共查询到20条相似文献,搜索用时 15 毫秒
1.
Muhammad Sohail 《Molecular biotechnology》1998,10(2):191-193
Molecular epidemiologic and other studies may require preparation of genomic DNA from large numbers of bacteria in sufficiently
pure form for restriction endonuclease digestion, cloning, RAPD-PCR, Southern hybridization, and so on.Staphylococcus and other Gram-positive bacteria have a rigid cell wall and can be difficult to lyse. Here, a simple and rapid method for
the preparation of genomic DNA from multiple samples is reported. This method produces clean DNA for use in most molecular
biology methods in <90 min. 相似文献
2.
Efficient insertional mutagenesis in lactococci and other gram-positive bacteria. 总被引:27,自引:5,他引:22
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
In lactococci, the study of chromosomal genes and their regulation is limited by the lack of an efficient transposon mutagenesis system. We associated the insertion sequence ISS1 with the thermosensitive replicon pG+ host to generate a mutagenic tool that can be used even in poorly transformable strains. ISS1 transposition is random in different lactococcal strains as well as in Enterococcus faecalis and Streptococcus thermophilus. High-frequency random insertion (of about 1%) obtained with this system in Lactococcus lactis allows efficient mutagenesis, with typically one insertion per cell. After ISS1 replicative transposition, the chromosome contains duplicated ISS1 sequences flanking pG+ host. This structure allows cloning of the interrupted gene. In addition, efficient excision of the plasmid leaves a single ISS1 copy at the mutated site, thus generating a stable mutant strain with no foreign markers. Mutants obtained by this transposition system are food grade and can thus be used in fermentation processes. 相似文献
3.
4.
Glutathione-independent isomerization of maleylpyruvate by Bacillus megaterium and other gram-positive bacteria. 总被引:2,自引:1,他引:1
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Maleylpyruvate, the ring fission product of gentisic acid, was found to be isomerized to fumarylpyruvate without a requirement for glutathione by an enzyme activity found in cell extracts of m-hydroxybenzoate-grown Bacillus megaterium 410. The isomerization reaction was detected as a shift in the absorbance maximum from 330 nm, the maximum for maleylpyruvate, to 345 nm, the maximum for fumarylpyruvate, when assayed at pH 8.0. Ammonium sulfate precipitation and dialysis of B. megaterium cell extracts resolved the isomerase activity from low-molecular-weight compounds such as glutathione but did not eliminate the isomerase activity. Iodoacetate and p-chloromercuribenzoate were potent inhibitors of the isomerase from B. megaterium. However, N-ethylmaleimide and iodoacetamide did not significantly inhibit this activity. In addition, fumaric acid was demonstrated as a product of gentisate oxidation by dialyzed cell extracts of B. megaterium. Glutathione-independent maleylpyruvate isomerases with properties similar to the isomerase found in B. megaterium were also found in other genera of gram-positive organisms. Eleven different organisms representing the genera Bacillus, Arthrobacter, Corynebacterium, Nocardia, and Rhodococcus were all found to possess this novel type of glutathione-independent maleylpyruvate isomerase. 相似文献
5.
A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR. 总被引:21,自引:0,他引:21
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification of intermediate products. This method which we have named one-step overlap extension PCR (OOE-PCR) can in principle be applied to every DNA fragment which can be cloned into a multiple cloning site of any common cloning vector. 相似文献
6.
The antibacterial activities of isoflavonoid (kievitone and phaseollin), flavonoid (hydroxyflavans), furanoacetylenic (wyerone), and sesquiterpenoid (capsidiol and rishitin), phytoalexins against eight Gram-negative and six Gram-positive bacteria were examined using the paper-disc antibiotic assay method. With the exception of capsidiol, which was inactive at the highest concentration tested (200,μg/disc) all of the phytoalexins were selectively toxic towards Gram-positive species. Wyerone and kievitone were generally more toxic than other phytoalexins; rishitin was the least active inhibitor. 相似文献
7.
A new procedure for efficient recovery of DNA, RNA, and proteins from Listeria cells by rapid lysis with a recombinant bacteriophage endolysin. 总被引:6,自引:2,他引:4
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A method for the rapid lysis of Listeria cells, employing a recombinant Listeria bacteriophage A118 lytic enzyme (PLY118), is described. The procedure can be used with all listerial species. It enables fast, efficient, and gentle recovery of DNA, RNA, or native cellular proteins from small-scale (2- to 5-ml) cultures. Moreover, this approach should be very useful in analytical detection and differentiation of Listeria strains when the release of native nucleic acids or proteins is required. 相似文献
8.
9.
10.
In recent years, a group of antibacterial proteins produced by gram-positive bacteria have attracted great interest in their potential use as food preservatives and as antibacterial agents to combat certain infections due to gram-positive pathogenic bacteria. They are ribosomally synthesized peptides of 30 to less than 60 amino acids, with a narrow to wide antibacterial spectrum against gram-positive bacteria; the antibacterial property is heat stable, and a producer strain displays a degree of specific self-protection against its own antibacterial peptide. In many respects, these proteins are quite different from the colicins and other bacteriocins produced by gram-negative bacteria, yet customarily they also are grouped as bacteriocins. Although a large number of these bacteriocins (or bacteriocin-like inhibitory substances) have been reported, only a few have been studied in detail for their mode of action, amino acid sequence, genetic characteristics, and biosynthesis mechanisms. Nevertheless, in general, they appear to be translated as inactive prepeptides containing an N-terminal leader sequence and a C-terminal propeptide component. During posttranslational modifications, the leader peptide is removed. In addition, depending on the particular type, some amino acids in the propeptide components may undergo either dehydration and thioether ring formation to produce lanthionine and beta-methyl lanthionine (as in lantibiotics) or thio ester ring formation to form cystine (as in thiolbiotics). Some of these steps, as well as the translocation of the molecules through the cytoplasmic membrane and producer self-protection against the homologous bacteriocin, are mediated through specific proteins (enzymes). Limited genetic studies have shown that the structural gene for such a bacteriocin and the genes encoding proteins associated with immunity, translocation, and processing are present in a cluster in either a plasmid, the chromosome, or a transposon. Following posttranslational modification and depending on the pH, the molecules may either be released into the environment or remain bound to the cell wall. The antibacterial action against a sensitive cell of a gram-positive strain is produced principally by destabilization of membrane functions. Under certain conditions, gram-negative bacterial cells can also be sensitive to some of these molecules. By application of site-specific mutagenesis, bacteriocin variants which may differ in their antimicrobial spectrum and physicochemical characteristics can be produced. Research activity in this field has grown remarkably but sometimes with an undisciplined regard for conformity in the definition, naming, and categorization of these molecules and their genetic effectors. Some suggestions for improved standardization of nomenclature are offered. 相似文献
11.
12.
D.J.-M. Vidon S. Donze C. Muller A. Entzmann & P. Andre 《Journal of applied microbiology》2001,90(6):988-993
AIMS: Listeria monocytogenes is capable, under certain conditions, of producing chemiluminescence which is amplified by luminol. This property was used to detect and count microcolonies of Listeria spp. in a few hours, without the use of a microscope. METHODS AND RESULTS: After trapping Listeria cells on polyvinylidene fluoride membranes, a chemiluminescence mixture was sprayed onto the membrane. The chemiluminescent spots emitted were analysed by a charge-coupled device camera connected to a data-processing system, which restored the intensity of the signals into three dimensional images. The intensity of the luminescence of microcolonies was improved by addition of cellobiose, and by brief exposure to u.v. light. CONCLUSION: Microcolonies of Listeria spp. can be imaged and counted by luminol-enhanced chemiluminescence with a photon-counting system. SIGNIFICANCE AND IMPACT OF THE STUDY: This method can be applied to the rapid detection and counting of Listeria spp. in raw milk. 相似文献
13.
M. I. Marks 《CMAJ》1975,112(2):170-173
Summary: Seven antimicrobials--clindamycin, penicillin, ampicillin, cloxacillin, erythromycin, lincomycin and cephalexin--were found to have a high degree of activity in vitro against 256 isolates of gram-positive bacteria and Hemophilus influenzae. Clindamycin was clearly superior against staphylococci and 3.12 mug/ml or less of clindamycin inhibited all 35 isolates of H. influenzae. Synergism was not demonstrated when clindamycin was tested in combination with sulfisoxazole or sulfamethoxazole by either the agar dilution or 24-hour growth curve method. This was true for penicillin as well, and the effect was independent of sulfonamide sensitivity. The erythromycin-sulfonamide combination was synergistic against 6 of 10 strains studied by the growth curve method; this effect was not demonstrable by the agar dilution method. 相似文献
14.
A rapid, guantitative, and selective estimation of radioactively labeled peptidoglycan in gram-positive bacteria 总被引:24,自引:0,他引:24
The Park and Hancock procedure for the isolation of bacterial cell wall peptidoglycans has been modified. The method developed permits rapid and accurate determination of small quantities of radioactively labeled material. The principal modifications consist of carrying out the procedure on glass-fiber filters after a vigorous hydrolysis of the cells with TCA (96°C, 30 min) and using Pronase in place of trypsin. 相似文献
15.
16.
A combined PCR and selective enrichment method for rapid detection of Listeria monocytogenes. 总被引:9,自引:0,他引:9
Development of a routine detection assay for Listeria monocytogenes in foods that uses the polymerase chain reaction (PCR) and enrichment cultures was investigated. Oligonucleotide primers were chosen to amplify a 3' region of L. monocytogenes hlyA gene spanning a conserved HindIII site. PCR detection sensitivity for L. monocytogenes in dilutions of pure enrichment cultures was between 50 and 500 colony forming units. A short enrichment period before PCR amplification allowed detection of the organisms in a range of complex foods contaminated with 10(4) cfu/g. Detection sensitivity for the assay in the presence of chicken skin and soft cheese was determined at 10-100 cfu/g. Utilization of enrichment cultures and PCR allowed identification of the organism within 24 h or 2 days. 相似文献
17.
S Wakisaka T Tachiki H C Sung H Kumagai T Tochikura S Matsui 《Analytical biochemistry》1987,163(1):117-122
A rapid enzymatic assay method for ammonia was developed by using glutamine synthetase from glutamate-producing bacteria together with pyruvate kinase, lactate dehydrogenase, and NADH. The time required for determination of 25 nmol of ammonia was 5 min with 1 unit of glutamine synthetase, as opposed to 14-30 min with 1 unit of glutamate dehydrogenases from various sources. The present method was used to determine ammonia in serum, microbiol-culture broth, and waste water. The method can be modified for spectrophotometry in the visible region by substituting pyruvate oxidase, peroxidase, and appropriate chromogens for lactate dehydrogenase and NADH. With 4-aminoantipyrine (4AA) and phenol, and with 4AA and N-ethyl-N-2-hydroxyethyl-m-toluidine as chromogens, the sensitivity of ammonia determination was 0.65 and 1.7 times that with glutamate dehydrogenase, respectively. The present method was also applicable to the continuous detection of the activity of some ammonia-forming enzymes such as guanase, adenosine deaminase, and urease and to the determination of 0.5-30 microM ATP-ADP after some modification of the mixture. 相似文献
18.
19.
A general method for cloning recA genes of gram-positive bacteria by polymerase chain reaction.
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
An internal fragment of the recA gene from eight gram-positive organisms has been amplified by using degenerate primers in a polymerase chain reaction. The internal 348- or 360-bp recA DNA segments from Bacillus subtilis, Clostridium acetobutylicum, Lactobacillus bulgaricus, Lactobacillus helveticus, Leuconostoc mesanteroides, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus salivarus subsp. thermophilus were amplified, cloned, and sequenced. The G + C contents of the DNA from these species range from 28 to 52%. The sequences of the bacterial recA genes show strong relatedness. This method is particularly useful for the recovery of the recA genes of gram-positive bacteria and avoids the difficulties of using a genetic complementation test for cloning. 相似文献