scholarly journals Development of a Markerless Genetic Exchange Method for Methanosarcina acetivorans C2A and Its Use in Construction of New Genetic Tools for Methanogenic Archaea

2004 ◽  
Vol 70 (3) ◽  
pp. 1425-1433 ◽  
Author(s):  
Matthew A. Pritchett ◽  
Jun Kai Zhang ◽  
William W. Metcalf
Keyword(s):  
Reporter Gene ◽  
Shuttle Vector ◽  
Methanogenic Archaea ◽  
Methanosarcina Acetivorans ◽  
Gene Encoding ◽  
Exchange Method ◽  
Vitro Assay ◽  
Glucuronidase Activity ◽  
Base Analog

ABSTRACT A new genetic technique for constructing mutants of Methanosarcina acetivorans C2A by using hpt as a counterselectable marker was developed. Mutants with lesions in the hpt gene, encoding hypoxanthine phosphoribosyltransferase, were shown to be >35-fold more resistant to the toxic base analog 8-aza-2,6-diaminopurine (8ADP) than was the wild type. Reintroduction of the hpt gene into a Δhpt host restored 8ADP sensitivity and provided the basis for a two-step strategy involving plasmid integration and excision for recombination of mutant alleles onto the M. acetivorans chromosome. We have designated this method markerless exchange because, although selectable markers are used during the process, they are removed in the final mutants. Thus, the method can be repeated many times in the same cell line. The method was validated by construction of ΔproC Δhpt mutants, which were recovered at a frequency of 22%. Additionally, a Methanosarcina-Escherichia shuttle vector, encoding the Escherichia coli proC gene as a new selectable marker, was constructed for use in proC hosts. Finally, the markerless exchange method was used to recombine a series of uidA reporter gene fusions into the M. acetivorans proC locus. In vitro assay of β-glucuronidase activity in extracts of these recombinants demonstrated, for the first time, the utility of uidA as a reporter gene in Methanosarcina. A >5,000-fold range of promoter activities could be measured by using uidA: the methyl-coenzyme M reductase operon fusion displayed ∼300-fold-higher activity than did the serC gene fusion, which in turn had 16-fold-higher activity than did a fusion to the unknown orf2 gene.

scholarly journals Physiology and Posttranscriptional Regulation of Methanol:Coenzyme M Methyltransferase Isozymes in Methanosarcina acetivorans C2A

2009 ◽  
Vol 191 (22) ◽  
pp. 6928-6935 ◽  
Author(s):  
Rina B. Opulencia ◽  
Arpita Bose ◽  
William W. Metcalf
Keyword(s):  
Methane Production ◽  
Posttranscriptional Regulation ◽  
Promoter Strength ◽  
Methanosarcina Acetivorans ◽  
Vitro Assay ◽  
Phenotypic Differences ◽  
Fusion Data ◽  
Encoding Genes ◽  
Similar Growth

ABSTRACT Methanosarcina species possess three operons (mtaCB1, mtaCB2, and mtaCB3) encoding methanol-specific methyltransferase 1 (MT1) isozymes and two genes (mtaA1 and mtaA2) with the potential to encode a methanol-specific methyltransferase 2 (MT2). Previous genetic studies showed that these genes are differentially regulated and encode enzymes with distinct levels of methyltransferase activity. Here, the effects of promoter strength on growth and on the rate of methane production were examined by constructing strains in which the mtaCB promoters were exchanged. When expressed from the strong PmtaC1 or PmtaC2 promoter, each of the MtaC and MtaB proteins supported growth and methane production at wild-type levels. In contrast, all mtaCB operons exhibited poorer growth and lower rates of methane production when PmtaC3 controlled their expression. Thus, previously observed phenotypic differences can be attributed largely to differences in promoter activity. Strains carrying various combinations of mtaC, mtaB, and mtaA expressed from the strong, tetracycline-regulated PmcrB(tetO1) promoter exhibited similar growth characteristics on methanol, showing that all combinations of MtaC, MtaB, and MtaA can form functional MT1/MT2 complexes. However, an in vitro assay of coupled MT1/MT2 activity showed significant variation between the strains. Surprisingly, these variations in activity correlated with differences in protein abundance, despite the fact that all the encoding genes were expressed from the same promoter. Quantitative reverse transcriptase PCR and reporter gene fusion data suggest that the mtaCBA transcripts show different stabilities, which are strongly influenced by the growth substrate.

scholarly journals Short-Form Bomanins Mediate Humoral Immunity in Drosophila

2018 ◽  
Vol 10 (4) ◽  
pp. 306-314 ◽  
Author(s):  
Scott A. Lindsay ◽  
Samuel J.H. Lin ◽  
Steven A. Wasserman
Keyword(s):  
Humoral Immunity ◽  
Short Form ◽  
Family Members ◽  
Gene Encoding ◽  
Vitro Assay ◽  
Gram Positive Bacteria ◽  
Toll Receptor ◽  
Secreted Peptides

The Bomanins (Boms) are a family of a dozen secreted peptides that mediate the innate immune response governed by the Drosophila Toll receptor. We recently showed that deleting a cluster of 10 Bom genes blocks Toll-mediated defenses against a range of fungi and gram-positive bacteria. Here, we characterize the activity of individual Bom family members. We provide evidence that the Boms overlap in function and that a single Bom gene encoding a mature peptide of just 16 amino acids can act largely or entirely independent of other family members to provide phenotypic rescue in vivo. We further demonstrate that the Boms function in Drosophila humoral immunity, mediating the killing of the fungal pathogen Candida glabrata in an in vitro assay of cell-free hemolymph. In addition, we find that the level of antifungal activity both in vivo and in vitro is linked to the level of Bom gene expression. Although Toll dictates expression of the antimicrobial peptides (AMPs) drosomycin and metchnikowin, we find no evidence that Boms act by modifying the expression of the mature forms of these antifungal AMPs.

scholarly journals Enzymatic reconstitution of ribosomal peptide backbone thioamidation

2018 ◽  
Vol 115 (12) ◽  
pp. 3030-3035 ◽  
Author(s):  
Nilkamal Mahanta ◽  
Andi Liu ◽  
Shihui Dong ◽  
Satish K. Nair ◽  
Douglas A. Mitchell
Keyword(s):  
Posttranslational Modifications ◽  
Active Site ◽  
Methanogenic Archaea ◽  
Methanosarcina Acetivorans ◽  
Binding Studies ◽  
Peptide Backbone ◽  
Α Subunit ◽  
Essential Enzyme ◽  
Methanopyrus Kandleri

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans. Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii. Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri. Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.

scholarly journals Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids

2021 ◽  
Vol 22 (13) ◽  
pp. 6927
Author(s):  
Maša Kenda ◽  
Jan Vegelj ◽  
Barbara Herlah ◽  
Andrej Perdih ◽  
Přemysl Mladěnka ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Firefly Luciferase ◽  
Qsar Model ◽  
In Vitro Assay ◽  
Biochanin A ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Vitro Assay ◽  
Reporter Gene Assays

Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure–activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.

scholarly journals Studies of the CobA-Type ATP:Co(I)rrinoid Adenosyltransferase Enzyme of Methanosarcina mazei Strain Gö1

2006 ◽  
Vol 188 (10) ◽  
pp. 3543-3550 ◽  
Author(s):  
Nicole R. Buan ◽  
Kimberly Rehfeld ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Biochemical Analysis ◽  
Methanogenic Archaea ◽  
Striking Difference ◽  
Resonance Spectroscopy ◽  
Gene Encoding ◽  
Reading Frame ◽  
Methanosarcina Mazei ◽  
Coenzyme B ◽  
Better Than

ABSTRACT Although methanogenic archaea use B12 extensively as a methyl carrier for methanogenesis, little is known about B12 metabolism in these prokaryotes or any other archaea. To improve our understanding of how B12 metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain Gö1 (open reading frame MM3138, referred to as cobAMm here) was cloned and used to restore coenzyme B12 synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme. cobAMm protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca2+ ions, but the activity was not enhanced by Mg2+ and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn2+. The CobA Mm enzyme had a Km ATP of 3 μM and a Km HOCbl of 1 μM. Unlike the S. enterica enzyme, CobA Mm used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the β phosphate of ATP was required for binding to the enzyme. A striking difference between CobA Se and CobA Mm was the use of ADP as a substrate by CobA Mm , suggesting an important role for the γ phosphate of ATP in binding. The results from 31P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPPi) is the reaction by-product; no cleavage of PPPi was observed, and the enzyme was only slightly inhibited by pyrophosphate (PPi). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobA Se and CobA Mm enzymes.

scholarly journals Validation and assessment of preanalytical factors of a fluorometric in vitro assay for glucocerebrosidase activity in human cerebrospinal fluid

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Linn Oftedal ◽  
Jodi Maple-Grødem ◽  
Marthe Gurine Gunnarsdatter Førland ◽  
Guido Alves ◽  
Johannes Lange
Keyword(s):  
Cerebrospinal Fluid ◽  
Enzymatic Activity ◽  
Lewy Bodies ◽  
Activity Assay ◽  
Limit Of Quantification ◽  
Gene Encoding ◽  
Vitro Assay ◽  
Freeze Thaw ◽  
Human Cerebrospinal Fluid

AbstractLysosomal dysfunction is an emerging feature in the pathology of Parkinson’s disease and Dementia with Lewy bodies. Mutations in the GBA gene, encoding the enzyme Glucocerebrosidase (GCase), have been identified as a genetic risk factor for these synucleinopathies. As a result, there has been a growing interest in the involvement of GCase in these diseases. This GCase activity assay is based on the catalytic hydrolysis of 4-methylumbelliferyl β-d-glucopyranoside that releases the highly fluorescent 4-methylumbelliferyl (4-MU). The final assay protocol was tested for the following parameters: Lower limit of quantification (LLOQ), precision, parallelism, linearity, spike recovery, number of freeze–thaw events, and sample handling stability. The GCase activity assay is within acceptable criteria for parallelism, precision and spike recovery. The LLOQ of this assay corresponds to an enzymatic activity of generating 0.26 pmol 4-MU/min/ml. The enzymatic activity was stable when samples were processed and frozen at − 80 °C within 4 h after the lumbar puncture procedure. Repetitive freeze–thaw events significantly decreased enzyme activity. We present the validation of an optimized in vitro GCase activity assay, based on commercially available components, to quantify its enzymatic activity in human cerebrospinal fluid and the assessment of preanalytical factors.

A murine transgenic model for transcriptional regulation of the Na/Pi-IIa major renal phosphate cotransporter

2007 ◽  
Vol 292 (5) ◽  
pp. F1617-F1625 ◽  
Author(s):  
Tzur Rosenberg ◽  
Catherine Shachaf ◽  
Maty Tzukerman ◽  
Karl Skorecki
Keyword(s):  
Reporter Gene ◽  
Genomic Fragment ◽  
Gene Encoding ◽  
Specific Expression ◽  
Early Postnatal Development ◽  
Protein Levels ◽  
Clear Cut ◽  
Lac Z

Levels of the type IIa Na/Pi (Na/Pi-IIa) cotransporter, which serves as the principal mediator of phosphate reabsorption in the kidney, can be modulated through posttranscriptional or posttranslational mechanisms by dietary, hormonal, and pharmacological influences. Previous studies have not demonstrated clear-cut evidence for modulation of Na/Pi-IIa cotransporter levels through transcriptional mechanisms. We have previously demonstrated that a 4.7-kb rat genomic fragment upstream of the rodent Npt2 gene encoding the Na/Pi-IIa cotransporter, is sufficient to mediate its transcriptional activity in vitro (Shachaf C, Skorecki KL, Tzukerman M. Am J Physiol Renal Physiol 278: F406–F416, 2000). Accordingly, we have established an in vivo experimental model in which this Npt2 genomic fragment fused upstream of a Lac Z reporter gene was expressed as a transgene in mice. The nine independent transgenic founder lines generated exhibited Lac Z reporter gene expression specifically in the renal cortex. This renal cortical-specific expression driven by the Npt2 promoter was confirmed at the mRNA and protein levels using RT-PCR, histochemistry, and Lac Z enzymatic activity. Furthermore, the expression of the transgene correlated with expression of the endogenous Npt2 gene during embryonic and early postnatal development. Thus we have generated a transgenic mouse model which will enable in vivo investigation of the contribution of transcriptional mechanisms to the overall regulation of Na/Pi-IIa expression under physiological and pathophysiological conditions.

Integration of embryonic nephrogenic cells carrying a reporter gene into functioning nephrons

1991 ◽  
Vol 261 (3) ◽  
pp. C550-C554 ◽  
Author(s):  
C. Koseki ◽  
D. Herzlinger ◽  
Q. al-Awqati
Keyword(s):  
Reporter Gene ◽  
Fluorescein Isothiocyanate ◽  
Mesenchymal Cells ◽  
Gene Encoding ◽  
Nephron Segments ◽  
Apical Vesicles ◽  
Foreign Genes ◽  
Beta Galactosidase ◽  
Lac Z

We developed a procedure to introduce and stably express foreign genes into the kidney. The Lac Z reporter gene encoding the bacterial protein beta-galactosidase was introduced by retrovirus-mediated gene transfer into rat nephrogenic mesenchymal cells, which were induced for 24 h with embryonic spinal cord in vitro. The Lac Z-tagged mesenchymal cells were subsequently transplanted underneath the capsule of the neonatal kidney. Two weeks after transplantation, the Lac Z-tagged cells derived from transplants were identified by their beta-galactosidase expression. Well-differentiated Lac Z positive cells were observed in glomerulus and proximal and distal nephron segments. To determine if the tagged mesenchymal cells developed into functional nephrons, fluorescein isothiocyanate-labeled dextran was infused into transplanted animals before death. We observed that fluorescent apical vesicles were colocalized to beta-galactosidase positive proximal tubular cells, indicating that the transplanted mesenchymal cells were integrated into reabsorbing nephrons. These results show the feasibility of introducing foreign genes into epithelia of functioning nephron segments.

scholarly journals Cloning of the Streptococcus mutans Gene Encoding Glucan Binding Protein B and Analysis of Genetic Diversity and Protein Production in Clinical Isolates

2001 ◽  
Vol 69 (11) ◽  
pp. 6931-6941 ◽  
Author(s):  
Renata O. Mattos-Graner ◽  
Song Jin ◽  
William F. King ◽  
Tsute Chen ◽  
Daniel J. Smith ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Group B Streptococcus ◽  
Clinical Isolates ◽  
Dna Polymorphisms ◽  
Gene Encoding ◽  
Vitro Assay ◽  
Functional Conservation ◽  
Dental Biofilm ◽  
Group B

ABSTRACT Streptococcus mutans, the primary etiological agent of dental caries, produces several activities that promote its accumulation within the dental biofilm. These include glucosyltransferases, their glucan products, and proteins that bind glucan. At least three glucan binding proteins have been identified, and GbpB, the protein characterized in this study, appears to be novel. The gbpB gene was cloned and the predicted protein sequence contained several unusual features and shared extensive homology with a putative peptidoglycan hydrolase from group B streptococcus. Examination of gbpB genes from clinical isolates ofS. mutans revealed that DNA polymorphisms, and hence amino acid changes, were limited to the central region of the gene, suggesting functional conservation within the amino and carboxy termini of the protein. The GbpB produced by clinical isolates and laboratory strains showed various distributions between cells and culture medium, and amounts of protein produced by individual strains correlated positively with their ability to grow as biofilms in an in vitro assay.

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