9046 mucc  (ATCC)

Journal:

Article Title: Role of Azotobacter vinelandii mucA and mucC Gene Products in Alginate Production

doi:

Figure Lengend Snippet: Schematic representation of the strategy followed to construct mucA and mucC mutants. (A and B) Insertional inactivation of the mucA (A) and mucC (B) genes producing the respective polar and nonpolar mutations. (C) Southern blot hybridization of total genomic DNA digested with EcoRV endonuclease with plasmid pRLA4 as a probe. Lanes: 1, ATCC 9046; 2, JRA8 (mucA); 3, JRA4 (mucABCD); 4, MLC2 (mucC); 5, MLC4 (mucCD). Identical hybridization patterns were found for strain AEIV and its corresponding muc mutants (data not shown).

Article Snippet: The increased algD expression in the ATCC 9046 mucC and mucCD mutant background (mutants WIC2 and WIC4 [Fig. D and E]) may indicate that in this highly mucoid strain MucC also exerts a direct negative role on AlgU activity, even though this effect is not so strong as to be reflected in the amount of alginate produced by the mutants. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 3 caption a7 Growth (open symbols) and β-galactosidase activity (closed symbols), on Burke's medium supplemented with 2% sucrose, of strains.

Techniques: Construct, Southern Blot, Hybridization, Plasmid Preparation

Journal:

Article Title: Role of Azotobacter vinelandii mucA and mucC Gene Products in Alginate Production

doi:

Figure Lengend Snippet: Alginate production in different A. vinelandii strains

Article Snippet: The increased algD expression in the ATCC 9046 mucC and mucCD mutant background (mutants WIC2 and WIC4 [Fig. D and E]) may indicate that in this highly mucoid strain MucC also exerts a direct negative role on AlgU activity, even though this effect is not so strong as to be reflected in the amount of alginate produced by the mutants. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 3 caption a7 Growth (open symbols) and β-galactosidase activity (closed symbols), on Burke's medium supplemented with 2% sucrose, of strains.

Techniques:

Journal:

Article Title: Role of Azotobacter vinelandii mucA and mucC Gene Products in Alginate Production

doi:

Figure Lengend Snippet: Bacterial strains and plasmids used in this work

Article Snippet: Antibiotic concentrations used for A. vinelandii and E. coli were as follows: ampicillin, not used and 200 μg/ml; chloramphenicol, not used and 30 μg/ml; gentamicin, 1.5 and 10 μg/ml; kanamycin, 2 μg/ml and not used; and tetracycline, 20 and 20 μg/ml, respectively. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant characteristics Source or reference Strains A. vinelandii AEIV Wild type, mucoid Svein Valla AEA8 AEIV with a nonpolar mucA ::Gm mutation This work AEA4 AEIV with a polar mucA ::Gm mutation This work AEC2 AEIV with a nonpolar mucC ::Tc mutation This work ATCC 9046 Highly mucoid due to the muc-1 spontaneous mutation 26 JRA8 ATCC 9046 with a nonpolar mucA ::Gm mutation This work JRA4 ATCC 9046 with a polar mucA ::Gm mutation This work MLC2 ATCC 9046 with a nonpolar mucC ::Tc mutation This work MLC4 ATCC 9046 with a polar mucC ::Tc mutation This work WI12 algD :: lacZ derivative of ATCC 9046 6 WIA8 WI12 with a nonpolar mucA ::Gm mutation This work WIA4 WI12 with a polar mucA ::Gm mutation This work WIC2 WI12 with a nonpolar mucC ::Tc mutation This work WIC4 WI12 with a polar mucC ::Tc mutation This work E. coli DH5α supE44 ΔlacU169 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 18 CAG16037 MC1061 λ( rpoH p3- lacZ ) 29 {"type":"entrez-protein","attrs":{"text":"CAG22216","term_id":"46915444","term_text":"CAG22216"}} CAG22216 CAG16037; rpoE ::ΩCm 39 Plasmids pBluescript SK(+) Plasmid used for subcloning DNA Stratagene pMOS Blue Plasmid used for subcloning PCR products Amersham pHP45Ω-Tc Plasmid used to obtain the Tc r cassette 15 pBSL141 Plasmid used to obtain the Gm r cassette 1 pLRA pMOS Blue derivative carrying a 1.3-kb DNA fragment containing A. vinelandii mucA gene amplified by PCR This work pLRA8 pLRA derivative containing mucA ::Gm nonpolar mutation This work pLRA4 pLRA containing mutation mucA ::Gm polar to mucBCD This work pLRC pMOS Blue derivative carrying a 1.3-kb DNA fragment containing A. vinelandii mucC gene amplified by PCR This work pLRC2 pLRC derivative containing mucC ::Tc nonpolar mutation This work pLRC4 pLRC derivative containing mutation mucC ::Tc polar to mucD This work pJMSAT1 Plasmid containing A. vinelandii algU-mucA genes; Ap 34 Open in a separate window Bacterial strains and plasmids used in this work Triparental or biparental A. vinelandii matings were done as reported previously ( 6 ).

Techniques: Plasmid Preparation, Mutagenesis, Subcloning, Amplification

Journal:

Article Title: Role of Azotobacter vinelandii mucA and mucC Gene Products in Alginate Production

doi:

Figure Lengend Snippet: Schematic representation of the strategy followed to construct mucA and mucC mutants. (A and B) Insertional inactivation of the mucA (A) and mucC (B) genes producing the respective polar and nonpolar mutations. (C) Southern blot hybridization of total genomic DNA digested with EcoRV endonuclease with plasmid pRLA4 as a probe. Lanes: 1, ATCC 9046; 2, JRA8 (mucA); 3, JRA4 (mucABCD); 4, MLC2 (mucC); 5, MLC4 (mucCD). Identical hybridization patterns were found for strain AEIV and its corresponding muc mutants (data not shown).

Article Snippet: Antibiotic concentrations used for A. vinelandii and E. coli were as follows: ampicillin, not used and 200 μg/ml; chloramphenicol, not used and 30 μg/ml; gentamicin, 1.5 and 10 μg/ml; kanamycin, 2 μg/ml and not used; and tetracycline, 20 and 20 μg/ml, respectively. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant characteristics Source or reference Strains A. vinelandii AEIV Wild type, mucoid Svein Valla AEA8 AEIV with a nonpolar mucA ::Gm mutation This work AEA4 AEIV with a polar mucA ::Gm mutation This work AEC2 AEIV with a nonpolar mucC ::Tc mutation This work ATCC 9046 Highly mucoid due to the muc-1 spontaneous mutation 26 JRA8 ATCC 9046 with a nonpolar mucA ::Gm mutation This work JRA4 ATCC 9046 with a polar mucA ::Gm mutation This work MLC2 ATCC 9046 with a nonpolar mucC ::Tc mutation This work MLC4 ATCC 9046 with a polar mucC ::Tc mutation This work WI12 algD :: lacZ derivative of ATCC 9046 6 WIA8 WI12 with a nonpolar mucA ::Gm mutation This work WIA4 WI12 with a polar mucA ::Gm mutation This work WIC2 WI12 with a nonpolar mucC ::Tc mutation This work WIC4 WI12 with a polar mucC ::Tc mutation This work E. coli DH5α supE44 ΔlacU169 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 18 CAG16037 MC1061 λ( rpoH p3- lacZ ) 29 {"type":"entrez-protein","attrs":{"text":"CAG22216","term_id":"46915444","term_text":"CAG22216"}} CAG22216 CAG16037; rpoE ::ΩCm 39 Plasmids pBluescript SK(+) Plasmid used for subcloning DNA Stratagene pMOS Blue Plasmid used for subcloning PCR products Amersham pHP45Ω-Tc Plasmid used to obtain the Tc r cassette 15 pBSL141 Plasmid used to obtain the Gm r cassette 1 pLRA pMOS Blue derivative carrying a 1.3-kb DNA fragment containing A. vinelandii mucA gene amplified by PCR This work pLRA8 pLRA derivative containing mucA ::Gm nonpolar mutation This work pLRA4 pLRA containing mutation mucA ::Gm polar to mucBCD This work pLRC pMOS Blue derivative carrying a 1.3-kb DNA fragment containing A. vinelandii mucC gene amplified by PCR This work pLRC2 pLRC derivative containing mucC ::Tc nonpolar mutation This work pLRC4 pLRC derivative containing mutation mucC ::Tc polar to mucD This work pJMSAT1 Plasmid containing A. vinelandii algU-mucA genes; Ap 34 Open in a separate window Bacterial strains and plasmids used in this work Triparental or biparental A. vinelandii matings were done as reported previously ( 6 ).

Techniques: Construct, Southern Blot, Hybridization, Plasmid Preparation

Journal:

Article Title: Role of Azotobacter vinelandii mucA and mucC Gene Products in Alginate Production

doi:

Figure Lengend Snippet: Growth (open symbols) and β-galactosidase activity (closed symbols), on Burke's medium supplemented with 2% sucrose, of strains. WI12 (parental strain) (A), WIA8 (mucA) (B), WIA4 (mucABCD) (C), WIC2 (mucC) (D), and WIC4 (mucCD) (E).

Article Snippet: Antibiotic concentrations used for A. vinelandii and E. coli were as follows: ampicillin, not used and 200 μg/ml; chloramphenicol, not used and 30 μg/ml; gentamicin, 1.5 and 10 μg/ml; kanamycin, 2 μg/ml and not used; and tetracycline, 20 and 20 μg/ml, respectively. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant characteristics Source or reference Strains A. vinelandii AEIV Wild type, mucoid Svein Valla AEA8 AEIV with a nonpolar mucA ::Gm mutation This work AEA4 AEIV with a polar mucA ::Gm mutation This work AEC2 AEIV with a nonpolar mucC ::Tc mutation This work ATCC 9046 Highly mucoid due to the muc-1 spontaneous mutation 26 JRA8 ATCC 9046 with a nonpolar mucA ::Gm mutation This work JRA4 ATCC 9046 with a polar mucA ::Gm mutation This work MLC2 ATCC 9046 with a nonpolar mucC ::Tc mutation This work MLC4 ATCC 9046 with a polar mucC ::Tc mutation This work WI12 algD :: lacZ derivative of ATCC 9046 6 WIA8 WI12 with a nonpolar mucA ::Gm mutation This work WIA4 WI12 with a polar mucA ::Gm mutation This work WIC2 WI12 with a nonpolar mucC ::Tc mutation This work WIC4 WI12 with a polar mucC ::Tc mutation This work E. coli DH5α supE44 ΔlacU169 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 18 CAG16037 MC1061 λ( rpoH p3- lacZ ) 29 {"type":"entrez-protein","attrs":{"text":"CAG22216","term_id":"46915444","term_text":"CAG22216"}} CAG22216 CAG16037; rpoE ::ΩCm 39 Plasmids pBluescript SK(+) Plasmid used for subcloning DNA Stratagene pMOS Blue Plasmid used for subcloning PCR products Amersham pHP45Ω-Tc Plasmid used to obtain the Tc r cassette 15 pBSL141 Plasmid used to obtain the Gm r cassette 1 pLRA pMOS Blue derivative carrying a 1.3-kb DNA fragment containing A. vinelandii mucA gene amplified by PCR This work pLRA8 pLRA derivative containing mucA ::Gm nonpolar mutation This work pLRA4 pLRA containing mutation mucA ::Gm polar to mucBCD This work pLRC pMOS Blue derivative carrying a 1.3-kb DNA fragment containing A. vinelandii mucC gene amplified by PCR This work pLRC2 pLRC derivative containing mucC ::Tc nonpolar mutation This work pLRC4 pLRC derivative containing mutation mucC ::Tc polar to mucD This work pJMSAT1 Plasmid containing A. vinelandii algU-mucA genes; Ap 34 Open in a separate window Bacterial strains and plasmids used in this work Triparental or biparental A. vinelandii matings were done as reported previously ( 6 ).

Techniques: Activity Assay