| Summary: | Escherichia coli is the best characterized bacterium; it grows rapidly, and it is easy to
manipulate genetically. An increased knowledge about the physiology of this model organism
will facilitate the development of engineered E.coli strains for applications such as production of
biofuels and biofilm control. The aims of this work were the application of protein engineering
to increase E. coli hydrogen production, the identification of the proteins regulating extracellular
DNA production (eDNA), and the evaluation of the effect of the proteins synthesizing the signal
3'-5'-cyclic diguanylic acid (c-di-GMP) on biofilm formation.
The Escherichia coli hydrogen production rate was increased 9 fold through random
mutagenesis of fhlA. Variant FhlA133 (Q11H, L14V, Y177F, K245R, M288K, and I342F)
enhances hydrogen production by increasing transcription of the four transcriptional units
regulated by FhlA. The amino acid replacements E363G and L14G in FhlA increased hydrogen
production 6 fold and 4 fold, respectively.
The complete E. coli genome was screened to identify proteins that affect eDNA
production. The nlpI, yfeC, and rna mutants increased eDNA production and the hns and rfaD
mutants decreased eDNA production. Deletion of nlpI increases eDNA 3 fold while
overexpression of nlpI decreases eDNA 16 fold. Global regulator H-NS is required for eDNA with E. coli since deletion of hns abolished eDNA production while overexpression of hns
restored eDNA to 70 percent of the wild-type levels. Our results suggest that eDNA production in E.
coli is related to direct secretion.
Deletions of the genes encoding the diguanylate cyclases YeaI, YedQ, and YfiN
increased swimming motility and eDNA as expected for low c-di-GMP levels. However,
contrary to the current paradigm, early biofilm formation increased dramatically for the yeaI (30
fold), yedQ (12 fold), and yfiN (18 fold) mutants. Hence, our results suggest that c-di-GMP
levels should be reduced for initial biofilm formation because motility is important for initial
attachment to a surface.
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