Characterization of the first eukaryotic cold-adapted patatin-like phospholipase from the psychrophilic Euplotes focardii: Identification of putative determinants of thermal-adaptation by comparison with the homologous protein from the mesophilic Euplotes crassus

The ciliated protozoon Euplotes focardii, originally isolated from the coastal seawaters of Terra Nova Bay in Antarctica, shows a strictly psychrophilic phenotype, including optimal survival and multiplication rates at 4–5 °C. This characteristic makes E. focardii an ideal model species for identifying the molecular bases of cold adaptation in psychrophilic organisms, as well as a suitable source of novel cold-active enzymes for industrial applications. In the current study, we characterized the patatin-like phospholipase from E. focardii (EfPLP), and its enzymatic activity was compared to that of the homologous protein from the mesophilic congeneric species Euplotes crassus (EcPLP). Both EfPLP and EcPLP have consensus motifs conserved in other patatin-like phospholipases.     

Polymer hydrolysis in a cold climate

In this review we discuss the activity of an ecologically significant group of psychrophilic bacteria, which are involved in the hydrolysis of plant cell wall polymers. Until now these organisms have been largely overlooked, despite the key role they play in releasing organic carbon fixed by primary producers in permanently cold environments such as Antarctica. This review details a specific group of plant cell wall polymer-degrading enzymes known as β-glycanases. Studies on "cold" enzymes in general are in their infancy, but it has been shown that many exhibit structural and functional modifications that enable them to function at low temperature. β-Glycanases in particular are intriguing because their substrates (cellulose and xylan) are very refractile, which may indicate that their "cold" modifications are pronounced. In addition, mesophilic β-glycanases have been extensively studied and the current state of our knowledge is reviewed. This body of information can be exploited to enable meaningful comparative studies between mesophilic and psychrophilic β-glycanases. The aim of such investigations is to obtain a deeper insight into those structural and functional modifications that enable these enzymes to function at low temperature and to examine the evolutionary relationship between mesophilic and psychrophilic β-glycanases. Received: December 21, 1998 / Accepted: February 3, 1999     

Purification and properties of a new psychrophilic metalloprotease (Fpp2) in the fish pathogen Flavobacterium psychrophilum

To go further into the characterization of the proteolysis exocellular system of the salmonid pathogen Flavobacterium psychrophilum, the purification and characterization of a novel protease designated Fpp2 (F. psychrophilum protease 2) was undertaken. A protease (Fpp2) hydrolyzing azocasein was purified. The Fpp2 can be defined as a metalloprotease, it had an estimated molecular mass of 62 kDa with calcium playing an important role in the thermostability of the enzyme. Proteolytic activity was optimal at pH 6.0-7.0 and 24 degrees C and activation energy for the hydrolysis of azocasein was determined to be 5.4 kcal mol(-1), being inactive at temperatures above 42 degrees C. All these results are characteristic of 'cold adapted enzymes'. Fpp2 proved to be a broad range hydrolytic enzyme because in optimal conditions it was able to hydrolyze matrix and muscular proteins. It can be concluded that the Fpp1, a previously characterized 55 kDa metalloprotease, and the Fpp2 protease were produced under different physiological conditions and were immunologically as well as biochemically different.     

Anaerobic treatment of 2,4,6-trichlorophenol in an expanded granular sludge bed-anaerobic filter (EGSB-AF) bioreactor at 15 degrees C

Expanded granular sludge bed-anaerobic filter (EGSB-AF) bioreactors were operated at 15 degrees C for the treatment of 2,4,6-trichlorophenol (TCP)-containing volatile fatty acid (VFA)-based wastewaters. The seed sludge used as inoculum for the control (no TCP) and test reactor was unexposed to chlorophenols (CPs) prior to the 425-day trial. TCP supplementation to the feed at 50 mg TCPl(-1) partially inhibited the anaerobic degradation of the VFA feed measured as COD removal efficiency. However, the withdrawal and subsequent application of stepwise increments to the TCP loading resulted in steady COD removal. Terminal restriction fragment length polymorphism analysis showed Methanosaeta-like Archaea in the control reactor over the experimental period. Different methanogenic populations were detected in the test reactor and responded to the changes in feed composition. Bacterial community analyses indicated changes in the community structure over time and suggested the presence of Campylobacter-like, Acidimicrobium-like and Heliophilum-like organisms in the samples. TCP mineralisation was by a reductive dechlorination pathway through 2,4-dichlorophenol (DCP) and 4-chlorophenol (4-CP) or 2-chlorophenol (2-CP). CP degradation rates in sludge granules from the lower chamber of the hybrid EGSB-AF reactor was in the order TCP > DCP > 4-CP > 2-CP. However, a biodegradability order of lower CPs > TCP was observed in fixed-film biomass taken from the upper reactor chamber, thus reflecting the role of this reactor section in the metabolism of residual lower CPs from the lower sludge-bed stage of operation.     

Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans

In the present study, psychrophilic yeasts, which grow on lactose as a sole carbon source at low temperature and under acidic conditions, were isolated from soil from Hokkaido, Japan. The phenotypes and sequences of 28S rDNA of the isolated strains indicated a taxonomic affiliation to Guehomyces pullulans. The isolated strains were able to grow on lactose at below 5 degrees C, and showed cold-active acid beta-galactosidase activity even at 0 degrees C and pH 4.0 in the extracellular fractions. Moreover, K(m) of beta-galactosidase activity for lactose in the extracellular fraction from strain R1 was found to be 50.5 mM at 10 degrees C, and the activity could hydrolyze lactose in milk at 10 degrees C. The findings in this study indicate the possibility that the isolated strains produce novel acid beta-galactosidases that are able to hydrolyze lactose at low temperature.     

Degradation of 2,4,6-tribromophenol and 2,4,6-trichlorophenol by aerobic heterotrophic bacteria present in psychrophilic lakes

Halogenated compounds have been incorporated into the environment, principally through industrial activities. Nonetheless, microorganisms able to degrade halophenols have been isolated from neither industrial nor urban environments. In this work, the ability of bacterial communities from oligotrophic psychrophilic lakes to degrade 2,4,6-tribromophenol and 2,4,6-trichlorophenol, and the presence of the genes tcpA and tcpC described for 2,4,6-trichlorophenol degradation were investigated. After 10 days at 4°C, the microcosms showed the ability to degrade both halophenols. Nonetheless, bacterial strains isolated from the microcosms did not degrade any of the halophenols, suggesting that the degradation was done by a bacterial consortium. Genes tcpA and tcpC were not detected. Results demonstrated that the bacterial communities present in oligotrophic psycrophilic lakes have the ability to degrade halophenolic compounds at 4°C and the enzymes involved in their degradation could be codified in genes different to those described for bacteria isolated from environments contaminated by industrial activities.     

Characterization of the pheromone gene family of an Antarctic and Arctic protozoan ciliate, Euplotes nobilii

Allelic genes encoding water-borne signal proteins (pheromones) were amplified and sequenced from the somatic (macronuclear) sub-chromosomic genome of Antarctic and Arctic strains of the marine ciliate, Euplotes nobilii. Their open reading frames appeared to be specific for polypeptide sequences of 83 to 94 amino acids identifiable with cytoplasmic pheromone precursors (pre-pro-pheromones), requiring two proteolytic steps to remove the pre- and pro-segments and secrete the mature pheromones. Differently from most of the macronuclear genes that have so far been characterized from Euplotes and other hypotrich ciliates, the 5′ and 3′ non-coding regions of all the seven E. nobilii pheromone genes are much longer than the coding regions (621 to 700 versus 214 to 285 nucleotides), and the 5′ regions in particular show nearly identical sequences across the whole set of pheromone genes. These structural peculiarities of the non-coding regions are likely due to the presence of intron sequences and provide presumptive evidence that they are site of basic, conserved activities in the mechanism that regulates the expression of the E. nobilii pheromone genes.     

Structural and denaturation studies of two mutants of a cold adapted superoxide dismutase point to the importance of electrostatic interactions in protein stability

A peculiar feature of the psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis (PhSOD) is the presence in its amino acid sequence of a reactive cysteine (Cys57). To define the role of this residue, a structural characterization of the effect of two PhSOD mutations, C57S and C57R, was performed. Thermal and denaturant-induced unfolding of wild type and mutant PhSOD followed by circular dichroism and fluorescence studies revealed that C→R substitution alters the thermal stability and the resistance against denaturants of the enzyme, whereas C57S only alters the stability of the protein against urea. The crystallographic data on the C57R mutation suggest an involvement of the Arg side chain in the formation of salt bridges on protein surface. These findings support the hypothesis that the thermal resistance of PhSOD relies on optimization of charge–charge interactions on its surface. Our study contributes to a deeper understanding of the denaturation mechanism of superoxide dismutases, suggesting the presence of a structural dimeric intermediate between the native state and the unfolded state. This hypothesis is supported by the crystalline and solution data on the reduced form of the enzyme.     

New species of psychrophilic acetogens: Acetobacterium bakii sp. nov., A. paludosum sp. nov., A. fimetarium sp. nov.

Three strains of new acetogenic bacteria were isolated from several low temperature environments. Cells were gram-positive, oval-shaped flagellated rods. The organisms fermented H₂/CO₂, CO, formate, lactate, and several sugars to acetate. Strains Z-4391 and Z-4092 grew in the temperature range from 1 to 30°C with an optimum at 20°C; strain Z-4290 grew in the range from 1 to 35°C with an optimum at 30°C. The DNA G+C content of strains Z-4391, Z-4092, and Z-4290 was 42.1, 41.7, and 45.8 mol% respectively.