Heavy-metal resistance in Marinobacter aquaeolei 617 insights into copper resistance

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

Heavy metal resistance in Marinobacter aquaeolei (Ma.aq) 617 in aerobic conditions was studied for three different ions, cadmium, cobalt and copper. The main aim of this work was the study of a putative copper resistance operon, copSRXAB, located in the chromosome of Marinobacter and the biochemical characterization of a unique copper binding protein CopX (proposed designation), associated with the copper resistance system. Growth under heavy metal ion stress was performed for those three heavy metals and the Minimum Inhibitory Concentration (MIC) / Maximum Tolerant Concentration (MTC) was determined using two different approaches, solid artificial sea water (ASW) plates and liquid ASW medium, supplemented with lactate and yeast extract, as carbon sources. The MIC/MTC of cadmium, cobalt and copper ions was found to be 200 μM, 4-6 mM, and 1.6 mM, respectively. These values classify Ma.aq strain 617 as cadmium, cobalt and copper resistant strain. Moreover, during the cobalt resistance studies we observed the production of an unknown protein or compound, which is proposed be a cobalamine containing protein and/or cobalamine itself. Under the scope of copper resistance, preliminary proteomics analysis of the Ma.aq periplasmic fraction was performed. CopX, identified by MALDI TOF-TOF mass spectrometry, was shown to be differentially expressed under copper stress. This demonstrated that the proposed copper operon, copSRXAB, has a role in the Ma.aq copper resistance. CopX was successfully heterologously expressed in Escherichia coli (Es.coli), and purified for the first time using usually two chromatographic steps (anionic exchangeand size exclusion) with a yield of 5.7 mg or 1.8 mg of purified CopX, per L of LB or M9 medium, respectively. Mass spectrometry Electron Spray Ionisation (ESI) and N-terminus analysis revealed that the signal peptide of CopX comprises 21 residues, and is efficiently processed by the Sec system of Es.coli. Biochemical characterization of CopX proved that it is a periplasmic monomeric type 1 copper protein, with a molecular weight of 17253.25 ± 0.30 Da, determined by mass spectrometry (ESI), that binds approximately 1 copper ion per polypeptide chain. The apparent molecular weight of CopX, 20.4 kDa, determined by size-exclusion chromatography does not depend on the ionic strength. Spectroscopic characterization showed that it presents an intense charge transfer (Scys – Cu ion) band at 440 nm and 580 nm and 720 nm. The extinction coefficient at 580 nm was found to be 3.8 mM-1cm-1, according to the copper content. CopX EPR spectrum is axial. The 15N HSQC NMR spectra of CopX confirms that it is folded, with 131 out of 147 backbone amide resonances identified, showing that it is amenable to NMR solution structure determination. CopX presents some unique features, such as, a ratio between A440nm and A580nm of 0.94 and a high hyperfine coupling constant, 170 G. Taking into account the biochemical properties, CopX is proposed to be part of a new class of the type 1 copper proteins, shown preliminarily for the first time to be associated with copper resistance.