scholarly journals Overexpression of the Victoriocin Gene in Helminthosporium (Cochliobolus) victoriae Enhances the Antifungal Activity of Culture Filtrates

2010 ◽  
Vol 100 (9) ◽  
pp. 890-896 ◽  
Author(s):  
Patricia B. de Sá ◽  
Hua Li ◽  
Wendy M. Havens ◽  
Mark L. Farman ◽  
Said A. Ghabrial
Keyword(s):  
Antifungal Activity ◽  
Type Strain ◽  
Wild Type Strain ◽  
In Vitro Translation ◽  
Signal Peptidase ◽  
Wild Type Virus ◽  
Wild Type ◽  
Cochliobolus Victoriae ◽  
Isolation And Characterization ◽  
Culture Filtrates

We have previously reported the isolation and characterization of the broad-spectrum antifungal protein, victoriocin, from culture filtrates of a virus-infected isolate of the plant-pathogenic fungus Helminthosporium (teleomorph: Cochliobolus) victoriae. We predicted that the 10-kDa mature victoriocin is derived in vivo from a preprotoxin precursor that is processed by a signal peptidase and kexin-like endopeptidase. We also presented evidence that the victoriocin precursor is encoded by a host gene, designated the victoriocin (vin) gene. In the present study, an H. victoriae genomic DNA library was constructed in the cosmid vector pMLF-2, and a cosmid clone carrying the vin gene and flanking sequences was isolated and used to generate constructs for transformation of virus-free and virus-infected H. victoriae isolates with the vin gene. Culture filtrates of the virus-free vin transformants exhibited high levels of antifungal activity compared with that revealed by the nontransformed virus-free wild-type strain, which exhibited little or no antifungal activity. Moreover, transformation of the wild-type virus-infected H. victoriae strain with the vin gene resulted in still higher production of victoriocin and higher antifungal activity in the culture filtrates of the vin transformants compared with the virus-infected wild-type strain. As previously predicted, the presence in the vin transformants of the preprovictoriocin and its post-translationally generated products, the provictoriocin and the mature victoriocin, was clearly demonstrated. Processing of the victoriocin preprotoxin requires eukaryotic host factors because no processing occurred in an in vitro translation system or in bacteria. It is of interest that some of the virus-free isolates transformed with the vin gene exhibited some features of the virus-induced disease phenotype, including moderate stunting and sectoring. Present data suggests that victoriocin may play an indirect role in disease development. Taken together, these results indicate that victoriocin is the primary protein responsible for the antifungal activity in culture filtrates of virus-infected H. victoriae isolates and that virus infection upregulates the expression of victoriocin. Overproduction of victoriocin may give the slower-growing virus-infected fungal strains some competitive advantage by inhibiting the growth of other fungi.

scholarly journals In the absence of Lgt, lipoproteins are shed from Streptococcus uberis independently of Lsp

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 134-141 ◽  
Author(s):  
E. L. Denham ◽  
P. N. Ward ◽  
J. A. Leigh
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Bovine Mastitis ◽  
Signal Peptidase ◽  
Wild Type ◽  
Streptococcus Uberis ◽  
Degradation Analysis ◽  
In The Wild ◽  
Full Length Gene

The role of lipoprotein diacylglyceryl transferase (Lgt) and lipoprotein signal peptidase (Lsp) responsible for processing lipoproteins was investigated in Streptococcus uberis, a common cause of bovine mastitis. In the absence of Lgt, three lipoproteins [MtuA (SUB0473), Hap (SUB1625) and an extracellular solute-binding protein (SUB0365)] were detected in extracellular locations. All were shown by Edman degradation analysis to be cleaved on the carboxy side of the LXXC lipobox. Detection of MtuA, a lipoprotein shown previously to be essential for infectivity and virulence, was used as a surrogate lipoprotein marker to locate and assess processing of lipoproteins. The absence of Lgt did not prevent location of MtuA to the cell membrane, its location in the wild-type strain but, in contrast to the situation with wild-type, did result in a widespread location of this protein. In the absence of both Lgt and Lsp, MtuA was similarly released from the bacterial cell. In such strains, however, the cell-associated MtuA represented the full-length gene product, indicating that Lsp was able to cleave non-lipidated (lipo)proteins but was not responsible for their release from this bacterium.

scholarly journals Mutation of waaC, Encoding Heptosyltransferase I in Campylobacter jejuni 81-176, Affects the Structure of both Lipooligosaccharide and Capsular Carbohydrate

2006 ◽  
Vol 188 (9) ◽  
pp. 3273-3279 ◽  
Author(s):  
Margaret I. Kanipes ◽  
Erzsebet Papp-Szabo ◽  
Patricia Guerry ◽  
Mario A. Monteiro
Keyword(s):  
Campylobacter Jejuni ◽  
Type Strain ◽  
Insertional Mutagenesis ◽  
Lipid A ◽  
Wild Type Strain ◽  
Capsular Polysaccharide ◽  
Resonance Spectroscopy ◽  
Gas Liquid Chromatography ◽  
Wild Type ◽  
Isolation And Characterization

ABSTRACT Campylobacter jejuni 81-176 lipooligosaccharide (LOS) is composed of two covalently linked domains: lipid A, a hydrophobic anchor, and a nonrepeating core oligosaccharide, consisting of an inner and outer core region. We report the isolation and characterization of the deepest rough C. jejuni 81-176 mutant by insertional mutagenesis into the waaC gene, encoding heptosyltransferase I that catalyzes the transfer of the first l-glycero-d-manno-heptose residue to 3-deoxy-d-manno-octulosonic residue (Kdo)-lipid A. Tricine gel electrophoresis, followed by silver staining, showed that site-specific mutation in the waaC gene resulted in the expression of a severely truncated LOS compared to wild-type strain 81-176. Gas-liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy showed that the waaC LOS species lacked all sugars distal to Kdo-lipid A. Parallel structural studies of the capsular polysaccharides of the wild-type strain 81-176 and waaC mutant revealed loss of the 3-O-methyl group in the waaC mutant. Complementation of the C. jejuni mutant by insertion of the wild-type C. jejuni waaC gene into a chromosomal locus resulted in LOS and capsular structures identical to those expressed in the parent strain. We also report here the presence of O-methyl phosphoramidate in wild-type strain 81-176 capsular polysaccharide.

scholarly journals The Burkholderia contaminans MS14ocfCGene Encodes a Xylosyltransferase for Production of the Antifungal Occidiofungin

2013 ◽  
Vol 79 (9) ◽  
pp. 2899-2905 ◽  
Author(s):  
Kuan-Chih Chen ◽  
Akshaya Ravichandran ◽  
Adam Guerrero ◽  
Peng Deng ◽  
Sonya M. Baird ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Gene Cluster ◽  
Mass Spectroscopy ◽  
Type Strain ◽  
Wild Type Strain ◽  
Activity Level ◽  
Antifungal Compound ◽  
Wild Type ◽  
Content Type ◽  
Burkholderia Contaminans

ABSTRACTBurkholderia contaminansstrain MS14 produces the antifungal compound occidiofungin, which is responsible for significant antifungal activities against a broad range of plant and animal fungal pathogens. Occidiofungin is a cyclic glycolipopeptide made up of eight amino acids and one xylose. A 56-kbocfgene cluster was determined to be essential for occidiofungin production. In this study, theocfCgene, which is located downstream ofocfDand upstream of theocfBgene in theocfgene cluster, was examined. Antifungal activity of theocfCgene mutant MS14KC1 was reduced against the indicator fungusGeotrichum candidumcompared with that of the wild-type strain. Furthermore, the analysis of the protein sequence suggests that theocfCgene encodes a glycosyltransferase. Biochemical analyses using nuclear magnetic resonance (NMR) and mass spectroscopy revealed that theocfCmutant produced the occidiofungin without the xylose. The purifiedocfCmutant MS14KC1 product had a level of bioactivity similar to that of the wild-type product. The revertant MS14KC1-R of theocfCmutant produced the same antifungal activity level on plate assays and the same antifungal compound based on high-performance liquid chromatography (HPLC) and mass spectroscopy analysis as wild-type strain MS14. Collectively, the study demonstrates that theocfCgene encodes a glycosyltransferase responsible to add a xylose to the occidiofungin molecule and that the presence of the xylose is not important for antifungal activity againstCandidaspecies. The finding provides a novel variant for future studies aimed at evaluating its use for inhibiting clinical and agricultural fungi, and the finding could also simplify the chemical synthesis of occidiofungin variants.

scholarly journals The ComQXPA Quorum Sensing System May Play an Important Role in the Synthesis of Bacillomycin D in Bacillus Amyloliquefaciens Q-426

2020 ◽  
Author(s):  
chunshan quan ◽  
liming jin ◽  
wei zhou ◽  
jialu liu ◽  
xian shi ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Quorum Sensing ◽  
Gene Cluster ◽  
Bacillus Amyloliquefaciens ◽  
Type Strain ◽  
Wild Type Strain ◽  
Early Stage ◽  
Tryptophan Residue ◽  
Wild Type ◽  
Bacillomycin D

Abstract Background: Bacillus amyloliquefaciens Q-426 can secrete numerous cyclic lipopeptides that have antifungal and antitumor activities. ComQXPA is a common quorum sensing (QS) system in Bacillus species. Most B. amyloliquefaciens strains are encoding the QS gene cluster comQXPA, however, the biological function of the ComQXPA system in B. amyloliquefaciens has not been well studied. In this study, we identified the comQXPA gene locus and the chemical structure of ComXQ-426 in B. amyloliquefaciens Q-426, and explored the function of ComXQ-426 in regulating lipopeptide production.Results: We identified and analyzed the comQXPA locus in Q-426. The full length of the comQXPA gene cluster was 4,014 bp, including 912 bp of comQ, 165 bp of comX, 2292 bp of comP, and 645 bp of comA. The comQXPA locus belongs to group B, as comQ and comX overlap by only one base pair. ComXQ-426 consists of six amino acids (GGDWKY) that contain a modified tryptophan residue. The antifungal activity of Q426ΔcomX was significantly affected, and almost no antifungal activity was observed, while the antifungal activity of strain Q426ΔcomX /comQX was restored to the same level as that of the wild-type strain. When the ComXQ-426 was added to the culture medium at a final concentration of 8 μg/L at the early stage of the log-phase, the antifungal activity of the wild-type strain Q-426 was significantly improved. Knocking out the comX gene did not affect the growth of the bacteria, however, the strain Q426ΔcomX lost its swimming ability, was unable to form colonies when spread on a solid surface, and could not form biofilms on the interface between the gas and liquid medium.Conclusions: Disruption of the ComPA signaling pathway in the Q-426 strain resulted in significant effects on bacillomycin D production, morphology, and motility.

scholarly journals Isolation and Characterization of Methanobacterium thermoautotrophicum ΔH Mutants Unable To Grow under Hydrogen-Deprived Conditions

1998 ◽  
Vol 180 (10) ◽  
pp. 2676-2681 ◽  
Author(s):  
Jeroen L. A. Pennings ◽  
Jan T. Keltjens ◽  
Godfried D. Vogels
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Methanobacterium Thermoautotrophicum ◽  
Mrna Levels ◽  
Wild Type ◽  
Physiological Characterization ◽  
Isolation And Characterization ◽  
Hydrogen Supply ◽  
In The Wild

ABSTRACT By using random mutagenesis and enrichment by chemostat culturing, we have developed mutants of Methanobacterium thermoautotrophicum that were unable to grow under hydrogen-deprived conditions. Physiological characterization showed that these mutants had poorer growth rates and growth yields than the wild-type strain. The mRNA levels of several key enzymes were lower than those in the wild-type strain. A fed-batch study showed that the expression levels were related to the hydrogen supply. In one mutant strain, expression of both methyl coenzyme M reductase isoenzyme I and coenzyme F420-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase was impaired. The strain was also unable to form factor F390, lending support to the hypothesis that the factor functions in regulation of methanogenesis in response to changes in the availability of hydrogen.

scholarly journals Biochemical genetics of Neurospora nuclease I: Isolation and characterization of nuclease (nuc) mutants

1983 ◽  
Vol 41 (3) ◽  
pp. 271-286 ◽  
Author(s):  
A. M. Forsthoefel ◽  
N. C. Mishra
Keyword(s):  
Nucleic Acids ◽  
Type Strain ◽  
Wild Type Strain ◽  
Extracellular Enzymes ◽  
Spontaneous Mutation ◽  
Subsequent Paper ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Phosphorus Source

SUMMARYIsolation and characterization of five new nuclease (nuc) deficient mutants ofNeurosporahave been described. The new mutants are unable to utilize nucleic acids as the sole phosphorus source and possess growth characteristics similar to thosenuc(nuc-1andnuc-2) mutants described previously. Two new mutants (nuc-4andnuc-5) were able to use RNA or predigested DNA (but not intact DNA) as phosphorus source and showed temperature sensitive growth at 37 °C. Based on the data from complementation and genetic analyses the five new nuc mutants (nuc-3, nuc-4, nuc-5, nuc-6andnuc-7) were found nonallelic to each other and to previously describednuc(nuc-1andnuc-2) mutants; the newnucmutants mapped to the right ofarg-12on linkage group II. On biochemical analyses, thesenucmutants were found to possess a lower level of extracellular nucleases and alkaline phosphatase as compared to the wild type strain. The ds DNase activity of the new mutants was only about 2–12% of that of the wild type strain; thus, the low level of these extracellular enzymes in thenucmutants causes their inability to utilize nucleic acids as the sole phosphorus source. Wild type levels of these enzymes were restored in the complementing heterokaryons capable of full growth on the DNA medium. Data from intercrosses, mutagen sensitivity and spontaneous mutation-frequency studies (as discussed in a subsequent paper) indicated the involvement of thenucgenes in DNA repair and recombination.

Isolation and characterization ofCryphonectria parasiticamutants that mimic a specific effect of hypovirulence-associated dsRNA on laccase activity

1995 ◽  
Vol 73 (10) ◽  
pp. 1655-1661 ◽  
Author(s):  
Daniel Rigling
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Laccase Activity ◽  
Specific Effect ◽  
Chestnut Blight ◽  
Malt Extract ◽  
Wild Type ◽  
Fungal Virulence ◽  
Isolation And Characterization ◽  
Endothia Parasitica

Hypovirulent, double-stranded (ds) RNA-containing strains of the chestnut blight fungus Cryphonectria parasitica were previously shown to produce less laccase activity than virulent strains when grown on malt extract agar containing tannic acid (Bavendamm reaction). Three mutants that lacked this specific laccase activity were selected after UV mutagenesis of a dsRNA-free, wild-type strain. Complementation tests and sexual crosses showed that all mutations were recessive, two were allelic (lacR1-1 and lacR1-2), and one (lacR2) was nonallelic. No linkage was detected between the two loci. None of the known C. parasitica laccases (LAC1, LAC2, and LAC3) was substantially reduced in the lacR1 mutants. The lacR2 mutant, in contrast, produced about 10-fold less extracellular LAC1 and LAC3 activities than the wild-type strain. Intracellular LAC2 was reduced to about 50% in this mutant. These results suggest a role for both LAC1 and LAC3 in the Bavendamm reaction. The three mutations had no significant effect on fungal virulence, pigmentation, and sporulation, all phenotypes that were suppressed in an isogenic dsRNA-containing strain. Key words: Endothia parasitica, genetics, hypovirulence, dsRNA, chestnut blight.

scholarly journals Isolation and Characterization of a Vibrio vulnificus Mutant Deficient in Both Extracellular Metalloprotease and Cytolysin

2001 ◽  
Vol 69 (9) ◽  
pp. 5943-5948 ◽  
Author(s):  
Jong-Jin Fan ◽  
Chung-Ping Shao ◽  
Ya-Chi Ho ◽  
Chun-Keung Yu ◽  
Lien-I Hor
Keyword(s):  
Type Strain ◽  
Alimentary Tract ◽  
Wild Type Strain ◽  
Vibrio Vulnificus ◽  
Wild Type ◽  
Allelic Exchange ◽  
Isolation And Characterization

ABSTRACT We isolated a Vibrio vulnificus mutant that was deficient in both metalloprotease and cytolysin by allelic exchange. The virulence of this mutant in mice and its cytotoxicity for HEp-2 cells were comparable to those of the wild-type strain, indicating that neither factor was essential for these properties. The cytolysin, but not the protease, seemed to be important for causing damage in the alimentary tract of the mice.

Isolation and characterization of a mutator strain of Streptomyces ambofaciens ATCC23877 exhibiting an increased level of genetic instability

1996 ◽  
Vol 42 (6) ◽  
pp. 562-570 ◽  
Author(s):  
Dominique Vandewiele ◽  
Jean-Nicolas Volff ◽  
Bertrand Aigle ◽  
Jean-Marc Simonet ◽  
Bernard Decaris
Keyword(s):  
Genetic Instability ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Analysis ◽  
Wild Type ◽  
Mutator Strain ◽  
Isolation And Characterization ◽  
Streptomyces Ambofaciens ◽  
In The Wild

In Streptomyces ambofaciens ATCC23877, 0.7% of pigment-defective mutants (Pig−) can be observed in the progeny of wild-type colonies. A mutator (Mut−) strain was isolated from the offspring of the wild-type strain. The Mut− strain produced colonies that sported nonpigmented papillae. Furthermore, the frequency of Pig− colonies obtained in the progeny of this strain was fivefold higher than in the wild-type strain. This strain showed the same level of sensitivity to ultraviolet light and mitomycin C as the wild-type strain. This Mut− phenotype was found to be reversible at high frequency (3 × 10−3). Genomic analysis using pulsed-field gel electrophoresis (PFGE) showed that the Pig− mutants arisen from the Mut− strain were less frequently rearranged (32% were deleted) compared with the mutants arising from the wild type (59% were deleted). Moreover, the Pig− papillae mutants possessed no visible rearrangement as revealed by PFGE analyses.Key words: Streptomyces, genetic instability, mutator strain, papillae.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

Sign in / Sign up

Export Citation Format

Share Document