Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of beta-galactosidase activity. beta-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.
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PMID:Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity. 1105 Jun 56

The functionality of the putative Mycobacterium tuberculosis phosphate transport operon was studied by operon- lacZ promoterless fusions in Mycobacterium smegmatis. The expression of the operon genes was evaluated in transformed M. smegmatis growing in medium with low and high phosphate concentration. Although the gene fusions expressed beta-galactosidase in medium with phosphate, a higher activity was detected in bacteria growing in medium with low phosphate. In contrast, alkaline phosphatase activity from M. smegmatis was detected only in bacteria growing in medium with low phosphate. The expression of the operon genes was driven by a promoter located 5' upstream from the start codon of the pstB gene. A second putative internal promoter 5' upstream of the pstS-1 gene was also detected. Furthermore, comparative analysis between the native and recombinant PstS-1 proteins showed that they were very similar. Like the native protein, the recombinant protein was also secreted to the culture medium as a glycosylated band. The results show that M. smegmatis recognized phosphate regulatory signals of the M. tuberculosis phosphate transport operon genes, and open the possibility to study gene phosphate regulation in mycobacteria.
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PMID:Molecular analysis of Mycobacterium tuberculosis phosphate specific transport system in Mycobacterium smegmatis. Characterization of recombinant 38 kDa (PstS-1). 1137 23

The TrcRS two-component system of Mycobacterium tuberculosis is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators. Reverse transcription-PCRs with total RNA showed that the trcR and trcS two-component system genes are transcribed in broth-grown M. tuberculosis. Analysis of the trcR and trcS genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of M. tuberculosis growth in cultured human primary macrophages. To determine if the TrcR response regulator is autoregulated, a trcR-lacZ fusion plasmid and a TrcR expression plasmid were cotransformed into Escherichia coli. Upon induction of the TrcR protein, there was a >500-fold increase in beta-galactosidase activity from the trcR-lacZ fusion, indicating that TrcR is involved in transcriptional autoactivation. Gel mobility shift assays with the trcR promoter and TrcR established that the response regulator was autoregulating via direct binding. By use of a delimiting series of overlapping trcR PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the trcR promoter was shown to be essential for TrcR binding. Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays. To further analyze the role of the AT-rich region in TrcR autoregulation, the trcR promoter was mutated and analyzed in lacZ transcriptional fusions in the presence of TrcR. Alteration of the AT-rich sequence in the trcR promoter resulted in the loss of trcR transcriptional activation in the presence of TrcR. This report indicates that the M. tuberculosis TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the trcR promoter.
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PMID:Expression, autoregulation, and DNA binding properties of the Mycobacterium tuberculosis TrcR response regulator. 1191 51

mce3 is one of the four mce operons in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence of this bacterium. Upstream of mce3 there is a putative regulatory gene (Rv1963) that harbours a double tetR-family signature. To study the role of this putative regulatory gene in the transcriptional regulation of the mce3 operon, Mycobacterium smegmatis mc(2)155 and M. tuberculosis H37Rv strains that harboured gene fusions between the mce3 promoter region and the Escherichia coli lacZ gene, either containing or not containing the Rv1963 gene, were used. The presence of the Rv1963 gene in the strains greatly reduced beta-galactosidase activity, suggesting that the Rv1963-encoded protein is a transcriptional repressor of the mce3 operon. Expression of mce3 by recombinant M. tuberculosis was increased when it was grown in a macrophage-like cell line (J774), compared to the level of expression seen when the recombinant bacterium was grown under in vitro conditions. However, no lifting of repression was induced. The mce3 promoter was defined by deletion and cloning of the Rv1963-Rv1964 intergenic region in a 200 bp DNA fragment harbouring the region upstream of the Rv1964 start codon. Gel-shift experiments determined that the Rv1963-binding site was located in this region. These results indicate that the mce3 operon is transcriptionally regulated and that under certain, unknown, conditions repression of gene expression could be lifted.
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PMID:Negative transcriptional regulation of the mce3 operon in Mycobacterium tuberculosis. 1236 33

The rel gene is responsible for the maintenance of the level of (p)ppGpp in bacteria under nutrient starvation. This phenomenon known as stringent response plays an important role during survival of the microorganisms in stationary phase. We have cloned 1.6 kb upstream sequence of rel gene of Mycobacterium tuberculosis in a shuttle vector pSD5B containing promoterless lacZ gene and promoter activity was observed in Mycobacterium smegmatis cells by blue/white selection and was measured by beta-galactosidase assay. In order to delineate the minimal promoter element of rel gene, a 200 bp fragment from this 1.6 kb upstream sequence was further cloned in promoterless lacZ shuttle vector pSD5B and promoter activity was observed in M. smegmatis cells in similar way. The 200 bp promoter fragment was found to be mycobacterium specific and did not respond when transformed in Escherichia coli. The +1 transcription start site was determined by primer extension method. The -10 promoter region was identified to be TATCCT. The three T bases when mutated, showed a remarkable decrease in the lacZ expression thus confirming the -10 region. The translation start site has also been identified by site directed frame shift mutagenesis. It appears that this rel promoter can be used for expression of proteins in mycobacteria.
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PMID:Identification and characterization of rel promoter element of Mycobacterium tuberculosis. 1592 71

A Mycobacterium tuberculosis membrane protein called Mycobacterium cell entry protein (Mce1A) was previously shown to mediate the uptake of nonpathogenic Escherichia coli and latex beads by nonphagocytic mammalian cells. Here we characterize further the in vitro invasive activity of Mce1A using colloidal gold nanoparticles and fluorescent latex microspheres. Mce1A-coated colloidal gold particles induced plasma membrane invagination and entered membrane-bound compartments inside HeLa cells. Few of the protein-coated particles were also found in the cytosol compartment. Cytochalasin D and nocodazole inhibited the uptake by HeLa cells, indicating that rearrangement of both microtubules and microfilaments was necessary for the uptake. The functional domain of Mce1A for invasion was narrowed to a highly basic 22-amino acid sequence termed Inv3. A synthetic Inv3 peptide stimulated uptake of colloidal gold particles as well as latex microspheres by HeLa cells. A chimeric protein composed of Inv3 sequence at the N terminus of beta-galactosidase appeared to stain the nuclear membrane, suggesting that it entered the HeLa cell cytoplasm. These observations suggest that the cell uptake activity of Mce1A is confined to a small peptide domain located in the core region of the protein. Inv3 could be used to ferry any protein in fusion with it into mammalian cells and may serve as a potent nonviral delivery system.
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PMID:A cell-penetrating peptide derived from mammalian cell uptake protein of Mycobacterium tuberculosis. 1662 Jul 48

Once across the barrier of the epithelium, macrophages constitute the primary defense against microbial invasion. For most microbes, the acidic, hydrolytically competent environment of the phagolysosome is sufficient to kill them. Despite our understanding of the trafficking events that regulate phagosome maturation, our appreciation of the lumenal environment within the phagosome is only now becoming elucidated through real-time functional assays. The assays quantify pH change, phagosome/lysosome fusion, proteolysis, lipolysis, and beta-galactosidase activity. This information is particularly important for understanding pathogens that successfully parasitize the endosomal/lysosomal continuum. Mycobacterium tuberculosis infects macrophages through arresting the normal maturation process of the phagosome, retaining its vacuole at pH 6.4 with many of the characteristics of an early endosome. Current studies are focusing on the transcriptional response of the bacterium to the changing environment in the macrophage phagosome. Manipulation of these environmental cues, such as preventing the pH drop to pH 6.4 with concanamycin A, abrogates the majority of the transcriptional response in the bacterium, showing that pH is the dominant signal that the bacterium senses and responds to. These approaches represent our ongoing attempts to unravel the discourse that takes place between the pathogen and its host cell.
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PMID:Mycobacterium tuberculosis and the environment within the phagosome. 1785 Apr 80

The survival of a bacterium with a depleted oxygen or nutrient supply is important for its long-term persistence inside the host under stressful conditions. We studied a gene, dps, from Mycobacterium smegmatis, encoding a protein, Dps (for DNA binding protein from starved cells), which is overexpressed under oxidative and nutritional stresses and provides bimodal protection to the bacterial DNA. Characterization of the dps promoter in vivo is therefore important. We cloned a 1-kb putative promoter region of the dps gene of M. smegmatis in an Escherichia coli-Mycobacterium shuttle vector, pSD5B, immediately upstream of the lacZ gene. Promoter activities were assayed in vivo both in solid medium and in liquid cultures by quantitative beta-galactosidase activity measurements. To characterize the minimal promoter region, a 200-bp fragment from the whole 1-kb sequence was further cloned in the same vector, and in a similar way, beta-galactosidase activity was quantitated. Primer extension analysis was performed to determine the +1 transcription start site of the gene. Point mutations were inserted in the putative promoter sequences in the -10 and -20 regions, and the promoter sequence was confirmed. The promoter was not recognized by purified M. smegmatis core RNA polymerase reconstituted with purified Mycobacterium tuberculosis sigmaA or sigmaB during multiple- and single-round in vitro transcription assays. Promoter-specific in vivo pull-down assays with an immobilized 1-kb DNA fragment containing the dps promoter established that extracellular function sigma factors were associated with this starvation-inducible promoter. Single-round transcription at the dps promoter further supported the idea that only core RNA polymerase reconstituted with sigmaF or sigmaH can generate proper transcripts.
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PMID:Identification and characterization of the dps promoter of Mycobacterium smegmatis: promoter recognition by stress-specific extracytoplasmic function sigma factors sigmaH and sigmaF. 1792 Dec 87

Tuberculosis (TB) remains a major global health problem, and successful genetic manipulation of mycobacteria is crucial for developing new approaches to study the mechanism of pathogenesis of Mycobacterium tuberculosis (M.tb) and to combat TB. In this study, a series of M.tb furA gene operator/promoter (pfurA) mutants were generated aiming at optimization of the promoter activities in mycobacterial strains. Measured by the lacZ gene-fusion reporter system, change of the initial codon GTG to the preferred ATG resulted in a double increase of beta-galactosidase activity, while a 6-bp substitution in the conserved FurA binding AT-rich region upstream of furA gene led to 4-6 folds increase of the activity. It is significant that combination of both mutations showed about 10 folds of beta-galactosidase activity higher than that of the prototype pfurA. Furthermore, all of the furA promoters were expressed continuously in vivo during intracellular growth of Mycobacterium bovis BCG, and were induced early upon infection in macrophages. Employing the series of pfurA-based differential expression vectors, M.tb chimeric antigen Ag856A2 known for its excellent immunogenicity, was shown to be expressed at different levels in the recombinant Mycobacterium smegmatis and BCG strains. These results indicated that this differential expression system is feasible to express any target antigen of interest in a modular fashion for the study of gene regulation in mycobacterial strains, and also for the development of different recombinant BCG vaccine candidates against TB or other infectious diseases, which would be beneficial for elicitation of optimal immune response.
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PMID:A novel differential expression system for gene modulation in Mycobacteria. 1883 6

The mce2 operon is one of the four mce operons present in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence mechanisms of this bacterium. In the present study we demonstrated that Rv0586, which encodes a putative GntR-like regulator, is part of the mce2 operon. By using a promoter-lacZ fusion approach and bioinformatics tools, we found that Rv0586 represses the expression of Mce2 proteins and of a putative endonuclease IV, encoded by end (Rv0670) gene. For this reason, we have re-named the repressor protein Mce2R. By gel-shift experiments Mce2R binding was determined to be located within the mce2 promoter region. In addition, two FadR-like operator motifs were identified within the promoter regions of both the mce2 operon and the end gene. These motifs overlap putative -10 and -35 promoter boxes. M. tuberculosis carrying mce2 and end promoter-lacZ fusions were used to infect J774 macrophage-like cells. Expression of beta-galactosidase was induced after phagocytocis, suggesting that some cellular factor could be a key component of the molecular switch regulation expression of the mce2 operon. In conclusion, these results add novel evidence of the complex regulation of mce operon expression.
Tuberculosis (Edinb) 2009 Jan
PMID:Mce2R from Mycobacterium tuberculosis represses the expression of the mce2 operon. 1902 63


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