7 research outputs found
Homeostasis of metabolites in Escherichia coli on transition from anaerobic to aerobic conditions and the transient secretion of pyruvate
We have developed a method for rapid quenching of samples
taken from chemostat cultures of Escherichia coli that gives
reproducible and reliable measurements of extracellular and
intracellular metabolites by 1H NMR and have applied it
to study the major central metabolites during the transition
from anaerobic to aerobic growth. Almost all metabolites
showed a gradual change after perturbation with air, consistent
with immediate inhibition of pyruvate formate-lyase, dilution
of overflow metabolites and induction of aerobic enzymes.
Surprisingly, although pyruvate showed almost no change
in intracellular concentration, the extracellular concentration
transiently increased. The absence of intracellular accumulation
of pyruvate suggested that one or more glycolytic enzymes
might relocate to the cell membrane. To test this hypothesis,
chromosomal pyruvate kinase (pykF) was modified to express
either PykF-green fluorescent protein or PykF-FLAG fusion
proteins. Measurements showed that PykF-FLAG relocates to
the cell membrane within 5 min of aeration and then slowly
returns to the cytoplasm, suggesting that on aeration, PykF
associates with the membrane to facilitate secretion of pyruvate
to maintain constant intracellular levels
Effects of N-Glycosylation Site Removal in Archaellins on the Assembly and Function of Archaella in Methanococcus maripaludis
In Methanococcus maripaludis S2, the swimming organelle, the archaellum, is composed of three archaellins, FlaB1S2, FlaB2S2 and FlaB3S2. All three are modified with an N-linked tetrasaccharide at multiple sites. Disruption of the N-linked glycosylation pathway is known to cause defects in archaella assembly or function. Here, we explored the potential requirement of N-glycosylation of archaellins on archaellation by investigating the effects of eliminating the 4 N-glycosylation sites in the wildtype FlaB2S2 protein in all possible combinations either by Asn to Glu (N to Q) substitution or Asn to Asp (N to D) substitutions of the N-glycosylation sequon asparagine. The ability of these mutant derivatives to complement a non-archaellated ΔflaB2S2 strain was examined by electron microscopy (for archaella assembly) and swarm plates (for analysis of swimming). Western blot results showed that all mutated FlaB2S2 proteins were expressed and of smaller apparent molecular mass compared to wildtype FlaB2S2, consistent with the loss of glycosylation sites. In the 8 single-site mutant complements, archaella were observed on the surface of Q2, D2 and D4 (numbers after N or Q refer to the 1st to 4th glycosylation site). Of the 6 double-site mutation complementations all were archaellated except D1,3. Of the 4 triple-site mutation complements, only D2,3,4 was archaellated. Elimination of all 4 N-glycosylation sites resulted in non-archaellated cells, indicating some minimum amount of archaellin glycosylation was necessary for their incorporation into stable archaella. All complementations that led to a return of archaella also resulted in motile cells with the exception of the D4 version. In addition, a series of FlaB2S2 scanning deletions each missing 10 amino acids was also generated and tested for their ability to complement the ΔflaB2S2 strain. While most variants were expressed, none of them restored archaellation, although FlaB2S2 harbouring a smaller 3-amino acid deletion was able to partially restore archaellation
A way to identify archaellins in Halobacterium salinarum archaella by FLAG-tagging
In the current study, haloarchaea Halobacterium salinarum cells were transformed individually with each of the modified archaellin genes (flaA1, flaA2 and flaB2) containing an oligonucleotide insert encoding the FLAG peptide (DYKDDDDK). The insertion site was selected to expose the FLAG peptide on the archaella filament surface. Three types of transformed cells synthesizing archaella, containing A1, A2, or B2 archaellin modified with FLAG peptide were obtained. Electron microscopy of archaella has demonstrated that in each case the FLAG peptide is available for the specific antibody binding. It was shown for the first time that the B2 archaellin, like archaellins A1 and A2, is found along the whole filament length
Flagellar filament bio-templated inorganic oxide materials-towards an efficient lithium battery anode
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