Expression of theunc genes inEscherichia coli

Theunc (or atp) operon ofEscherichia coli comprises eight genes encoding the known subunits of the proton-translocating ATP synthase (H⁺-ATPase) plus a ninth gene (uncI) of unknown function. The subunit stoichiometry of the H⁺-ATPase ( ₃₃₁₁₁a₁b₂c_10–15) requires that the respectiveunc genes be expressed at different rates. This review discusses the experimental methods applied to determining how differential synthesis is achieved, and evaluates the results obtained. It has been found that the primary level of control is translational initiation. The translational efficiencies of theunc genes are determined by primary and secondary mRNA structures within their respective translational initiation regions. The respective rates of translation are matched to the subunit requirements of H⁺-ATPase assembly. Finally, points of uncertainty remain and experimental strategies which will be important in future work are discussed.     

Expression of a human proenkephalin a cDNA inEscherichia coli

A 1000 base pair cDNA coding for the entire human proenkephalin A(proA) polypeptide was subcloned into the multifunctional pMPV 2911/ME. coli vector. The recombinant plasmid was found to express an approximately 30 kDa prohormone, which was recognized by a Met-Arg⁶-Phe² antibody, directed against the C-terminal part of the enkephalin A prohormone. The expression of human proenkephalin A cDNA should thus permit the rapid purification of unfused recombinant enkephalin A prohormone, which itself may provide a model substrat to identify endoproteolytic processing activities.     

Antigenic relationship between reactivity to hepatitis B e antigen and 19 kDa protein ofMycobacterium tuberculosis among the Tibetan settlers in Karnataka

McGlynn and her co-workers have reported that among the Vietnamese refugees in Philadelphia and among Alaskan natives who are hepatitis B carriers, there is a statistically significant association between anegative tuberculin test and the presence of hepatitis B e antigen. A repetition of this work among the population of Bangalore did not yield any significant results because of the very low incidence of hepatitis found among this population. However, on the basis of available data that hepatitis B infection is more prevalent among the Mongolian population than among people of other populations, the work was repeated among Tibetans who had settled down in Karnataka. This set of experiments showed that, contrary to the report of McGlynnet al, there is a statistically significant association between apositive tuberculin test and the presence of hepatitis B e antigen and that those individuals who showed the presence of hepatitis B e antigen exhibited less severe form of the disease than those who were negative to this antigen. These findings suggested that immunity to tuberculosis and hepatitis B infections may have a common underlying principle. Data bank search revealed a stretch of amino acid sequences which is common to hepatitis B e antigen and 19 kDa antigen ofMycobacterium tuberculosis. The significance of these results is discussed.     

Immunodiagnosis of pulmonary tuberculosis by concomitant detection of antigen and antibodies of excretory-secretory protein ofMycobacterium tuberculosis H37Ra

With a view to diagnosing tuberculosis in populations in endemic areas, excretory-secretory antigen fraction(Mtb EST-6) of purifiedMycobacterium tuberculosis H₃₇Ra and affinity purified polyclonal antibodies againstMtb EST were used to detect both antibodies and circulating antigen in the sera of patients and disease-free individuals. Indirect stick penicillinase ELISA system usingMtb EST-6 detected antigen-specific IgG antibody in 84% of sputum positive, 77% of sputum negative pulmonary tuberculosis patients and 7% of healthy and 11% of subjects with nontub~rculosis diseases. Similarly, a sandwich penicillinase ELISA system using affinity purified antiMtb EST antibodies detected circulating antigen in 83% and 61% of sputum positive and negative pulmonary tuberculosis subjects. In contrast only 24% of healthy and 18% of disease controls showed seropositivity. Antibody assay showed higher sensitivity and specificity (83% and 91% respectively) compared to antigen detection (sensitivity of 79% and specificity of 79%). However, by concomitant use of both assays it was possible to enhance the specificity of detection to 98%, though sensitivity was reduced marginally to 70%. The present study confirms the presence of both antigen and specific antibodies in the circulation during clinical disease and draws attention to the utility ofMtb EST-6 as a diagnostic marker of pulmonary tuberculosis.     

Deoxyribonucleic acid replication time in Mycobacterium tuberculosis H37 Rv

The DNA increment method, designed for measuring the increment in the amount of DNA after inhibition of initiation of fresh rounds of replication initiation was employed to measure the rate of deoxyribonucleic acid (DNA) chain growth in Mycobacterium tuberculosis H37Rv growing in Youman and Karlson's medium at 37°C with a generation time of 24 h and also in relatively fast growing species like Mycobacterium smegmatis and Escherichia coli. From the results obtained, the time required for a DNA replication fork to traverse the chromosome from origin to terminus (C period) was calculated. The chain elongation rates of DNA of the three organisms was determined from the C period and the known genome sizes assuming that all these genomes have a single replication origin and bidirectional replication fork. The rate for M. tuberculosis was 3,200 nucleotides per min about 11 times slower than that of M. smegmatis and about 13–18 times slower than that of E. coli.Abbreviations DNA deoxyribonucleic acid - td delay in initiation - OD optical density - CAM chloramphenicol - RIF rifampicin     

Expression of genes from the marine bacteriumAlteromonas haloplanktis 214 inEscherichia coli K-12

DNA from the marine bacteriumAlteromonas haloplanktis 214 was partially digested withSau 3A and inserted into theBam HI site of the cloning vector pBR322. The ligation mixture was used to transformEscherichia coli HB101. The gene bank plasmid preparation obtained was used to transformEscherichia coli K-12 strain EO2717, an organism auxotrophic for histidine, arginine, adenine, uracil and thiamin. Prototrophic transformants for each of the five metabolites were isolated using appropriate minimal media for selection. Plasmids isolated from each of the transformants were shown by hybridization to containA. haloplanktis DNA and to be capable of complementing the appropriate mutation inE. coli EO2717. Restriction maps showed that each of the plasmids was different.     

N-[2-Naphthyl]-glycine hydrazide,a potent inhibitor of DNA-dependent RNA polymerase ofMycobacterium tuberculosis H37RV

N-[2-Naphthyl]-glycine hydrazide has been shown for the first time as a potent inhibitor of the DNA-dependent RNA polymerase (EC 2.7.7.6) ofMycobacterium tuberculosis H₃₇R_v. At a concentration of 10^-9 M, the compound shows maximum inhibition of the enzyme, the inhibition being less at higher concentrations. It is suggested that the novel type of inhibition pattern may be due to hydrophobic interactions occurring between the molecules of the compound at higher concentrations. The finding that there is a shift in the λ_max of the compound could also account for this phenomenon. The effect of this compound was also tested on DNA-dependent RNA polymerases from an eukaryotic fungus,Microsporum canis. At a concentration of 10⁻⁹ M it inhibits RNA polymerase II (32%) but not RNA polymerasesI andIII     

Expression and characterization of Rv2430c, a novel immunodominant antigen of Mycobacterium tuberculosis

About 10% of the coding sequence of Mycobacterium tuberculosis corresponds to hitherto unknown members of the PE and PPE protein families which display significant sequence and length variation at their C-terminal region. It has been suggested that this could possibly represent a rich source of antigenic variation within the pathogen. We describe the purification and biophysical characterization of the recombinant PPE protein coded by hypothetical ORF Rv2430c, a member of the PPE gene family that was earlier shown to induce a strong B cell response. Expression of the recombinant PPE protein in Escherichia coli led to its localization in inclusion bodies and subsequent refolding using dialysis after its extraction from the same resulted in extensive precipitation. Therefore, an on-column refolding strategy was used, after which the protein was found to be in the soluble form. CD spectrum of the recombinant protein displayed predominantly alpha helical content (81%) which matched significantly with in silico and web-based secondary structure predictions. Furthermore, fluorescence emission spectra revealed that aromatic amino acids are buried inside the protein, which are exposed to aqueous environment under 8M urea. These results, for the first time, provide evidence on the structural features of PPE family protein which, viewed with its reported immunodominant characteristics, have implications for other proteins of the PE/PPE family.     

Expression of cGMP-dependent protein kinase inEscherichia coli

Cyclic GMP-dependent protein kinase (cGMP kinase) is involved in the relaxation of smooth muscle. The enzyme has been cloned and expressed in eukaryotic cell lines but so far not in prokaryotic cells. Three vectors were constructed for the expression of I cGMP kinase inEscherichia coli. Transformation with the pET3a/cgk vector which uses the T7 RNA polymerase/promotor system resulted in efficient accumulation of cGMP kinase. Most of the protein was in an insoluble and catalytic inactive form. Various solubilization and refolding conditions did not yield an active enzyme. A small fraction of the cGMP kinase was present in the soluble cell extract. This fraction bound cGMP with high affinity but had no cGMP stimulated kinase activity. To prevent aggregation two additional vectors were constructed. (I) A bacterial leader sequence, which directs the export of proteins into the periplasmic space, was fused to the aminoterminus of the cGMP kinase. (II) A gram/gram⁺ shuttle vector for expression under the control of the tac promotor was used. Both constructs directed the synthesis of an isoluble and inactive cGMP kinase. These results suggest that large amounts of cGMP kinase can be expressed inE. coli, but mainly in an isoluble and inactive form. In contrast to eukaryotic cells, bacteria may lack systems for correct protein folding and/or posttranslational modification that are crucial for the productive folding and/or activation of cGMP kinase.     

Effect of oral exposure ofMycobacterium avium intracellular on the protective immunity induced by BCG

The relative protective efficacy of oral administration of mycobacteria as compared to the conventional intradermal route of vaccination has been assessed in guinea pigs. Skin test reactivity to partially purified protein derivative and protective immunity to challenge with virulentMycobacterium tuberculosis were used as parameters of protective immunity. Oral immunisation of guinea pigs either with BCG or withMycobacterium avium intracellulare induces skin test reactivity and protective immunity comparable to that induced by intradermal route of vaccination. Oral exposure ofMycobacterium avium intracellulare prior to oral or intradermal dose of BCG did not interfere with the protective immunity induced by BCG in guinea pigs challenged withMycobacterium tuberculosis H₃₇Rv.     

An approach for studying the mediators of pathogenesis inMycobacterium tuberculosis

Mycobacterium tuberculosis is an example of an intracellular pathogen that mediates the disease state through complex interactions with the host’s immune system. Not only does this organism replicate in the hostile environment prevailing within the infected macrophage, but it has also developed intricate mechanisms to inhibit several defence mechanisms of the host’s immune system. It is postulated here that the mediators of these interactions with the host are products of differentially expressed genes in the pathogen. B and T cell responses of the host are hence to be used as tools to identify such gene products from an expression library of theMycobacterium tuberculosis genome. The various pathways of generating a productive immune response that may be targeted by the pathogen are discussed     

Expression inEscherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation

Human alcohol dehydrogenase (ADH, tiff isozyme of class I) was expressed in Escherichia coli, purified to homogeneity, and characterized regarding N-terminal processing. The expression system was obtained by ligation of a cDNA fragment corresponding to the fl-subunit of human liver alcohol dehydrogenase into the vector pKK 223-3 containing the tac promoter. The enzyme, detected by Western-blot analysis and ethanol oxidizing activity, constituted up to 3 ~o of the total amount of protein. Recombinant ADH was separated from E. coli ADH by ion-exchange chromatography and the isolated enzyme was essentially pure as judged by SDS-polyacrylamide gel electrophoresis and sequence analysis. The N-terminal sequence was identical to that of the authentic fl-subunit except that the N-terminus was non-acetylated, indicating a correct removal of the initiator methionine, but lack of further processing.     

Characterization of pea chloroplastic carbonic anhydrase. Expression inEscherichia coli and site-directed mutagenesis

A cDNA encoding the mature, chloroplast-localized carbonic anhydrase in pea has been expressed inE. coli. The enzyme is fully active and yields of up to 20% of the total soluble protein can be obtained from the bacteria. This expression system was used to monitor the effects of site-directed mutagenesis of seven residues found within conserved regions in the pea carbonic anhydrase amino acid sequence. The effects of these modifications are discussed with respect to the potential of various amino acids to act as sites for zinc coordination or intramolecular proton shuttles.     

Expression of the naphthoate synthase gene in Mycobacterium tuberculosis in a self-generated oxygen depleted liquid culture system

Mycobacterium tuberculosis has been classified for decades as a strict aerobic species. Whole genome sequencing of the type culture strain H37Rv has revealed the presence of a full set of genes allowing for anaerobic metabolism. Naphthoate synthase (menB) is a key enzyme required for the synthesis of menaquinone, which plays a crucial role in anaerobic electron transport, ultimately resulting in the formation of energy generating intermediates. Interrupting the synthesis of this enzyme will interfere with the production of menaquinone and therefore this enzyme is a potential drug target. This study serves to investigate the role of naphtoate synthase in the survival of M. tuberculosis H37Rv when incubated under oxygen limiting conditions of unagitated liquid culture over 15 weeks. M.?tuberculosis H37Rv was grown in Middlebrook 7H9 media. The tubes were kept undisturbed at 37?°C for up to 15 weeks. At selected time points, aliquots of cells were removed and frozen. RNA was simultaneously extracted from all aliquots. The RNA was converted to cDNA for Real-Time PCR on the ABI 7000 SDS. Gene expression was normalized against 16S RNA quantities at each time point. A systematic increase in the expression of the menB gene product was observed over the incubation period with a 4.3-fold increase seen at week 6 (P?相似文献   

Analysis of DevR regulated genes in Mycobacterium tuberculosis

The DevRS two component system of Mycobacterium tuberculosis is responsible for its dormancy in host and becomes operative under hypoxic condition. It is experimentally known that phosphorylated DevR controls the expression of several downstream genes in a complex manner. In the present work we propose a theoretical model to show role of binding sites in DevR mediated gene expression. Individual and collective role of binding sites in regulating DevR mediated gene expression has been shown via modeling. Objective of the present work is twofold. First, to describe qualitatively the temporal dynamics of wild type genes and their known mutants. Based on these results we propose that DevR controlled gene expression follows a specific pattern which is efficient in describing other DevR mediated gene expression. Second, to analyze behavior of the system from information theoretical point of view. Using the tools of information theory we have calculated molecular efficiency of the system and have shown that it is close to the maximum limit of isothermal efficiency.     

Use of a short A/T-rich cassette for enhanced expression of cloned genes inEscherichia coli

A short (43-bp) A/T-rich stretch of DNA located in The intergenic region between thebaiA2 andbaiF genes fromEubacterium sp. strain VPI 12708 was amplified by polymerase chain reaction (PCR) and inserted in front of the Shine-Dalgarno (SD) sequences of three inefficiently-expressedEubacterium sp. strain VPI 12708 genes cloned inEschcrichia coli plasmids. Insertion of this A/T-rich cassette increased gene expression in all cases tested. Deletion of part of the A/T-rich region from abaiF clone in pUC19 resulted in decreased gene expression. Synthesis of specific mRNA was increased with addition of the A/T-rich cassette to constructs containing thebaiC gene from Eubacterium sp. strain VPI 12708, but mRNA synthesis was not significantly changed in cells containing plasmid constructs with thebaiF andbaiG genes. Enhanced translation resulting from a decrease in mRNA secondary structure in the ribosome binding site region is discussed as a possible reason for increased gene expression with the A/T-rich cassette.     

Formation of Nonculturable Cells of Mycobacterium tuberculosis and Their Resuscitation

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long poststationary phase. These cells were small (0.6–0.8 m) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosisculture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.     

Growth ofMycobacterium smegmatis in minimal and complete media

The growth patterns ofMycobacterium smegmatis SN2 in a minimal medium and in nutrient broth have been compared. The growth was monitored by absorbancy (Klett readings), colony forming units, wet weight and content of DNA, RNA and protein. During the early part of the growth cycle, the bacteria had higher wet weight and macromolecular content in nutrient broth than in minimal media. During the latter half of the growth cycle however, biosynthesis stopped much earlier in nutrient broth and the bacteria had a much lower content of macromolecules than in the minimal medium. In both the media, a general pattern of completing biosynthesis rapidly in the initial phase and a certain amount of cell division at a later time involving the distribution of preformed macromolecules was seen. The possible adaptive significance of this observation has been discussed.     

Nitrate reductases inEscherichia coli

Escherichia coli expresses two different membrane-bound respiratory nitrate reductases, nitrate reductase A (NRA) and nitrate reductase Z (NRZ). In this review, we compare the genetic control, biochemical properties and regulation of these two closely related enzyme systems. The two enzymes are encoded by distinct operons located within two different loci on theE. coli chromosome. ThenarGHJI operon, encoding nitrate reductaseA, is located in thechlC locus at 27 minutes, along with several functionally related genes:narK, encoding a nitrate/nitrite antiporter, and thenarXL operon, encoding a nitrate-activated, two component regulatory system. ThenarZYWV operon, encoding nitrate reductase Z, is located in thechlZ locus located at 32.5 minutes, a region which includes anarK homologue,narU, but no apparent homologue to thenarXL operon. The two membrane-bound enzymes have similar structures and biochemical properties and are capable of reducing nitrate using normal physiological substrates. The homology of the amino acid sequences of the peptides encoded by the two operons is extremely high but the intergenic regions share no related sequences. The expression of both thenarGHJI operon and thenarK gene are positively regulated by two transacting factors Fnr and NarL-Phosphate, activated respectively by anaerobiosis and nitrate, while thenarZYWV operon and thenarU gene are constitutively expressed. Nitrate reductase A, which accounts for 98% of the nitrate reductase activity when fully induced, is clearly the major respiratory nitrate reductase inE. coli while the physiological role of the constitutively expressed nitrate reductase Z remains to be defined.Abbreviations NR nitrate reductaseOn leave from Department of Biochemistry and Molecular Biology, The University of Texas Medical school at Houston, Houston, Texas, 77225, USA     

Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro

This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1β in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1β partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.