CRYSTAL STRUCTURE AND EXPRESSION OF A PUTATIVE PHAGE-LIKE PROTEIN CODED IN THE GENOME OF A MARINE ANTARCTIC BACTERIA

LEONARDO PELLIZZA; MARTÍN ARAN; SEBASTIÁN KLINKE; JIMENA RINALDI; GABRIELA SYCZ; CLARA SMAL; GASTÓN PARIS; FERNANDO A. GOLDBAUM; DANIEL O. CICERO; JOSÉ L. LOPEZ; BEATRIZ G. GUIMARAES; ESTEBAN LANZAROTTI; ADRIAN G. TURJANSKI; SUSANA C. VAZQUEZ; WALTER P. MAC CORMACK

České Budějovice

Congreso; 6TH INTERNATIONAL CONFERENCE ON POLAR AND ALPINE MICROBIOLOGY; 2015

INTRODUCTIONBizionia argentinensis JUB59T (B.a) was isolated from surface marine water in Potter Cove, (62° 149´ S; 58° 409´W), Antarctica and described as a new species. JUB59 is a psychrotolerant marine bacteria; Gram-negative, nonmotile rods forming yellowish orange colonies on marine agar (Bercovich et al. 2008). The sequencing of JUB59Tgenome (GenBank AFXZ00000000) suggested interesting features such as UV resistance, hydrolytic exoenzymes,and nitrogen metabolism. Several putative gene sequences, of unknown biological function and with possible newfolds were selected, cloned, expresed and its products were purified from the culture broth. One of the B.a gene products, named C24, showed structural homology with a T4 phage tail fiber protein. In this work, we investigatethe structure and expresion of C24.METHODOLOGYC24 gene was amplified by PCR from B.a chromosomal DNA, cloned into the expression plasmid pDEST-527 andexpressed in E. coli BL21(DE3). The protein was purified using a HisTrap HP column, dialyzed and concentrated.Rhomboidal-shaped crystals with a maximum size of 0.3 mm x 0.2 mm x 0.1 mm were obtained and analyzed at 100 K at the PROXIMA 1 protein crystallography beamline at the SOLEIL synchrotron, France. Superpositions and rmsd calculations were done with the PDBeFold server and the study of interfaces, monomers and assemblieswith the PDBePISA server at the EBI. The likely induction by stress of C24 expression in B.a was tested in Erlenmeyer flasks with 30 ml of marine broth (10°C and 200 rpm). When cultures reached stationary phase, onewas left as a control and the others were subjected to different treatments [shift to high (28 °C) and low (0 °C) temperature, high (9 g l-1) and low (0.8 g l-1) salinity, oligotrophy and presence of other related (B. myxarmorumADA-4T) and non-related (Cellulophaga sp. S03-8, Pseudomonas sp. P96-50 and Shewanella sp. Ag06-30) Antarctic bacteria]. Samples were taken after 1h and 7h, total RNA was extracted and the presence of C24 transcripts wasdetected by PCR using specific primers and the products obtained were sequenced to confirm their identity. The same primers were used to detect the presence of C24 gene in other Antarctic bacterial isolates.RESULTSA construct harboring the bzarg_797 gene from B.a was expressed and thestructure of the 277-residue protein called C24 was solved. A unique 89.1 kDa trimer was found in the asymmetric unit of the crystal, which corresponds to the expected biological assembly of the protein. The protein binds a unique Mn (II) ion which is octahedrally coordinated by six histidine residues. C24 bears four distinct domains, with contribution of the three chains in each of them: i) a globular, unique proximal shoulder domain, ii) a globular collar domain, iii) an elongated, intertwined metal-binding needle domain, and iv) a globular,receptor-binding-like unique distal head domain. The structure of C24 partially resembles that of the receptor-binding tip from the bacteriophage T4 long tail fiber, yet there are marked differences between both in their domain organization, size, sequence identity and metal binding nature. Both structures present a similar overall shape, but they can be superimposed only in the collar, intertwined and needle regions, with the metal binding sites being different in number and also slightly shifted between proteins. The structure similarity search on the globular proximal shoulder and distal head domains returned no homologue partners. The bacteriophage T4 long tail fiber head domain, despite showing ananalogous threaded architecture, fails to superimpose both in structure and in primary sequence to C24. In the latter viral domain, exposed aromatic and positively charged residues are proposed to interact with sugar residuesand their attached phosphate groups, respectively, in E. coli host cell receptors. Remarkably, the head domain from C24 also presents several positive and aromatic exposed residues with unknown function so far.The C24 gene was not detected in other Bizionia type strains nor in 18 Antarctic isolates of different genus, amongwhich 11 were obtained from Potter Cove. After growing B.a at 10 °C, C24 was expressed and could be detectedby RT-PCR in the control culture and in all of the other stress-induced conditions tested, suggesting a constitutiveexpression of C24 gene at least under the culture conditions used.DISCUSSIONC24 expression seems not to be related with response to stress induced by sudden shifts to extreme environmental change or to the presence of other possible competitor bacteria. It seems to be constitutively expressed at least at the culture conditions used. At present, the role of C24 in B.a could not be elucidated. One possibility is that C24 be a relict from a former lysogenic phage, as at least three different prophages were detected in B.a genome, that were excised after mitomicin C treatment. It could also be related to a type VI transport system or be involved in ?social? behavior like biofilm fomation or attachment to surfaces or even to the growth in other environmental situations non tested so far. Further studies will be conducted with the aim of knowing the role C24 expression has in B.a strain and its likely presence in other bacteria.