of one carbon unit from formate allows Fhs to convert tetrahydrofolate to N-10-formyl-THF, and PurT to convert glycinamide ribotide (GAR) to fGAR. Our bioinformatics analysis of fhs and purT genes (encoding Fhs and PurT) showed that in a majority of bacteria (similar to 94%), their presence was mutually exclusive. A large number of organisms possessing fhs lacked purT and vice versa. The phenomenon is so penetrating that even within a genus (Bacillus) if a species possessed fhs it lacked purT and vice versa. To investigate physiological importance of this phenomenon, we used Escherichia coli, which naturally lacks fhs (and possesses purT) as model. We generated strains, which possessed fhs and purT genes in singles or together. Deletion of purT from E. coli in the presence or absence of fhs did not confer a detectable growth disadvantage in pure cultures. However, growth competition assays revealed that the strains possessing either of the single genes outcompeted those possessing both the genes suggesting that mutual exclusion of purT and fhs in organisms confers fitness advantage in mixed cultures

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

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

Microscopic observations of <i>E. coli Δssb</i>::<i>kan</i> supported by various SSB constructs.

<p>Cultures of <i>E. coli</i> RDP317 (Δ<i>ssb::kan</i>) transformants harboring various SSB constructs were grown in the presence of indicated concentrations of arabinose and analyzed by phase contrast microscopy. Bars at the lower left of each panel indicate a scale of 2 μm.</p

Asymmetric catalysis : ligand design and microwave acceleration

This thesis deals partly with the design and synthesis ofligands for use in asymmetric catalysis, and partly with theapplication of microwave heating on metal-based asymmetriccatalytic reactions. Enantiomerically pure pyridyl alcohols and bipyridylalcohols were synthesized from the chiral pool for future usein asymmetric catalysis. Lithiated pyridines were reacted withseveral chiral electrophiles, yielding diastereomeric mixturesthat could be separated without the use of resolutiontechniques. New pyridino- and quinolinooxazolines were synthesized andtested in palladium-catalyzed asymmetric allylation using1,3-diphenyl-2-propenyl acetate and dimethyl malonate. Theconformational preferences of the ligands in palladiumcomplexes were studied with crystallography, 2D-NMR techniquesand DFT calculations. Conclusions about how the chirality wastransferred from the ligand to the substrate could be drawnfrom the conformational analysis. The effect of heating Pd- and Mo-catalyzed asymmetricallylic substitution reactions was investigated with oil bathheating and microwave irradiation. With a few exceptions,ligands with high room temperature selectivity were shown toretain their selectivity on heating. Reaction rates, catalyststability and product selectivities of microwave-heatedreactions were compared with those of reactions performed inoil bath. Palladium-catalyzed asymmetric allylation was studied withseveral ligand types, allylic substrates and nucleophiles. Someof the experimental procedures had to be adapted to microwaveheating conditions. The procedure for asymmetric allylation catalyzed bybispyridylamide molybdenum complexes was developed into aone-pot microwave-mediated reaction. With microwaves, Mo(CO)6could be used as an easily-handled metal sourceand inert conditions could be omitted. Derivatives of thebispyridylamide ligandswere synthesized and tested withmolybdenum as catalysts to investigate the effects ofsubstituents on the pyridine ring. Keywords: ligand, asymmetric catalysis, pyridylalcohols, oxazolines, conformational study, Pd-allyl, fastchemistry, microwave chemistry, Mo-allyl, bispyridylamides.NR 2014080

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

Electrophoretic mobility shift assays using ³²P labeled 79mer ssDNA.

<p>DNA oligomer (1 pmol) was mixed with 0.2 pmol, 2 pmol or 10 pmol SSB tetramer (as indicated) for 30 min and analyzed on native PAGE (8%). DNA binding resulted in ‘Complex I’ at lower protein concentrations and ‘Complex II’ at higher protein concentrations.</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