Preparation of Genomic <scp>DNA</scp> from Bacteria

Wilson, Kerry‐Jayne

doi:10.1002/0471142727.mb0204s56

Cited by 1,237 publications

(917 citation statements)

References 2 publications

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“…Plasmids of L. lactis were isolated in a similar way, except that degradation of the cell wall was carried out by prior addition of 30 mg of lysozyme per ml and incubation for 30 min at 37°C. Chromosomal DNA was isolated as described elsewhere (46). Restriction enzymes and other DNA-modifying enzymes from various sources were used according to the suppliers' recommendations.…”

Section: Methodsmentioning

confidence: 99%

Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

Gaeng

Scherer

Neve³

et al. 2000

Appl Environ Microbiol

128

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Bacteriophage lysins (Ply), or endolysins, are phage-encoded cell wall lytic enzymes which are synthesized late during virus multiplication and mediate the release of progeny virions. Bacteriophages of the pathogen Listeria monocytogenes encode endolysin enzymes which specifically hydrolyze the cross-linking peptide bridges in Listeria peptidoglycan. Ply118 is a 30.8-kDa L-alanoyl-D-glutamate peptidase and Ply511 (36.5 kDa) acts as N-acetylmuramoyl-L-alanine amidase. In order to establish dairy starter cultures with biopreservation properties against L. monocytogenes contaminations, we have introduced ply118 and ply511 into Lactococcus lactis MG1363 by using a pTRKH2 backbone. The genes were expressed under control of the lactococcal promoter P32, which proved superior to other promoters (P21 and P59) tested in this study. High levels of active enzymes were produced and accumulated in the cytoplasmic cell fractions but were not released from the cells at significant levels. Therefore, ply511 was genetically fused with the SP slpA nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide. Expression of SP slpA-ply511 from pSL-PL511 resulted in secretion of functional Ply511 enzyme from L. lactis cells. One clone expressed an unusually strong lytic activity, which was found to be due to a 115-bp deletion that occurred within the 3 -end coding sequence of SP slpA-ply511, which caused a frameshift mutation and generated a stop codon. Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511⌬(S262-K341) mutant polypeptide strongly increased its lytic activity. Proteolytic processing of the secretion competent SP SlpA-Ply511 propeptide following membrane translocation had no influence on enzyme activity. Immunoblotting experiments using both cytoplasmic and supernatant fractions indicated that the enzyme was quantitatively exported from the cells and secreted into the surrounding medium, where it caused rapid lysis of L. monocytogenes cells. Moreover, transformation of pSL-PL511⌬C into L. lactis Bu2-129, a lactose-utilizing strain that can be employed for fermentation of milk, also resulted in secretion of functional enzyme and showed that the vector is compatible with the native lactococcal plasmids.

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Section: Methodsmentioning

confidence: 99%

Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

Gaeng

Scherer

Neve³

et al. 2000

Appl Environ Microbiol

128

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“…DNA fragments used in cloning procedures were excised from agarose gels and purified with a GeneClean III Kit (Q•BIOgene, Carlsbad, CA). Bacterial genomic DNA was prepared using a previously described protocol [22]. Plasmids were purified from overnight cultures using the Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI).…”

Section: Dna Manipulationmentioning

confidence: 99%

Aerogenic vaccination with a Burkholderia mallei auxotroph protects against aerosol-initiated glanders in mice

Ulrich

Amemiya

Waag

et al. 2005

Vaccine

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“…Genomic DNA (gDNA) from O. anthropi NCIMB 40321 was isolated by the SDS-proteinase K-cetyltrimethylammonium bromide method essentially as described previously (54). However, use of cultures in the early-logarithmic-growth phase (optical density at 620 nm, 0.4 to 0.5) instead of the stationary phase appeared to be essential for isolation of sufficient amounts of good-quality gDNA.…”

mentioning

confidence: 99%

l-Selective Amidase with Extremely Broad Substrate Specificity fromOchrobactrum anthropiNCIMB 40321

Sonke¹,

Ernste²,

Tandler³

et al. 2005

Appl Environ Microbiol

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An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60°C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn 2؉ (to 80%), Mn 2؉ (to 400%), and Mg 2؉ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of ␣-hydrogen-and (bulky) ␣,␣-disubstituted ␣-amino acid amides, ␣-hydroxy acid amides, and ␣-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, ␤-amino and ␤-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/ formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.Enantiomerically pure ␣-amino acids, both natural and unnatural, form an important class of chiral building blocks for the pharmaceutical and agrochemical industries. Examples of important ␣-hydrogen-␣-amino acids are D-phenylglycine (DPhg) and D-p-hydroxyphenylglycine, which are produced as side chains for the manufacture of semisynthetic ␤-lactam antibiotics such as ampicillin, amoxicillin, and cephalexin (14, 53), and D-valine, an intermediate for the pyrethroid insecticide fluvalinate (28). Another frequently used ␣-hydrogen-␣-amino acid is L-tert-leucine, which is used as a building block for a variety of antiviral (e.g., anti-human immunodeficiency virus), antiarthritis, and anticancer drugs under development and as a chiral auxiliary in chemical asymmetric synthesis (5, 10). Besides ␣-hydrogen-␣-amino acids, ␣,␣-disubstituted ␣-amino acids also constitute a group of compounds of increasing importance, as exemplified by the use of L-␣-methyl-3,4-dihydroxyphenylalanine (L-␣-methyldopa) as an antihypertensive drug (34, 42), ␣-methylvaline as an intermediate for the herbicide Arsenal and related herbicides (47, 49), and L-␣-methylphenylglycine as a building block for the new fungicide fenamidone (27).Enantiomerically pure ␣-amino acids can be produced on a commercial scale by a number of different processes, both chemical and enzymatic. Biocatalytic processes that have been commercialized include the acylase process operated by Degussa (8) a...

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Preparation of Genomic DNA from Bacteria

Cited by 1,237 publications

References 2 publications

Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

Aerogenic vaccination with a Burkholderia mallei auxotroph protects against aerosol-initiated glanders in mice

l-Selective Amidase with Extremely Broad Substrate Specificity fromOchrobactrum anthropiNCIMB 40321

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