Journal: MicrobiologyOpen

Article Title: Nitrogen regulation of protein–protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea

doi: 10.1002/mbo3.120

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: HM26 ( ΔpyrE2 ) was the parent strain used for all gene modifications and was derived from Hfx. mediterranei wild-type R4 (ATCC 33500) using plasmid pMH101N- ΔpyrE2 .

Techniques: Plasmid Preparation, Bacteria, Mutagenesis, Cloning, Knock-In

Journal: MicrobiologyOpen

Article Title: Nitrogen regulation of protein–protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea

doi: 10.1002/mbo3.120

Figure Lengend Snippet: Northern blot analysis of the amtB-glnK genomic region of Hfx. mediterranei . (A) Four different DIG-labeled PCR products were used as probes. Primers (listed in ) were amt1-mRNA-3F and amt1-mRNA-3R (probe A, 515 bp), glnK1-mRNA-1F and glnK1-mRNA-1R (probe B, 364 bp), amt2-mRNA-4F and amt2-mRNA-4R (probe C, 473), glnK2-mRNA-2F and glnK2-mRNA-2R (probe D, 337 bp). (B) 1 μg or 0.5 μg (‘samples) of RNA from Hfx. mediterranei wild-type cells grown with different nitrogen sources were probed with the four DIG-labeled PCR fragments. Lanes correspond to (1 and 1′) complex medium samples, (2 and 2′) 5 mmol/L NH 4 Cl samples, (3 and 3′) 5 mmol/L KNO 3 samples, (M) RiboRuler High Range RNA Ladder from Fermentas , (RNA) RNA sample stained with ethidium bromide and visualized using ultraviolet light.

Article Snippet: HM26 ( ΔpyrE2 ) was the parent strain used for all gene modifications and was derived from Hfx. mediterranei wild-type R4 (ATCC 33500) using plasmid pMH101N- ΔpyrE2 .

Techniques: Northern Blot, Labeling, Staining

Journal: MicrobiologyOpen

Article Title: Nitrogen regulation of protein–protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea

doi: 10.1002/mbo3.120

Figure Lengend Snippet: Anti-Flag immunoblot of Hfx. mediterranei strains with Flag-tag coding sequence insertions in amtB1/2 . (A) Anti-Flag immunoblot of whole cell lysate of HM26-F3 ( amtB1-flag ) strain grown with different sources of nitrogen. Lysate was obtained by boiling whole cells in reducing SDS-PAGE loading buffer prior to separation of proteins by 12% SDS-PAGE (with cells in OD 600 per lane of 0.325 units for HM26-F3 and HM26 and 0.065 units for the positive control [+, Hfx. volcanii H26-pJAM947 expressing Flag-SAMP1]). HM26 was grown in 75 mmol/L KNO 3 (1) and HM26-F3 was grown in (2) 75 mmol/L NH 4 Cl, (3) 75 mmol/L KNO 3 , and (4) complex medium. HM26-F3 strain was also grown in complex medium and nitrogen starved for (5) 24 h and (6) 48 h. (B) Cytoplasmic and membrane fractions of Hfx. mediterranei strains with Flag-tag coding sequence insertions in amtB1/2 . All four Flag-tagged strains (HM26-F1 to F4) and parent HM26 (as negative control) were grown in complex medium (to an OD 600 of 1) and transferred to minimal medium with 0.5% (w/v) glucose and no nitrogen source for 48 h. Protein (50 μg) from membrane or cytoplasmic fractions was separated by 12% SDS-PAGE and analyzed by anti-Flag immunoblotting. Lanes correspond to (+) Flag-SAMP1 from Hfx. volcanii as positive control, (1) HM26 strain, (2) HM26-F1 ( flag-amtB1 ), (3) HM26-F2 ( flag-amtB2 ), (4) HM26-F3 ( amtB1-flag ), (5) HM26-F4 ( amtB2-flag ). For all samples, proteins transferred to membranes were analyzed by staining with Ponceau Red to confirm equal protein loading prior to analysis by anti-Flag immunoblot. Migration of Kaleidoscope Molecular Weight Markers ( Bio-Rad ) indicated on the left.

Article Snippet: HM26 ( ΔpyrE2 ) was the parent strain used for all gene modifications and was derived from Hfx. mediterranei wild-type R4 (ATCC 33500) using plasmid pMH101N- ΔpyrE2 .

Techniques: Western Blot, FLAG-tag, Sequencing, SDS Page, Positive Control, Expressing, Membrane, Negative Control, Staining, Migration, Molecular Weight