Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil

Abstractω-Transaminases’ (ω-TAs) importance for synthesizing chiral amines led to the development of different methods to quickly identify and characterize new sources of these enzymes. Here we describe the optimization of growth and induction of such an enzyme in a wild type strain of Bacillus sp. strain BaH (IBRC-M 11337) isolated from Iranian soil in shaking flasks by the response surface methodology (RSM). Optimum conditions were set in a multiplexed bench-top bioreactor system (Sixfors). ω-TA activity of obtained biomass was checked by an innovative efficient colorimetric assay for localizing ω-TAs in crude extracts on acrylamide gel by using ortho-xylylenediamine (OXD) as amino donor. The application of the established OXD assay is thereby expanded from high-throughput activity screenings and colony-based screenings of heterologously expressed mutants to a direct identification of ω-TAs in wild-type strains: This assay can be used to detect the protein band of the respective enzyme in crude extracts of novel isolates by visual inspection of native PAGEs without any upstream protein purification, thus enabling subsequent further investigations of a newly discovered enzyme directly from the crude extract.

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Development of mutants of Gliocladium virens tolerant to benomyl

Canadian Journal of Microbiology ◽

10.1139/m90-084 ◽

1990 ◽

Vol 36 (7) ◽

pp. 484-489 ◽

Cited By ~ 12

Author(s):

G. C. Papavizas ◽

D. P. Roberts ◽

K. K. Kim

Keyword(s):

Carbon Source ◽

Type Strain ◽

Wild Type Strain ◽

Carbon Sources ◽

Sclerotium Rolfsii ◽

Wild Type ◽

Gliocladium Virens ◽

Rhizosphere Competence ◽

Filter Paper Cellulase ◽

Type Strains

Aqueous suspensions of conidia of Gliocladium virens strains Gl-3 and Gl-21 were exposed to both ultraviolet radiation and ethyl methanesulfonate. Two mutants of Gl-3 and three of Gl-21 were selected for tolerance to benomyl at 10 μg∙mL−1, as indicated by growth and conidial germination on benomyl-amended potato dextrose agar. The mutants differed considerably from their respective wild-type strains in appearance, growth habit, sporulation, carbon-source utilization, and enzyme activity profiles. Of 10 carbon sources tested, cellobiose, xylose, and xylan were the best for growth, galactose and glucose were intermediate, and arabinose, ribose, and rhamnose were poor sources of carbon. The wild-type strains and the mutants did not utilize cellulose as the sole carbon source for growth. Two benomyl-tolerant mutants of Gl-3 produced less cellulase (β-1,4-glucosidase, carboxymethylcellulase, filter-paper cellulase) than Gl-3. In contrast, mutants of Gl-21 produced more cellulase than the wild-type strain. Only Gl-3 provided control of blight on snapbean caused by Sclerotium rolfsii. Wild-type strain Gl-21 and all mutants from both strains were ineffective biocontrol agents. Key words: Gliocladium, benomyl tolerance, Sclerotium, rhizosphere competence.

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Infection Components of Wild-Type and Mutant Strains of Colletotrichum gloeosporioides f. sp. aeschynomene on Northern Jointvetch

Plant Disease ◽

10.1094/pdis.1997.81.4.404 ◽

1997 ◽

Vol 81 (4) ◽

pp. 404-409 ◽

Cited By ~ 12

Author(s):

Y. Luo ◽

D. O. TeBeest

Keyword(s):

Fungal Pathogens ◽

Wild Type Strain ◽

Colletotrichum Gloeosporioides ◽

Wild Type ◽

Biological Control Of Weeds ◽

Lesion Number ◽

Mutant Strains ◽

Controlled Environments ◽

Resistant Strains ◽

Type Strains

Colletotrichum gloeosporioides f. sp. aeschynomene causes an anthracnose of northern jointvetch, Aeschynomene virginica. Infection components, including lesion number, latent period, lesion expansion rate, and sporulation, were measured in experiments conducted in controlled environments. Two wild-type strains (3-1-3 and CLA 5A), four benomyl-resistant strains (B13, B15, B18 and B21), and four nitrate nonutilizing mutant strains (Nit A, Nit R, Nit L, and Nit T) of the pathogen were tested. Nitrate nonutilizing strains caused significantly fewer lesions on northern jointvetch than did wild-type and benomyl-resistant strains. Latent periods were significantly shorter for the wild-type strain CLA 5A than for most other strains. Lesion expansion rates of all benomyl-resistant strains were significantly slower than those of the wild- type strains. Large variations in sporulation were observed for most strains, and no differences in sporulation were found between wild-type and mutant strains. The usefulness of infection component analysis for the identification of competitiveness of strains of fungal pathogens for biological control of weeds is discussed.

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A Stable 44S Intermediate in the Autodigestion of 50S Ribosomal Subunits

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066206 ◽

1971 ◽

Vol 29 ◽

pp. 430-431

Author(s):

John H. Nisbet ◽

Henry S. Slayter

Keyword(s):

Escherichia Coli ◽

Molecular Weight ◽

Ribosomal Rna ◽

Gel Electrophoresis ◽

Type Strain ◽

Wild Type Strain ◽

Ribosomal Subunit ◽

Wild Type ◽

Ribosomal Subunits ◽

Type Strains

Wild - type strains of Escherichia coli are known to contain as many as four endogenous nucleases (Ref. 1). These are commonly found associated with the ribosomes after extraction from the cell, but may be removed, with the exception of RNase IV, by washing the ribosomes in NH4Cl (at 0.2 M and higher concentrations). We have examined the effect of these nucleases on the 50S ribosomal subunit of one wild-type strain, K12 (Hfr 3000), by incubating the unwashed particles at 37° in the presence of varying magnesium concentrations.At 10-4 molar magnesium (slower at 10-3 molar), the 50S particle is converted to a species sedimenting at about 44S. About 20% of the total O.D260 is liberated at the same time. Continued incubation leads to the release of more O.D260 material while the RNA remaining in the 44S (Fig. 1) particle is progressively cleaved, eventually to the point where it consists of one principal fragment of molecular weight 0.42 x 106 daltons and several lesser fragments. The ribosomal RNA and proteins have been characterized by acrylamide gel electrophoresis.

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The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis

Applied and Environmental Microbiology ◽

10.1128/aem.03640-13 ◽

2013 ◽

Vol 80 (2) ◽

pp. 751-756 ◽

Cited By ~ 15

Author(s):

María Claudia Abeijón Mukdsi ◽

Hélène Falentin ◽

Marie-Bernadette Maillard ◽

Victoria Chuat ◽

Roxana Beatriz Medina ◽

...

Keyword(s):

Fatty Acids ◽

Type Strain ◽

Wild Type Strain ◽

Lipolytic Activity ◽

Premature Stop Codon ◽

Propionibacterium Freudenreichii ◽

Wild Type ◽

Swiss Cheese ◽

Content Type ◽

Type Strains

ABSTRACTFree fatty acids are important flavor compounds in cheese.Propionibacterium freudenreichiiis the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis byP. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants ofP. freudenreichiiCIRM-BIA1Tfor each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of thepf279gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of thepf774gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of thepf279gene sequence, leading to a premature stop codon, whereas they harbored apf774gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase inP. freudenreichiiand a key agent of Swiss cheese lipolysis.

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The BfeR Regulator Mediates Enterobactin-Inducible Expression of Bordetella Enterobactin Utilization Genes

Journal of Bacteriology ◽

10.1128/jb.186.21.7302-7311.2004 ◽

2004 ◽

Vol 186 (21) ◽

pp. 7302-7311 ◽

Cited By ~ 32

Author(s):

Mark T. Anderson ◽

Sandra K. Armstrong

Keyword(s):

Wild Type Strain ◽

Transcriptional Regulator ◽

Sole Source ◽

Bordetella Bronchiseptica ◽

Transcriptional Analysis ◽

Respiratory Pathogens ◽

Wild Type ◽

Outer Membrane Receptor ◽

Reading Frame ◽

Type Strains

ABSTRACT Utilization of the enterobactin siderophore by the respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica is dependent on the BfeA outer membrane receptor. This study determined that production of BfeA was increased significantly in iron-starved bacteria upon supplementation of cultures with enterobactin. A 1.01-kb open reading frame, designated bfeR, encoding a predicted positive transcriptional regulator of the AraC family was identified upstream and divergently oriented from bfeA. In iron-depleted cultures containing enterobactin, a Bordetella bfeR mutant exhibited markedly decreased BfeA receptor production compared to that of the wild-type strain. Additionally, B. pertussis and B. bronchiseptica bfeR mutants exhibited impaired growth with ferric enterobactin as the sole source of iron, demonstrating that effective enterobactin utilization is bfeR dependent. Transcriptional analysis using bfeA-lacZ reporter fusions in wild-type strains demonstrated that bfeA transcription was stimulated in iron-depleted conditions in the presence of enterobactin, compared to modest expression levels in cultures lacking enterobactin. In contrast, bfeA transcription in B. pertussis and B. bronchiseptica bfeR mutants was completely unresponsive to the enterobactin inducer. bfeA transcriptional analyses of a bfeA mutant demonstrated that induction by enterobactin did not require BfeA receptor-mediated uptake of the siderophore. These studies establish that bfeR encodes an enterobactin-dependent positive regulator of bfeA transcription in these Bordetella species.

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Diffusible Signal Factor–Repressed Extracellular Traits Enable Attachment of Xylella fastidiosa to Insect Vectors and Transmission

Phytopathology ◽

10.1094/phyto-06-13-0151-r ◽

2014 ◽

Vol 104 (1) ◽

pp. 27-33 ◽

Cited By ~ 11

Author(s):

Clelia Baccari ◽

Nabil Killiny ◽

Michael Ionescu ◽

Rodrigo P. P. Almeida ◽

Steven E. Lindow

Keyword(s):

Type Strain ◽

Host Plants ◽

Wild Type Strain ◽

Culture Supernatant ◽

Culture Media ◽

Xylella Fastidiosa ◽

Xylem Sap ◽

Wild Type ◽

Diffusible Signal Factor ◽

Type Strains

The hypothesis that a wild-type strain of Xylella fastidiosa would restore the ability of rpfF mutants blocked in diffusible signal factor production to be transmitted to new grape plants by the sharpshooter vector Graphocephala atropunctata was tested. While the rpfF mutant was very poorly transmitted by vectors irrespective of whether they had also fed on plants infected with the wild-type strain, wild-type strains were not efficiently transmitted if vectors had fed on plants infected with the rpfF mutant. About 100-fewer cells of a wild-type strain attached to wings of a vector when suspended in xylem sap from plants infected with an rpfF mutant than in sap from uninfected grapes. The frequency of transmission of cells suspended in sap from plants that were infected by the rpfF mutant was also reduced over threefold. Wild-type cells suspended in a culture supernatant of an rpfF mutant also exhibited 10-fold less adherence to wings than when suspended in uninoculated culture media. A factor released into the xylem by rpfF mutants, and to a lesser extent by the wild-type strain, thus inhibits their attachment to, and thus transmission by, sharpshooter vectors and may also enable them to move more readily through host plants.

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The Role of Ethylene Production in Virulence of Pseudomonas syringae pvs. glycinea and phaseolicola

Phytopathology ◽

10.1094/phyto.2001.91.5.511 ◽

2001 ◽

Vol 91 (5) ◽

pp. 511-518 ◽

Cited By ~ 60

Author(s):

Helge Weingart ◽

Henriette Ullrich ◽

Klaus Geider ◽

Beate Völksch

Keyword(s):

Pseudomonas Syringae ◽

Ethylene Production ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

In Planta ◽

Population Sizes ◽

Soybean Plants ◽

Type Strains

The importance of ethylene production for virulence of Pseudomonas syringae pvs. glycinea and phaseolicola was assayed by comparing bacterial multiplication and symptom development in bean and soybean plants inoculated with ethylene-negative (efe) mutants and wild-type strains. The efe mutants of Pseudomonas syringae pv. glycinea were significantly reduced in their ability to grow in planta. However, the degree of reduction was strain-dependent. Population sizes of efe mutant 16/83-E1 that did not produce the phototoxin coronatine were 10- and 15-fold lower than those of the wild-type strain on soybean and on bean, and 16/83-E1 produced very weak symptoms compared with the wild-type strain. The coronatine-producing efe mutant 7a/90-E1 reached fourfold and twofold lower population sizes compared with the wild-type strain on soybean and bean, respectively, and caused disease symptoms typical of the wild-type strain. Experiments with ethylene-insensitive soybeans confirmed these results. The virulence of the wild-type strains was reduced to the same extent in ethylene-insensitive soybean plants as the virulence of the efe mutants in ethylene-susceptible soybeans. In contrast, the virulence of Pseudomonas syringae pv. phaseolicola was not affected by disruption of the efe gene.

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Biochemical genetics of Neurospora nuclease II: Mutagen sensitivity and other characteristics of the nuclease mutants

Genetics Research ◽

10.1017/s0016672300021340 ◽

1983 ◽

Vol 41 (3) ◽

pp. 287-297 ◽

Cited By ~ 6

Author(s):

N. C. Mishra ◽

A. M. Forsthoefel

Keyword(s):

High Frequency ◽

Wild Type Strain ◽

Spontaneous Mutation ◽

Wild Type ◽

Very High Frequency ◽

Mutagen Sensitivity ◽

Mutator Effect ◽

Extreme Sensitivity ◽

Type Strains ◽

Very High

SUMMARYFive newnucmutants ofNeurospora crassawere characterized for their relative sensitivities to different mutagens (UV, MNG, MMS), to mitomycin-C and to histidine; latter has been shown to inhibit the growth of certain UV sensitive mutants. These mutants were also compared for their capabilities for spontaneous mutation as determined by resistance to p-fluoro-phenylalanine. Based on these characterization, the mutants seem to belong to two groups. The first group includednuc-3andnuc-6which showed sensitivity to all mutagen tested and possessed capability for a very high frequency of spontaneous mutation (i.e. mutator effect). The second group includednuc-4, nuc-5andnuc-7; these were as resistant to different mutagens as the wild type strain, but possessed an antimutator effect (i.e. the frequency of spontaneous mutation by these three mutants were at least 0·5–100 × less than the wild type strains). There was some variation in these properties of mutants belonging to the two groups. Among all the fivenucmutants,nuc-3was characterized by extreme sensitivity to all mutagens. None of the fivenucmutants were sensitive to histidine. The properties ofnucmutants are discussed in relation to their possible role in DNA repair and recombination.

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Modulating action of cyclic AMP on the expression of two SOS genes in Escherichia coli K-12

Canadian Journal of Microbiology ◽

10.1139/m87-123 ◽

1987 ◽

Vol 33 (8) ◽

pp. 704-708 ◽

Cited By ~ 4

Author(s):

Jordi Barbé ◽

Isidre Gibert ◽

Ricardo Guerrero

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

Cultural Conditions ◽

Gene Coding ◽

K 12 ◽

Type Strains ◽

Lambda Prophage

Ultraviolet irradiation and cyclic AMP treatment produce a synergistic effect on the induction of the clel gene (coding for bacteriocin ColE1) in wild-type strains of Escherichia coli. On the other hand, cyclic AMP does not affect the uv-mediated induction of the recA, sfiA, and umuDC genes. Growth in the presence of glucose or glycerol does not affect the factor of amplification of the expression of the clel gene in uv-irradiated cells of the wild-type strain. Although, in cultures not treated with uv, the basal level of clel induction is about twice as high in cells grown with glycerol as in those using glucose as carbon source. In recA mutants neither simultaneous nor separate treatments with either cyclic AMP or uv irradiation induced transcription of the clel gene. Moreover, cyclic AMP induced a slight increase in clel gene expression in uv-irradiated cya strains, but not in the crp mutants. Nevertheless, the pattern of the uv-mediated induction of other SOS genes, such as umuDC, was the same in the cya and crp mutants, as in their parental wild-type strains. Furthermore, the uv-mediated induction of lambda prophage was decreased after either addition of cyclic AMP or growth in cultural conditions where the level of this nucleotide was low.

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GENETIC AND MOLECULAR ANALYSIS OF THE GAL3 GENE IN THE EXPRESSION OF THE GALACTOSE/MELIBIOSE REGULON OF SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/113.2.229 ◽

1986 ◽

Vol 113 (2) ◽

pp. 229-246

Author(s):

Timothy E Torchia ◽

James E Hopper

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Null Allele ◽

Northern Blot Analysis ◽

Wild Type Strain ◽

Adaptation Period ◽

Wild Type ◽

Naturally Occurring ◽

Type Strains ◽

Rate Of Accumulation

ABSTRACT During the galactose adaptation period of a Saccharomyces cerevisiae strain bearing a naturally occurring gal3 allele, we found a longer induction lag and slower rate of accumulation of GAL10 and MEL1 RNAs compared to wild-type strains. A strain of genotype gal3 gal1 gal7 is noninducible for MEL1 gene expression, but this expression block is bypassed by overexpression of the GAL4 gene or by deletion of the GAL80 gene, either of which causes a constitutive phenotype. An otherwise wild-type strain that bears a chromosomal gal3 gene disruption mutation does not produce wild-type GAL3 RNA and exhibits induction comparable to a strain bearing the naturally occurring gal3. Based on this array of results, we conclude that the GAL3 gene product executes its function at a point before GAL4 mediated transcription of the GAL1-10-7 and MEL1 genes. Thus, the data are consistent with the previously advanced hypothesis that the GAL3 gene product functions to synthesize the inducer or coinducer molecule. In experiments in which the presence of either the plasmid-carried cloned GAL3 gene or the plasmid-carried cloned GAL1-10-7 genes allows MEL1 induction of a gal3 gal1 gal7 cell, we find that loss of the plasmid results in the shutoff of MEL1 expression even when galactose is continuously present. Either GAL3 function or GAL1-10-7 functions are therefore required for both the initiation and the maintenance of the induced state. Since the strains bearing either the naturally occurring gal3 allele or the gal3 disruption (null) allele do induce, the plasmid loss experiments indicate the existence of two completely independent induction initiation-maintenance pathways, one requiring GAL3 function, the other requiring GAL1-10-7 function. Finally, Northern blot analysis reveals two major GAL3 transcripts that differ in size by approximately 500 nucleotides.

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