Isolation and Characterization of Burkholderia cenocepacia Mutants Deficient in Pyochelin Production: Pyochelin Biosynthesis Is Sensitive to Sulfur Availability

ABSTRACT The opportunistic pathogen Burkholderia cenocepacia produces the yellow-green fluorescent siderophore, pyochelin. To isolate mutants which do not produce this siderophore, we mutagenized B. cenocepacia with the transposon mini-Tn5Tp. Two nonfluorescent mutants were identified which were unable to produce pyochelin. In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter. The cysW gene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl. Sulfate uptake assays confirmed that both mutants were defective in sulfate transport. Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin. Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth. We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis. These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions.

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Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product

Microbiology ◽

10.1099/mic.0.26476-0 ◽

2003 ◽

Vol 149 (10) ◽

pp. 2931-2939 ◽

Cited By ~ 63

Author(s):

Alex K. Jaques ◽

Tamo Fukamizo ◽

Diana Hall ◽

Richard C. Barton ◽

Gemma M. Escott ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Wild Type Strain ◽

Chitinase Activity ◽

Gene Replacement ◽

Wild Type ◽

Gene Encoding ◽

Batch Cultures ◽

Putative Active Site ◽

Hydrolysis Of

The gene encoding a major, inducible 45 kDa chitinase of Aspergillus fumigatus was cloned and analysis of the deduced amino acid sequence identified a chitinase of the fungal/bacterial class which was designated ChiB1. Recombinant ChiB1, expressed in Pichia pastoris, was shown to function by a retaining mechanism of action. That is, the β-conformation of the chitin substrate linkage was preserved in the product in a manner typical of family 18 chitinases. Cleavage patterns with the N-acetylglucosamine (GlcNAc) oligosaccharide substrates GlcNAc4, GlcNAc5 and GlcNAc6 indicated that the predominant reaction involved hydrolysis of GlcNAc2 from the non-reducing end of each substrate. Products of transglycosylation were also identified in each incubation. Following disruption of chiB1 by gene replacement, growth and morphology of disruptants and of the wild-type strain were essentially identical. However, during the autolytic phase of batch cultures the level of chitinase activity in culture filtrate from a disruptant was much lower than the activity from the wild-type. The search for chitinases with morphogenetic roles in filamentous fungi should perhaps focus on chitinases of the fungal/plant class although such an investigation will be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A. fumigatus TIGR database (http://www.tigr.org/).

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CDC37 is required for p60v-src activity in yeast.

Molecular Biology of the Cell ◽

10.1091/mbc.7.9.1405 ◽

1996 ◽

Vol 7 (9) ◽

pp. 1405-1417 ◽

Cited By ~ 58

Author(s):

B Dey ◽

J J Lightbody ◽

F Boschelli

Keyword(s):

Protein Tyrosine Kinase ◽

Wild Type Strain ◽

Active Form ◽

Biologically Active ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Wild Type ◽

Isolation And Characterization ◽

Genes Encoding

Mutations in genes encoding the molecular chaperones Hsp90 and Ydj1p suppress the toxicity of the protein tyrosine kinase p60v-src in yeast by reducing its levels or its kinase activity. We describe isolation and characterization of novel p60v-src-resistant, temperature-sensitive cdc37 mutants, cdc37-34 and cdc37-17, which produce less p60v-src than the parental wild-type strain at 23 degrees C. However, p60v-src levels are not low enough to account for the resistance of these strains. Asynchronously growing cdc37-34 and cdc37-17 mutants arrest in G1 and G2/M when shifted from permissive temperatures (23 degrees C) to the restrictive temperature (37 degrees C), but hydroxyurea-synchronized cdc37-34 and cdc37-17 mutants arrest in G2/M when released from the hydroxyurea block and shifted from 23 to 37 degrees C. The previously described temperature-sensitive cdc37-1 mutant is p60v-src-sensitive and produces wild-type amounts of p60v-src at permissive temperatures but becomes p60v-src-resistant at its restrictive temperature, 38 degrees C. In all three cdc37 mutants, inactivation of Cdc37p by incubation at 38 degrees C reduces p60v-src-dependent tyrosine phosphorylation of yeast proteins to low or undetectable levels. Also, p60v-src levels are enriched in urea-solubilized extracts and depleted in detergent-solubilized extracts of all three cdc37 mutants prepared from cells incubated at the restrictive temperature. These results suggest that Cdc37p is required for maintenance of p60v-src in a soluble, biologically active form.

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

Genetics Research ◽

10.1017/s0016672300021339 ◽

1983 ◽

Vol 41 (3) ◽

pp. 271-286 ◽

Cited By ~ 6

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.

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Characterization of the Phthalate Permease OphD from Burkholderia cepacia ATCC 17616

Journal of Bacteriology ◽

10.1128/jb.181.19.6197-6199.1999 ◽

1999 ◽

Vol 181 (19) ◽

pp. 6197-6199 ◽

Cited By ~ 29

Author(s):

Hung-Kuang Chang ◽

Gerben J. Zylstra

Keyword(s):

Burkholderia Cepacia ◽

Type Strain ◽

Wild Type Strain ◽

Cell Uptake ◽

Uptake System ◽

Wild Type ◽

Knockout Mutant ◽

Gene Encoding ◽

Uptake Inhibition

ABSTRACT The ophD gene, encoding a permease for phthalate transport, was cloned from Burkholderia cepacia ATCC 17616. Expression of the gene in Escherichia coli results in the ability to transport phthalate rapidly into the cell. Uptake inhibition experiments show that 4-hydroxyphthalate, 4-chlorophthalate, 4-methylphthalate, and cinchomeronate compete for the phthalate permease. An ophD knockout mutant of 17616 grows slightly more slowly on phthalate but is still able to take up phthalate at rates equivalent to that of the wild-type strain. This means that 17616 must have a second phthalate-inducible phthalate uptake system.

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Molecular characterization of protein O-mannosyltransferase and its involvement in cell-wall synthesis in Aspergillus nidulans

Microbiology ◽

10.1099/mic.0.27005-0 ◽

2004 ◽

Vol 150 (6) ◽

pp. 1973-1982 ◽

Cited By ~ 58

Author(s):

Takuji Oka ◽

Tetsu Hamaguchi ◽

Yuka Sameshima ◽

Masatoshi Goto ◽

Kensuke Furukawa

Keyword(s):

Cell Wall ◽

Aspergillus Nidulans ◽

Protein Modification ◽

Wild Type Strain ◽

Wild Type ◽

Gene Encoding ◽

And Function ◽

Encoding Protein

Protein O-glycosylation is essential for protein modification and plays important roles in eukaryotic cells. O-Mannosylation of proteins occurs in the filamentous fungus Aspergillus. The structure and function of the pmtA gene, encoding protein O-d-mannosyltransferase, which is responsible for the initial O-mannosylation reaction in Aspergillus nidulans, was characterized. Disruption of the pmtA gene resulted in the reduction of in vitro protein O-d-mannosyltransferase activity to 6 % of that of the wild-type strain and led to underglycosylation of an extracellular glucoamylase. The pmtA disruptant exhibited abnormal cell morphology and alteration in carbohydrate composition, particularly reduction in the skeletal polysaccharides in the cell wall. The results indicate that PmtA is required for the formation of a normal cell wall in A. nidulans.

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

Infection and Immunity ◽

10.1128/iai.69.9.5943-5948.2001 ◽

2001 ◽

Vol 69 (9) ◽

pp. 5943-5948 ◽

Cited By ~ 66

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.

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Isolation and Characterization of a Slowly Milk-Coagulating Variant of Lactobacillus helveticus Deficient in Purine Biosynthesis

Applied and Environmental Microbiology ◽

10.1128/aem.67.4.1846-1850.2001 ◽

2001 ◽

Vol 67 (4) ◽

pp. 1846-1850 ◽

Cited By ~ 10

Author(s):

Elvira M. Hebert ◽

Graciela S. De Giori ◽

Raul R. Raya

Keyword(s):

Amino Acid ◽

Wild Type Strain ◽

Defined Medium ◽

Lactobacillus Helveticus ◽

Transport Systems ◽

Wild Type ◽

Isolation And Characterization ◽

Essential Components ◽

Amino Acid Requirements

ABSTRACT A slowly milk-coagulating variant (Fmc−) ofLactobacillus helveticus CRL 1062, designated S1, was isolated and characterized. Strain S1 possessed all the known essential components required to utilize casein as a nitrogen source, which include functional proteinase and peptidase activities as well as functional amino acid, di- and tripeptide, and oligopeptide transport systems. The amino acid requirements of strain S1 were similar to those of the parental strain. However, on a purine-free, chemically defined medium, the growth rate of the Fmc− strain was threefold lower than that of the wild-type strain. L. helveticusS1 was found to be defective in IMP dehydrogenase activity and therefore was deficient in the ability to synthesize XMP and GMP. This conclusion was further supported by the observation that the addition of guanine or xanthine to milk, a substrate poor in purine compounds, restored the Fmc+ phenotype of L. helveticusS1.

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

Canadian Journal of Microbiology ◽

10.1139/m96-076 ◽

1996 ◽

Vol 42 (6) ◽

pp. 562-570 ◽

Cited By ~ 5

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.

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