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.

本研究以表型鉴定的南极细菌希瓦氏菌（Shewanella gelidimarina）ACAM 456^T（16S rDNA登录号U85907）的小规模培养物为材料，于4℃下好氧振荡培养于添加了5mM硝酸钾的Difco海洋肉汤中。收集菌体并离心，分离得到周质组分，随后通过格里斯（Greiss）反应检测亚硝酸盐生成量，以此表征周质硝酸还原酶（periplasmic nitrate reductase）的潜在表达水平。后续蛋白质纯化实验中，获得了一种在格里斯检测中呈阳性反应的蛋白聚集体，其变性聚丙烯酰胺凝胶电泳（denaturing PAGE）结果显示分子量为42 kDa，这一特性与其他细菌中已鉴定的周质硝酸还原酶不符。对该蛋白进行N端测序（共20个氨基酸残基：A D P L T V Y G K L N V T A Q S N D V N），结果显示其与希瓦氏菌（Shewanella oneidensis）MR-1的推定外膜孔蛋白（登录号：gi:24347323）具有高度序列同源性。
后续研究已明确，在硝酸补充培养基中、铁限制条件下（即该菌属异化Fe(III)呼吸通路被下调的培养条件）培养的S. gelidimarina中，可稳定检测到周质硝酸还原酶的表达。本研究仍在进行中，旨在对冷适应型氧化还原活性金属蛋白开展化学（光谱学）与生化（酶动力学）层面的表征。
本研究的实验材料为此前采集的表型鉴定南极细菌S. gelidimarina与S. frigidimarina，相关样本采集信息可参考文献：《南极海冰嗜冷菌与南极海冰藻类群落磷脂：多不饱和脂肪酸（PUFA）新来源》[ASAC_708] 及《南极海冰细菌的多样性与生态生理学》[ASAC_1012]。
—— 项目公开摘要 ——
栖息于南极的冷适应型细菌所表达的蛋白质具有独特的理化特性。截至目前，仅有一种由嗜冷菌表达的金属蛋白完成了初步表征。针对冷适应型金属蛋白的系统性表征研究，将构成一项创新性的澳大利亚本土科研项目，有望催生新型生物技术及/或生物修复应用。