Moritella Profunda Research Articles

Expression, purification, crystallization and preliminary X-ray crystallographic studies of a cold-adapted aspartate carbamoyltransferase from Moritella profunda.

Aspartate carbamoyltransferase (ATCase) catalyzes the carbamoylation of the alpha-amino group of L-aspartate by carbamoyl phosphate (CP) to yield N-carbamoyl-L-aspartate and orthophosphate in the first step of de novo pyrimidine biosynthesis. Apart from its key role in nucleotide metabolism, the enzyme is generally regarded as a model system in the study of proteins exhibiting allosteric behaviour. Here, the successful preparation, crystallization and diffraction data collection of the ATCase from the psychrophilic bacterium Moritella profunda are reported. To date, there is no structural representative of a cold-adapted ATCase. The structure of M. profunda ATCase is thus expected to provide important insights into the molecular basis of allosteric activity at low temperatures. Furthermore, through comparisons with the recently reported structure of an extremely thermostable ATCase from Sulfolobus acidocaldarius, it is hoped to contribute to general principles governing protein adaptation to extreme environments. A complete native data to 2.85 A resolution showed that the crystal belongs to space group P3(2)21, with unit-cell parameters a = 129.25, b = 129.25, c = 207.23 A, alpha = beta = 90, gamma = 120 degrees, and that it contains three catalytic and three regulatory subunits per asymmetric unit. The three-dimensional structure of the Escherichia coli ATCase was sufficient to solve the structure of the M. profunda ATCase via the molecular-replacement method and to obtain electron density of good quality.

Moritella profunda sp. nov. and Moritella abyssi sp. nov., two psychropiezophilic organisms isolated from deep Atlantic sediments.

Strains 2674T (=LMG 21259T =JCM 11435T) and 2693T (=LMG 21258T =JCM 11436T) were isolated from Atlantic sediments at a temperature of 2 degrees C and a depth of 2815 m off the West African coast. Polyphasic evidence indicates that the two strains belong to the genus Moritella and represent distinct species, for which the names Moritella profunda sp. nov. (for strain 2674T) and Moritella abyssi sp. nov. (for strain 2693T) are proposed. The moderate piezophily of the two organisms is intermediate between that of the type species, Moritella marina, which is not piezophilic, and Moritella yayanosii, an obligate piezophile. Both are strict psychrophiles with slightly different cardinal temperatures: at 0.1 MPa, maximal growth rates are observed at 2 degrees C (M. profunda) and 4 degrees C (M. abyssi) with maximum temperatures of 12 degrees C (M. profunda) or 14 degrees C (M. abyssi). The optimal pressure is lower than that at the site of isolation, and raising the temperature to 10 degrees C makes the organisms more piezophilic.

Regulation of Arginine Biosynthesis in the Psychropiezophilic Bacterium Moritella profunda: in vivo Repressibility and in vitro Repressor–Operator Contact Probing

We report the cloning of the arginine repressor gene from the psychropiezophilic Gram-negative bacterium Moritella profunda, the purification of its product (ArgR Mp), the identification of the operator in the bipolar argECBFGH( A) operon, in vivo repressibility studies, and an in vitro analysis of the repressor–operator interaction, including binding to mutant and heterologous arginine operators. The ArgR Mp subunit shows about 70% amino acid sequence identity with Escherichia coli ArgR (ArgR Ec). Binding of purified hexameric ArgR Mp to the control region of the divergent operon proved to be arginine-dependent, sequence-specific, and significantly more sensitive to heat than complex formation with ArgR Ec. ArgR Mp binds E. coli arginine operators very efficiently, but hardly recognizes the operator from Bacillus stearothermophilus or Thermotoga maritima. ArgR Mp binds to a single site overlapping the −35 element of argC P, but not argE P. Therefore, the arrangement of promoter and operator sites in the bipolar argECBFGH( A) operon of M. profunda is very different from the organization of control elements in the bipolar argECBH operon of E. coli , where both promoters overlap the common operator and are equally repressible. We demonstrate that M. profunda argC P is about 44-fold repressible, whereas argE P is fully constitutive. A high-resolution contact map of the ArgR Mp–operator interaction was established by enzymatic and chemical footprinting, missing contact and base-specific premodification binding interference studies. The results indicate that the argC operator consists of two ARG box-like sequences (18 bp imperfect palindromes) separated by 3 bp. ArgR Mp binds to one face of the DNA helix and establishes contacts with two major groove segments and the intervening minor groove of each ARG box, whereas the minor groove segment facing the repressor at the center of the operator remains largely uncontacted. This pattern is reminiscent of complex formation with the repressors of E. coli and B. stearothermophilus , and suggests that each ARG box is contacted by two ArgR subunits belonging to opposite trimers. Moreover, the premodification interference patterns and mutant studies clearly indicate that the inner, center proximal halves of each ARG box in the M. profunda argC operator are more important for complex formation and repression than the outermost halves. A close inspection of sequence conservation and of single base-pair O c-type mutations indicate that the same conclusion can be generalized to E. coli operators.