Structure and function of an unusual flavodoxin from the domain Archaea.

Flavodoxins, electron transfer proteins essential for diverse metabolisms in microbes from the domain Bacteria, are extensively characterized. Remarkably, although genomic annotations of flavodoxins are widespread in microbes from the domain Archaea, none have been isolated and characterized. Herein is described the structural, biochemical, and physiological characterization of an unusual flavodoxin (FldA) from Methanosarcina acetivorans, an acetate-utilizing methane-producing microbe of the domain Archaea In contrast to all flavodoxins, FldA is homodimeric, markedly less acidic, and stabilizes an anionic semiquinone. The crystal structure reveals an flavin mononucleotide (FMN) binding site unique from all other flavodoxins that provides a rationale for stabilization of the anionic semiquinone and a remarkably low reduction potentials for both the oxidized/semiquinone (-301 mV) and semiquinone/hydroquinone couples (-464 mV). FldA is up-regulated in acetate-grown versus methanol-grown cells and shown here to substitute for ferredoxin in mediating the transfer of low potential electrons from the carbonyl of acetate to the membrane-bound electron transport chain that generates ion gradients driving ATP synthesis. FldA offers potential advantages over ferredoxin by (i) sparing iron for abundant iron-sulfur proteins essential for acetotrophic growth and (ii) resilience to oxidative damage.

Reducing cell wall feruloylation by expression of a fungal ferulic acid esterase in Festuca arundinacea modifies plant growth, leaf morphology and the turnover of cell wall arabinoxylans.

A feature of cell wall arabinoxylan in grasses is the presence of ferulic acid which upon oxidative coupling by the action of peroxidases forms diferuloyl bridges between formerly separated arabinoxylans. Ferulate cross-linking is suspected of playing various roles in different plant processes. Here we investigate the role of cell wall feruloyaltion in two major processes, that of leaf growth and the turnover of cell wall arabinoxylans on leaf senescence in tall fescue using plants in which the level of cell wall ferulates has been reduced by targeted expression of the Aspergillus niger ferulic acid esterase A (FAEA) to the apoplast or Golgi. Analysis of FAE expressing plants showed that all the lines had shorter and narrower leaves compared to control, which may be a consequence of the overall growth rate being lower and occurring earlier in FAE expressing leaves than in controls. Furthermore, the final length of epidermal cells was shorter than controls, indicating that their expansion was curtailed earlier than in control leaves. This may be due to the observations that the deposition of both ether and ester linked monomeric hydroxycinnamic acids and ferulate dimerization stopped earlier in FAE expressing leaves but at a lower level than controls, and hydroxycinnamic acid deposition started to slow down when peroxidase levels increased. It would appear therefore that one of the possible mechanisms for controlling overall leaf morphology such as leaf length and width in grasses, where leaf morphology is highly variable between species, may be the timing of hydroxycinnamic acid deposition in the expanding cell walls as they emerge from cell division into the elongation zone, controlled partially by the onset of peroxidase activity in this region.

Processing of cellulose synthase (AcsAB) from Gluconacetobacter hansenii 23769.

The cellulose synthase protein (AcsAB) is encoded by a single gene in Gluconacetobacter hansenii ATCC 23769. We have examined the processing pattern of this enzyme and the localization of the cleavage products by heterologously expressing the truncated portions of the AcsAB protein and using specific antibodies generated against these regions. We found that the AcsAB protein is processed into three polypeptide subunits of molecular masses 46kDa, 34kDa and 95kDa. The 46kDa polypeptide (AcsA(cat)) harbors the conserved glycosyltransferase domain and hence contains the catalytic subunit of the enzyme. This polypeptide is localized in the cytoplasmic membrane. The 34kDa polypeptide (AcsA(reg)) is the regulatory subunit with the cyclic diGMP-binding PilZ domain. This polypeptide is largely cytoplasmic. The 95kDa subunit (AcsB) is of unknown function and contains a predicted signal peptide at its N-terminus. This subunit is localized in the outer membrane. In addition to this, we have also localized the AcsC protein in the outer membrane, confirming its predicted localization based on the OM-signal sequence at its N-terminus.

Genome sequence of a cellulose-producing bacterium, Gluconacetobacter hansenii ATCC 23769.

The Gram-negative bacterium Gluconacetobacter hansenii is considered a model organism for studying cellulose synthesis. We have determined the genome sequence of strain ATCC 23769.