Shuttle vectors have also been
constructed from native see more plasmids isolated from other Z. mobilis strains, such as pNSW301 Selleckchem Eltanexor from the ZM6100 strain [26]; pZMPI from the Z. mobilis PROM Al strain [27] and pZA2 from the NCIMB 8827 strain [19]. Of these, the pZM2 (pZMO3) plasmid has been used most extensively for the construction of expression plasmids for physiological investigations or industrial applications in Z. mobilis, e.g. [9, 10, 12, 16, 28, 29]. Most notably, the pZM2-derived pZB5 plasmid, which houses four genes involved in pentose sugar metabolism, was used to broaden the substrate range of the CP4 strain, enabling it to utilize xylose for the bioproduction of ethanol [9, 10]. Plasmids derived from pZM2 have also been used to express green fluorescent protein reporters [30]; to AZD7762 cost produce proteins of biotechnological interest such as the InaZ ice-nucleation protein [28]; to express fungal carotenoid biosynthetic proteins to direct the production of beta-carotene [31]; and to produce and secrete cellulolytic enzymes to facilitate the utilization of lignocellulosic biomass [29]. In microbial cells, proteins often function
within hetero-multimeric complexes, or have activities that are directly modulated by protein-protein interactions [32, 33]. Approaches involving various combinations of affinity chromatography and mass spectrometry have previously been employed to establish large-scale protein interaction networks, known as ‘interactomes’, within prokaryotic and eukaryotic microorganisms [34, 35]. However, to the best of our knowledge, protein-protein interaction analyses have never been performed
in Z. mobilis or a related alphaproteobacterial species. The genome sequence for Z. mobilis NCIMB 11163 was recently published [36]. This included the sequences of three endogenous plasmids: p11163_1 (deposited as pZA1001; 53,380 bp), p11163_2 (pZA1002; 40,818 bp) and p11163_3 (pZA1003; 4,551 bp). This was consistent with results from our own Z. mobilis plasmid sequencing efforts, in which we had determined the sequences of the two smallest plasmids from NCIMB 11163: pZMO1A (1,647 bp) and pZMO7 (4,551 bp) (Figure 1) [37]. The sequences of pZMO7 and p11163_3 (pZA1003) are identical, and they correspond to the same plasmid. Due to its relatively small size and genetic Masitinib (AB1010) composition (see below), we hypothesized that pZMO7 may be suitable for shuttle vector development. Figure 1 Restriction maps of two native plasmids from Z. mobilis NCIMB 11163. (A) pZMO1A (B) pZMO7. The aim of this study was to develop an Escherichia coli-Z. mobilis shuttle-vector system based on pZMO7, and determine its potential for heterologous protein expression and proteomic applications within Z. mobilis. To achieve this, we constructed a shuttle vector backbone (pZ7C) that contained a ca. 1,900 bp replicon fragment from pZMO7.