Comparison of N-terminal affinity fusion domains: effect on expression level and product heterogeneity of recombinant restriction endonuclease EcoRV

The influence of different N-terminal affinity fusion domains on the product heterogeneity of recombinant proteins expressed in Escherichia coli was investigated. N-Terminal extended forms of the restriction endonuclease EcoRV with either glutathione-S-transferase [GST], histidine hexapeptide [(His)₆], or a combination of GST and (His)₆ [GST-(His)₆] were compared to native EcoRV with respect to expression level, susceptability to inclusion body formation and protein fragmentation. Fingerprinting of product heterogeneity was done by using two-dimensional (2-D) non equilibrium pH-gradient electrophoresis with subsequent immunoblotting. Fusion proteins containing GST were poorly expressed compared to native EcoRV. In addition, GST fusion proteins were highly susceptible to invivo aggregation and fragmentation and displayed more heterogeneity on 2-D immunoblots. However, the sole presence of oligohistidine at the N-terminus of EcoRV proved to be advantageous. Fragmentation of (His)₆-EcoRV was not observed and 2-D immunoblots did not show heterogenous forms of the recombinant protein. In addition, fusion of the histidine-hexapeptide to the N-terminus of native EcoRV increased the expression level of the recombinant protein twofold compared to native EcoRV. Inclusion body formation of the (His)₆-EcoRV fusion protein was intensive when cells were grown at 37°C but not at 30°C. The advantage of oligohistidine fusion to EcoRV was finally demonstrated by purifying soluble (His)₆-EcoRV in a single-step procedure from crude cell lysates using immobilized metal chelate affinity chromatography.     

Influence of temperature on the production of an archaeal thermoactive alcohol dehydrogenase from Pyrococcus furiosus with recombinant Escherichia coli

The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD₆₀₀ of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28°C, heat shock of the culture from 37 to 42°C and from 37 to 45°C, and variation of time of induction (induction at an OD₆₀₀ of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42°C, and IPTG induction of the adhC expression at an OD₆₀₀ of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular.     

Hypothermia protects H9c2 cardiomyocytes from H2O2 induced apoptosis

The purpose of our study was to investigate underlying basic mechanisms of hypothermia-induced cardioprotection during oxidative stress in a cardiomyocyte cell culture model. For hypothermic treatment we cooled H9c2 cardiomyocytes to 20 °C, maintained 20 min at 20 °C during which short-term oxidative damage was inflicted with 2 mM H₂O_2, followed by rewarming to 37 °C. Later on, we analyzed lactate dehydrogenase (LDH), caspase-3 cleavage, reactive oxygen species (ROS), mitochondrial activity, intracellular ATP production, cytoprotective signal molecules as well as DNA damage. Hypothermia decreased H₂O₂ damage in cardiomyocytes as demonstrated in a lower LDH release, less caspase-3 cleavage and less M30 CytoDeath staining. After rewarming H₂O₂ damaged cells demonstrated a significantly higher reduction rate of intracellular ROS compared to normothermic H₂O₂ damaged cardiomyocytes_. This was in line with a significantly greater mitochondrial dehydrogenase activity and higher intracellular ATP content in cooled and rewarmed cells. Moreover, hypothermia preserved cell viability by up-regulation of the anti-apoptotic protein Bcl-2 and a reduction of p53 phosphorylation. DNA damage, proven by PARP-1 cleavage and H2AX phosphorylation, was significantly reduced by hypothermia. In conclusion, we could demonstrate that hypothermia protects cardiomyocytes during oxidative stress by preventing apoptosis via inhibiting mitochondrial dysfunction and DNA damage.     

Identification of new peptides synthesized under the hydrogenase control system of Alcaligenes eutrophus

Total protein of Alcaligenes eutrophus was analyzed by two-dimensional protein map. Cells grown at 30° C expressed hydrogen-oxidizing (Hox) ability mediated by a soluble (Hos) and a particulate hydrogenase (Hop). Hox ability was not expressed at 37° C (HoxTs). The six subunits of the two hydrogenases were identified. Besides these six subunits eight peptides were not or hardly detected at 37° C. The mutant HF117 which expressed Hox ability at 37° C (HoxTr), formed the hydrogenase peptides and five of the eight peptides. These peptides designated B, C, E, F, and H were characterized by their isoelectric point and molecular mass (M _r); their M _r were 18 800, 45 400, 41 900, 39 400, and 40 600, respectively. The five peptides were not formed in regulatory Hox^– mutants, and not formed in mutants cured of plasmid pHG1, carrying the genetic information for hydrogenase formation. Strain HF160, carrying a Tn5 insertion in a gene essential for Hos expression specifically did not form the B-peptide. All peptides were found in the soluble fraction of cell extracts, the F-peptide was also detected in the particulate fraction. The function of the new Hox-peptides is presently unknown.Abbreviations PAGE polyacrylamide gelelectrophoresis - SDS sodium dodecylsulfate - Hox hydrogen oxidizing ability     

Two classes of isocitrate lyase genes are expressed during late embryogeny and postgermination in Brassica napus L.

Deletion of the SLT2 gene of Saccharomyces cerevisiae, which codes for a homologue of MAP (mitogen-activated) protein kinases, causes an autolytic lethal phenotype in cells grown at 37° C. The gene encodes domains characteristic of protein kinases, which include a lysine (at position 54) that lies 19 residues from a glycine-rich cluster, considered to be the putative ATP binding site. The ability of three mutant alleles of SLT2 generated by site-directed mutagenesis, namely E54 (glutamic acid), R54 (arginine) and F54 (phenylalanine), to complement slt2 mutants was tested. All three failed to complement the autolytic phenotype and were unable to restore growth and viability of cells. A strain obtained by transplacement of slt2-F54 also behaved as a thermosensitive autolytic mutant. By immunoprecipitation with polyclonal antibodies raised against Slt2 protein expressed in Escherichia coli, it was possible to confirm that alteration of the lysine-54 residue did not affect the stability of the protein, thus allowing us to conclude that activity of the Slt2 protein kinase is critically required for growth and morphogenesis of S. cerevisiae at 37° C. A significant fraction of the mutant cell population lysed at 24° C and the cells displayed a characteristic alteration of the surface consisting of a typical depression in an area of the cell wall. At 37° C, the cell surface was clearly disorganized.     

Expression of mutations and protein release by yeast conditional autolytic mutants in batch and continuous cultures

Thermosensitive mutant strains of Saccharomyces cerevisiae that fail to generate an osmotically stable cell wall when grown at a non-permissive temperature release their cell contents upon expression of the mutation. Therefore, they may represent an alternative for the production of homologous or heterologous protein preparations. In order to analyse the expression of two of these mutations, lyt2 and slt2, we grew the corresponding strains under precisely defined conditions in batch and continuous fermentors. A switch in the temperature of batch cultures from 24° C to 37° C determined lysis of the cells with a significant release of intracellular enzymes. These include alkaline phosphatase and periplasmic proteins such as glucan-degrading enzymes, the pattern of cell lysis and protein release being maintained for about 6 h. One-stage continuous cultures of a lyt2 mutant were maintained for long periods at 37° C; a fraction of the population lysed and released the indicated proteins, but eventually a revertant of the lytic phenotype was selected. To avoid this, a two-stage continuous culture system was developed by connecting two fermentors in series, the effluent from the first one at 24°C being fed to the second one adjusted to 37° C. A steady state of cell lysis and protein liberation was reached in the second-stage fermentor without any evidence of selection of revertants. This system can be very useful for developing conditions for the use of yeast strains to produce protein preparations. Correspondence to: C. Nombela     

Relationship Between Expression of Cell Surface Hydrophobicity Protein 1 (CSH1p) and Surface Hydrophobicity Properties of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>

Candida dubliniensis and Candida albicans cause most of the oral candidiasis infections in AIDS patients. Unlike C. albicans, which variably expresses cell surface hydrophobicity (CSH) depending on environmental conditions, C. dubliniensis is hydrophobic under all environmental conditions. C. dubliniensis produces CdCSH1p, a protein related to CaCSH1p that contributes to CSH expression of C. albicans. We investigated whether environmental conditions affect CdCSH1p expression, CSH avidity, and adhesion to fibronectin (Fn). C. dubliniensis CD36 was grown at 23°C and 37°C in four different media. CdCSH1p expression was affected by growth temperature, with cells grown at 37°C expressing the protein, but cells grown at 23°C did not. Hydrophobic avidity for two media was higher in cells grown at 37°C than at 23°C. Cells grown at 23°C were generally less adherent than 37°C-grown cells to Fn. The results suggest CdCSH1p but not hydrophobic avidity may have a role in adhesion of C. dubliniensis to Fn.     

Pellet forming and fragmentation in liquid culture of Streptomyces griseus

Pellets of Streptomyces griseus formed during submerged liquid culture were effectively dispersed in Tris/HCl buffer at 37 °C. However, inclusion of Tris/HCl in the culture medium did not lead to dispersed growth. In order to decrease the pellet formation in batch culture, a large inoculum of spores was required and dispersed growth was further favoured by culture at 37 °C. This method was also used to achieve dispersed culture of S. coelicolor A3(2). Autolytic enzyme activity was presumed to be responsible for fragmentation of pellets.     

Thermal instability of red blood cell membrane bilayers: Temperature dependence of hemolysis

Summary Rates of human red blood cell hemolysis were measured as a function of temperature. Three distinct temperature intervals for hemolysis were noted: a) At temperatures equal to or less than 37°C no hemolysis was observed for the duration of the incubation (30 hr). b) For temperatures exceeding 45°C hemolysis rates are rapid and are accompanied by gross changes in cellular morphology. The activation energy for hemolysis is 80 kcal/mole; this value is characteristic of protein denaturation and enzyme inactivation suggesting that these processes contribute to hemolysis at these high temperatures. c) Between 38 and 45°C the energy of activation is 29 kcal/mole, indicating that a fundamentally different process than protein inactivation is responsible for hemolysis at these relatively low temperatures. A mechanism based on the concept of the critical bilayer assembly temperature of cell membranes (N.L. Gershfeld,Biophys. J. 50:457–461, 1986) accounts for hemolysis at these relatively mild temperatures: The unilamellar state of the membrane is stable at 37°C, but is transformed to a multibilayer when the temperature is raised; hemolysis results because formation of the multibilayer requires exposing lipid-free areas of the erythrocyte surface. An analysis of the activation energy for hemolysis is presented that is consistent with the proposed unilamellar-multibilayer transformation.     

High-level expression of a soluble snake venom enzyme,gloshedobin, in E. coli in the presence of metal ions

The mature gene of gloshedobin, a snake venom thrombin-like enzyme from the snake, Gloydius shedaoensis, was cloned and expressed in strain E. coli BL21(DE3). Having been induced by IPTG, the recombinant gloshedobin was in both soluble and insoluble forms. To avoid inclusion body formation, expression was optimized at 25 °C. Furthermore, a 50% increase in solubilization of the target protein was obtained by adding 0.1 mM Mg²⁺ to the medium. The purified recombinant gloshedobin gave a 44 kDa band on SDS-PAGE gel.     

Molecular cloning and expression of the pyranose 2-oxidase cDNA from<Emphasis Type="Italic"> Trametes ochracea</Emphasis> MB49 in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

A cDNA of a structural gene encoding pyranose 2-oxidase (P2O) from Trametes ochracea strain MB49 was cloned into Escherichia coli strain BL21(DE3) on a multicopy plasmid under the control of the trc promoter. Synthesis of P2O was studied in batch cultures in LB or M9-based mineral medium at 28°C. While there was a low specific activity of P2O in LB medium, the enzyme was synthesised constitutively in mineral medium and represented 3% of the cell soluble protein (0.3 U mg^–1). The effect of isopropyl -d-thiogalactoside on the expression of P2O was studied in mineral medium at 25 and 28°C. The synthesis of P2O at 28°C corresponded to 39% of the cell soluble protein but the major portion of P2O (93%) was in the form of non-active inclusion bodies (activity of P2O equalled 0.19 U mg^–1). At 25°C, the amount of P2O represented 14% of the cell soluble protein and the activity of P2O was 1.1 U mg^–1. The soluble enzyme represented 70% of the total amount of P2O.     

Chemotactic behavior of Campylobacter spp. in function of different temperatures (37 °C and 42 °C)

The chemotactic behaviour of Campylobacter strains was determined in the presence of different amino acids at two temperatures (37 °C and 42 °C). Two strains of catalase positive (Campylobacter jejuni) and negative (Campylobacter sputurum) Campylobacter were isolated from river water in Tonekabon, Iran and identified by phenotyping and 16srRNA Gene sequencing methods. Chemotactic responses of the isolates were assessed toward a variety of amino acids viz., L-cystine, L-asparagine, L-histidine, L-aspartic acid, L-serine, L-phenylalanine, L-leucine and L-tryptophan by disc and capillary methods at two temperatures: 37 °C and 42 °C. C. jejuni showed positive chemotactic response towards L-cystine,L-tryptophan, L-phenylalanine, - L-leucine, L-asparagine and L-Serine at both, 37 °C and 42 °C however, it was greater at 37 °C. C. sputurum showed negative or weak response towards all of the amino acids. In addition, C. jejuni illustrated strong chemotactic response to L-asparagine follow by L-serine and weak chemotaxis response to L-phenylalanine and L-cysteine at 37 °C. Overall, C. jejuni showed relatively strong chemotactic response to some amino acids, likewise it was greater at 37 °C. Hence, the human body temperature (37 °C) in compared to avian body temperature (42 °C) probably promotes chemotactic response of C. jejuni, which it might be a reason for causing disease in human being compared to avian.     

Optimized production of the soluble human interferon a receptor (IFNAR) expressed in E. coli

An optimized procedure has been developed for production andpurification of the human interferon a receptor (IFNAR) expressed inE. coli as fusion protein with glutathione S-transferase (GST).Expression induced at 30° cell disruption performed at pH = 9.0in presence of detergents reduced the inclusion body formation andgenerated up to 20% of the fusion protein in the soluble form. Therecombinant IFNAR, recovered in soluble andrenaturated forms, wasable to block the antiviral and antiproliferative activities of IFNaB.     

Mannan structural complexity is decreased when Candida albicans is cultivated in blood or serum at physiological temperature

The Candida albicans cell wall provides an architecture that allows for the organism to survive environmental stress as well as interaction with host tissues. Previous work has focused on growing C. albicans on media such as Sabouraud or YPD at 30 °C. Because C. albicans normally colonizes a host, we hypothesized that cultivation on blood or serum at 37 °C would result in structural changes in cell wall mannan. C. albicans SC5314 was inoculated onto YPD, 5% blood, or 5% serum agar media three successive times at 30 °C and 37 °C, then cultivated overnight at 30 °C in YPD. The mannan was extracted and characterized using 1D and 2D ¹H NMR techniques. At 30 °C cells grown in blood and serum contain less acid-stable terminal β-(1→2)-linked d-mannose and α-(1→2)-linked d-mannose-containing side chains, while the acid-labile side chains of mannan grown in blood and serum contain fewer β-Man-(1→2)-α-Man-(1→ side chains. The decrement in acid-stable mannan side chains is greater at 37 °C than at 30 °C. Cells grown on blood at 37 °C show fewer →6)-α-Man-(1→ structural motifs in the acid-stable polymer backbone. The data indicate that C. albicans, grown on media containing host-derived components, produces less complex mannan. This is accentuated when the cells are cultured at 37 °C. This study demonstrates that the C. albicans cell wall is a dynamic and adaptive organelle, which alters its structural phenotype in response to growth in host-derived media at physiological temperature.     

Bacterial aspects associated with the expression of a single-chain antibody fragment in Escherichia coli

The bacterial expression of a single-chain antibody fragment, designated L6 sFv, was examined. Periplasmic targeting resulted in the production of a correctly folded protein that bound tumor antigen. However, immediately after induction at either 30°C or 37°C there was a significant loss in bacterial viability, which was followed by a loss in absorbance. The loss in absorbance correlated with cell lysis and release of the L6 sFv into the culture supernatant. The kinetics of appearance of L6 sFv in the supernatant paralleled that of periplasmic \-lactamase and confirmed an initial loss of cell-wall integrity prior to cell lysis. Bacteria incubated at 30°C produced approximately threefold more correctly folded antibody fragment because of an increase in the number of cells/A ₆₆₀ at the lower incubation temperature. More than 95% of the L6 sFv, made at either incubation temperature, was incorrectly folded. Osmotic-shock procedures did not release L6 sFv. However, in situ subtilisin susceptibility experiments with bacterial spheroplasts confirmed a periplasmic location. French press disruption resulted in the release of correctly but not incorrectly folded material. Membrane fractionation revealed that the incorrectly folded L6 sFv remained associated with both the inner and outer membrane. These results demonstrate that, in this system, antibody fragment expression resulted initially in cell death, which was followed by release of protein into the culture supernatant and eventually cell lysis. It is also suggested that membrane association in the periplasmic space may impede proper folding.     

Thermotolerant nalidixic acid-resistant mutants ofEscherichia coli

Nalidixic acid-resistant mutants ofEscherichia coli CGSC #6353 capable of growth at 48°C were obtained by mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine. Cotransductional analyses employing phage P1 indicated that the mutation resulting in the phenotype of growth at 48°C is an allele of thegyrA structural gene. Similar thermal inactivation kinetics were observed for ribosomes isolated from a thermotolerant (T/r) mutant grown at both 37°C and 48°C and from the parental strain grown at 37°C. Cell-free extracts prepared from the T/r mutant grown at 48°C exhibited a sharp increase in protein synthesis at 55°C, whereas this effect was not displayed by extracts from the mutant or parental strains grown at 37°C. In addition, preincubation at 55°C enhanced protein synthesis at 37°C up to 15-fold in an extract prepared from the T/r mutant grown at 48°C, whereas comparable values were 2.6- to 3.0-fold for extracts from the mutant and parental strains grown at 37°C.     

Inactivation of Candida albicans by ultraviolet radiation

Summary Inactivation of Candida albicans by ultraviolet (uv) light is markedly dependent upon (a) the cell division stage and (b) the nutrition and growth temperatures of cells both before and after irradiation. Cells grown at 37°C after irradiation show lower survivals than those grown at 25°C. At either recovery temperature, cells which had been cultured before irradiation at 37°C are able to sustain less uv damage prior to inactivation than those cultured at 25°C. The radiosensitivities of budding and non-budding cells are the same when survivals are scored at 25°C; at low uv dosages, cells show slightly poorer recoveries on enriched medium than on minimal medium whereas at higher dosages, their recoveries on both kinds of media are equivalent. In contrast, at 37°C, uv treated non-budding cells are much more susceptible to inactivation than budding cells; non-budding cells also express much poorer recovery on enriched medium than on minimal medium at 37°C whereas budding cells survive equally well on either medium. Though non-budding cells grown for irradiation on minimal or enriched media exhibit the same radiosensitivites, budding cells grown for irradiation on enriched medium are more susceptible to inactivation at 37°C than those grown on minimal medium.The particularly poor recovery by irradiated non-budding cells at 37°C is correlated with their unique tendency to undergo a transitory filamentation when initiating growth at that temperature. Evidence is presented that neither the filamentous growth per se nor the temporary inhibition of cell division associated with filamentation causes the poor recovery. Furthermore, while irradiated non-budding cells at 37°C exhibit singular susceptibility to inhibition of recovery by metabolic antagonists which disturb protein synthesis, the course of their filamentous growth is not affected by such agents. It is concluded that recovery from irradiation and the instigation of cytokinesis by non-budding cells of C. albicans result from different metabolic processes which may be related through a common temperature sensitive step. C. albicans does not photoreactivate and observations on recovery by cells prevented from undergoing immediate postirradiation replication do not indicate the existence of a system for dark repair of DNA damage comparable to that occurring in bacteria. Difficulties attending a valid demonstration of DNA dark repair in yeasts are discussed.     

The effect of hypothermic treatment on the repair or expression of X-ray damage measured in split dose experiments on L5178Y-S cells

Summary The nature of the post-irradiation lesions and processes leading to cellular reproductive death or survival were investigated in mouse lymphoblastic leukemia L5178Y-S (LY-S) cells. Post-(x-)irradiation incubation at 25° C protects LY-S cells against the fixation of biologically expressed damage which takes place at 37° C. An optimal condition for the repair of damage, assayed in split-dose experiments as split-dose recovery (SDR), is 1 h at 37° C followed by 4 h holding at 25° C prior to the second half of a split dose, or 5 h holding at 25° C without a 37° C incubation during the interval between doses. Longer incubations at 37° C resulted in progressively decreased survivals. Postirradiation inhibition of DNA synthesis at 37° C was observed only during the first 30 min; thereafter,³H-dThdR incorporation washigher than in unirradiated controls. Theexcess synthesis effect was removed by shifting irradiated cells to 25° C holding. The inhibition observed at 25° C was reversed by shifting to 37° C. Thus the degree of postirradiation DNA synthesis is inversely related to SDR. DNA filter elution shows complete strand break repair by 20 min at 37° C, and by 3 h at 25° C; DNA double-strand break (DSB) repair plateaus at 80% (37° C) and 60% (25° C) after 90 min. An inverse correlation was found between total strand break repair rate, as assayed by filter elution methods, and cell survival. This work was supported by a grant from The Mathers Charitable Foundation.A preliminary report of this work was presented at the 35th Annual Meeting of the Radiation Research Society, Atlanta, GA 1987, USA     

Adaptive response to cold temperatures and characterization of cspA in Salmonella typhimurium LT2

Salmonella typhimurium is a major foodborne microbial pathogen which primarily contaminates poultry products causing salmonellosis in humans. S. typhimurium LT2 cultures, when transferred from 37 °C to 5 °C or 10 °C, showed an initial lag period in growth with an approximate generation time of 10–25 h. Western blot assay using E. coli CS7.4 antibody and analysis of radiolabeled total cellular proteins from S. typhimurium cultures after exposure to 10 °C or 5 °C showed elevated expression of a major cold shock protein, CS7.4. Identification of a decreased level of CS7.4 at 37 °C suggests that the expression of this protein may require a large temperature downshift. Putative regulatory protein binding segment on the 5-untranslated region referred as Fragment 7 in S. typhimurium exhibited a 90.6% and a 56.25% nucleotide sequence identity when compared with the Fragment 7 of E. coli and S. enteritidis, respectively. The differences in the nucleotide sequence within the Fragment 7 between S. typhimurium and S. enteritidis may explain the differential expression of CspA at 37 °C. The nucleotide sequence of the open reading frame of S. typhimurium cspA gene showed a single base difference at 816 bp position from a G to a C which altered the amino acid residue from a glycine to an alanine. In addition to CspA, an elevated expression of a 105 kDa, and decreased expression of 6 proteins were evidenced when cultures of S. typhimurium were exposed to 10 °C or 5 °C. Differential expression of the CspA and other proteins in S. typhimurium following exposure to cold temperatures suggest that adaptation and continued growth and survival at cold temperatures in this pathogen may be aided by these cold-responsive proteins.     

Inaugural structure from the DUF3349 superfamily of proteins, Mycobacterium tuberculosis Rv0543c

The first structure for a member of the DUF3349 (PF11829) family of proteins, Rv0543c from Mycobacterium tuberculosis, has been determined using NMR-based methods and some of its biophysical properties characterized. Rv0543c is a 100 residue, 11.3 kDa protein that both size exclusion chromatography and NMR spectroscopy show to be a monomer in solution. The structure of the protein consists of a bundle of five α-helices, α1 (M1 – Y16), α2 (P21 – C33), α3 (S37 – G52), α4 (G58 – H65) and α5 (S72 – G87), held together by a largely conserved group of hydrophobic amino acid side chains. Heteronuclear steady-state {¹H}–¹⁵N NOE, T₁, and T₂ values are similar through-out the sequence indicating that the backbones of the five helices are in a single motional regime. The thermal stability of Rv0543c, characterized by circular dichroism spectroscopy, indicates that Rv0543c irreversibly unfolds upon heating with an estimated melting temperature of 62.5 °C. While the biological function of Rv0543c is still unknown, the presence of DUF3349 proteins predominately in Mycobacterium and Rhodococcus bacterial species suggests that Rv0543 may have a biological function unique to these bacteria, and consequently, may prove to be an attractive drug target to combat tuberculosis.