Metallated Proteins Expressed by Psychrophilic Bacteria

Small-scale cultures of a phenotyped Antarctic bacterium, Shewanella gelidimarina (ACAM 456T; Accession number U85907 (16S rDNA)), were grown aerobically with shaking at 4 degrees C in Difco Marine broth supplemented with potassium nitrate (5 mM). Cells were centrifuged and the periplasmic fraction harvested and assayed for nitrite production (using the Greiss reaction) as a measure of the potential expression of the enzyme, periplasmic nitrate reductase. Subsequent protein purification experiments identified a protein aggregate which gave a positive response in the Greiss assay with properties (denaturing PAGE: 42 kDa) that were inconsistent with periplasmic nitrate reductase enzymes characterized from alternate bacteria. N-Terminal sequencing (20 residues: A D P L T V Y G K L N V T A Q S N D V N) showed a high sequence homology to a putative outer membrane porin from Shewanella oneidensis MR-1 (Accession number: gi:24347323). The expression of periplasmic nitrate reductase has since been unambiguously established from cultures of S. gelidimarina grown under iron-limited conditions (i.e.; where the Fe(III) dissimilatory respiratory pathway of this genus is downregulated) in nitrate supplemented media. This work is ongoing and is aimed towards the chemical (spectroscopy) and biochemical (enzyme kinetics) characterisation of cold-adapted redox active metalloproteins.

This work is based upon phenotyped Antarctic bacteria (S. gelidimarina; S.frigidimarina) that was collected at another time (Refer: Psychrophilic Bacteria from Antarctic Sea-ice and Phospholipids of Antarctic sea ice algal communities new sources of PUFA [ASAC_708] and Biodiversity and ecophysiology of Antarctic sea-ice bacteria [ASAC_1012]).

---- Public Summary from Project ----
Cold-adapted bacteria resident in the Antarctic express proteins that have unusual properties. To date, only one metal containing protein (metalloprotein) expressed by cold-loving bacteria has been preliminarily characterised. The characterisation of cold-adapted metalloproteins will provide an innovative Australian-based research program that may lead to novel biotechnology and/or bioremediation applications.