Optimized generation of vectors for the construction of Haloferax volcanii deletion mutants

Hammelmann, M; Soppa, Jörg

doi:10.1016/j.mimet.2008.05.029

Cited by 39 publications

(50 citation statements)

References 17 publications

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“…The in-frame deletion mutants were generated using the so-called Pop-In/Pop-Out method which was developed by Bitan-Banin et al (2003) and subsequently optimized (Allers et al, 2004;Hammelmann & Soppa, 2008). Construction of suicide vectors including versions of the target genes carrying internal in-frame deletions has been described previously (pJO1, JO2 and pJO3;van Ooyen & Soppa, 2007).…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, a triple mutant was constructed which was totally devoid of a functional gene for an E1a subunit. An optimized procedure of the so-called Pop-In/ Pop-Out method was used for mutant construction (Hammelmann & Soppa, 2008). In all cases the genomic organization of the respective oadhc gene clusters was analysed by Southern blot analysis, and the successful construction of the four mutants was verified (data not shown).…”

Section: Construction Of In-frame Deletion Mutantsmentioning

confidence: 99%

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A 2-oxoacid dehydrogenase complex of Haloferax volcanii is essential for growth on isoleucine but not on other branched-chain amino acids

et al. 2010

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The halophilic archaeon Haloferax volcanii contains three operons encoding 2-oxoacid dehydrogenase complexes (OADHCs) OADHC1-OADHC3. However, the biological role of these OADHCs is not known as previous studies have demonstrated that they cannot use any of the known OADHC substrates. Even the construction of single mutants in all three oadhc operons, reported recently, could not identify a substrate. Therefore, all three possible double mutants and a triple mutant were generated, and single, double and triple mutants were compared to the wild-type. The four mutants devoid of a functional OADHC1 had a reduced growth yield during nitrate-respirative growth on tryptone. A metabolome analysis of the medium after growth of the triple mutant in comparison to the wild-type revealed that the mutant was unable to degrade isoleucine and leucine, in contrast to the wild-type. It was shown that oadhc1 mutants were unable to grow in synthetic medium on isoleucine, in contrast to the other mutants and the isogenic parent strain. However, all strains grew indistinguishably on valine and leucine. The transcript of the oadhc1 operon was highly induced during growth on isoleucine. However, attempts to detect enzymic activity were unsuccessful, while the branched-chain OADHC (BCDHC) of Pseudomonas putida could be measured easily. Therefore, the growth capability of the triple mutant and the wild-type on the two first degradation intermediates of isoleucine was tested and provided further evidence that OADHC is involved in isoleucine degradation. Taken together, the results indicate that OADHC1 is a specialized BCDHC that uses only one (or maximally two) of the three branched-chain 2-oxoacids, in contrast to BCDHCs from other species.

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Section: Methodsmentioning

confidence: 99%

Section: Construction Of In-frame Deletion Mutantsmentioning

confidence: 99%

A 2-oxoacid dehydrogenase complex of Haloferax volcanii is essential for growth on isoleucine but not on other branched-chain amino acids

et al. 2010

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“…Importantly, there is no N1-methyl pseudouridine modification present in any other position of the H. volcanii tRNAs. To enable the characterization of the biological role of Hvo_1989 in vivo Archaeal pseudouridine N1-methyltransferase www.rnajournal.org 415 an in-frame deletion mutant of the gene was constructed using the so-called Pop-In-Pop-Out strategy (Allers et al 2004;Hammelmann and Soppa 2008). The genomic organization of the mutant at the Hvo_1989 locus was verified by Southern blot analysis (Supplemental Fig.…”

Section: Mja_1640 Methylates T-arm Resembling Trnas In Vitromentioning

confidence: 99%

“…The vector for the construction of an in-frame deletion of gene hvo_1989 was generated using a recently described method (Hammelmann and Soppa 2008). In short, the upstream and the downstream regions of the gene were amplified by PCR using the following oligonucleotides:…”

Section: Construction Of a Knockout Strain For Hvo_1989 In H Volcaniimentioning

confidence: 99%

Identification of the enzyme responsible for N1-methylation of pseudouridine 54 in archaeal tRNAs

Wurm

Griese²,

Bahr³

et al. 2012

RNA

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ABSTRACTtRNAs from all three kingdoms of life contain a variety of modified nucleotides required for their stability, proper folding, and accurate decoding. One prominent example is the eponymous ribothymidine (rT) modification at position 54 in the T-arm of eukaryotic and bacterial tRNAs. In contrast, in most archaea this position is occupied by another hypermodified nucleotide: the isosteric N1-methylated pseudouridine. While the enzyme catalyzing pseudouridine formation at this position is known, the pseudouridine N1-specific methyltransferase responsible for this modification has not yet been experimentally identified. Here, we present biochemical and genetic evidence that the two homologous proteins, Mja_1640 (COG 1901, Pfam DUF358) and Hvo_1989 (Pfam DUF358) from Methanocaldococcus jannaschii and Haloferax volcanii, respectively, are representatives of the methyltransferase responsible for this modification. However, the in-frame deletion of the pseudouridine N1-methyltransferase gene in H. volcanii did not result in a discernable phenotype in line with similar observations for knockouts of other T-arm methylating enzymes.

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“…Haloferax volcanii is well suited for such an approach because sRNAs predicted by comparative genomics can subsequently be characterized using various experimental approaches. Notably, deletion mutants can be constructed very easily, 32,33 it can be grown in microtiter plates enabling phenotyping of mutants, 31 transcriptome analysis using DNA microarrays has been established, 34,35 proteome analysis is routinely performed, 36,37 and many molecular genetic tools are available. [38][39][40] Here we report the application of two bioinformatic approaches to predict sRNA genes in the genome in H. volcanii by comparative genomics of intergenic regions.…”

Section: Introductionmentioning

confidence: 99%

Bioinformatic prediction and experimental verification of sRNAs in the haloarchaeonHaloferax volcanii

Babski¹,

Tjaden²,

Voss³

et al. 2011

RNA Biology

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Optimized generation of vectors for the construction of Haloferax volcanii deletion mutants

Cited by 39 publications

References 17 publications

A 2-oxoacid dehydrogenase complex of Haloferax volcanii is essential for growth on isoleucine but not on other branched-chain amino acids

A 2-oxoacid dehydrogenase complex of Haloferax volcanii is essential for growth on isoleucine but not on other branched-chain amino acids

Identification of the enzyme responsible for N1-methylation of pseudouridine 54 in archaeal tRNAs

Bioinformatic prediction and experimental verification of sRNAs in the haloarchaeonHaloferax volcanii

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