The hormone-sensitive lipase from Psychrobacter sp. TA144: New insight in the structural/functional characterization

Cold-adapted esterases and lipases have been found to be dominant activities throughout the cold marine environment, indicating their importance in bacterial degradation of the organic matter. lip2 Gene from Psychrobacter sp. TA144, a micro-organism isolated from the Antarctic sea water, was cloned and over-expressed in Escherichia coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterised. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 °C at pH 8.0. The enzyme resulted to be active on pNP-esters of fatty acids with acyl chain length from C₂ to C₁₂ and the preferred substrate was pNP-pentanoate showing a k_cat = 26.2 ± 0.1 s⁻¹, K_M = 0.122 ± 0.006 mM and a k_cat/K_M = 215 ± 11 mM⁻¹ s⁻¹. The enzyme was strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Fe³⁺, Mn²⁺ ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C₅₀ values of 1.98 M and 0.92 M, respectively.     

Optimization of extracellular psychrophilic alkaline lipase produced by marine Pseudomonas sp. (MSI057)

An endosymbiotic Pseudomonas sp. (MSI057), which could produce high yields of lipase, was isolated from marine sponge Dendrilla nigra, collected from the peninsular coast of India. Maximum production of enzyme was obtained in minimal medium supplemented with 1% tributyrin. Catabolite repression was observed when the medium was supplemented with readily available carbon sources. The optimum temperature and pH for the enzyme production was 30 degrees C and 9.0, respectively. The enzyme exhibited maximum activity in pH range of 8-9 with an optimum pH 9.0. The activity of purified enzyme was optimum at 37 degrees C and showed 80% activity at 20 degrees C and the enzyme activity decreased dramatically above 50 degrees C. Based on the present findings, the enzyme was characterized as psychrophilic alkaline lipase, which can be developed for industrial applications.     

Rhodotorula himalayensis sp. nov., a novel psychrophilic yeast isolated from Roopkund Lake of the Himalayan mountain ranges, India

Twenty-five psychrophilic yeasts were isolated from the soil of Roopkund Lake, Himalayas, India. Two colony morphotypes were identified and representatives of ‘morphotype 1’ were identified as Cryptococcus gastricus. Representatives of ‘morphotype 2’, namely 3A^T, 4A, 4B and Rup4B, showed similar phenotypic properties and are identical with respect to the nucleotide sequence of the ITS1-5.8S rRNA gene-ITS2 region and D1/D2 domain of the 26S rRNA gene. The sequence of D1/D2 domain of 3A^T shows 97.6–98.8% similarity with Rhodotorula psychrophila CBS10440^T, Rhodotorula glacialis CBS10437^T and Rhodotorula psychrophenolica CBS10438^T and in the neighbour-joining phylogenetic tree strains; 3A^T, 4A, 4B and Rup4B form a cluster with Rhodotorula glacialis and Rhodotorula psychrophila. Strains 3A^T, 4A, 4B and Rup4B also differ from their nearest phylogenetic relatives in several biochemical characteristics such as in assimilation of d-galactose, l-sorbose, maltose, citrate, d-glucuronate and creatinine. Thus, based on the phylogenetic analysis and the phenotypic differences 3A^T, 4A, 4B and Rup 4B are assigned the status of a new species of Rhodotorula for which the name Rhodotorula himalayensis sp. nov. is proposed with 3A^T as the type strain (=CBS10539^T =MTCC8336^T). GenBank/EMBL accession numbers for (partial) 18SrRNA gene-ITS1-5.8S rRNA gene-ITS2-26S rRNA gene (partial) sequences of Rhodotorula himalayensis sp. nov. 3A^T is AM410635.     

Gene structure and transcriptional regulation of <Emphasis Type="Italic">dnaK</Emphasis> and <Emphasis Type="Italic">dnaJ</Emphasis> genes from a psychrophilic bacterium, <Emphasis Type="Italic">Colwellia maris</Emphasis>

The dnaK and dnaJ genes, encoding heat shock proteins, were cloned from a psychrophilic bacterium, Colwellia maris. Significant homology was evident comparing DnaK and DnaJ of the psychrophilile with the counterparts of mesophilic and thermophilic bacteria. In the DnaJ protein, three conserved regions of the Hsp40 family were observed. A putative promoter similar to the sigma32 consensus sequence was found upstream of the dnaK gene. The G+C content in the 5'-untranslated region of the dnaK gene was much lower than that in the corresponding region of mesophilic bacteria. Northern-blot analysis and primer-extension analysis showed that both genes were transcribed separately as monocistronic mRNAs. Following several temperature upshifts from 10 to 26 degrees C, maximum induction of the dnaK and dnaJ mRNAs was detected at 20 degrees C, suggesting that this temperature induces the heat shock response in this bacterium. In addition, the level of the induction of the dnaJ gene was much lower than that of the dnaK gene. These findings together revealed several specific features of the heat shock response at a relatively low temperature in psychrophiles.     

Toward a molecular understanding of cold activity of enzymes from psychrophiles

Despite the fact that a much greater proportion of the earth environment is cold rather than hot, much less is known about psychrophilic, cold-adapted microorganisms compared with thermophiles living at high temperatures. In particular, investigation of the molecular basis of cold-active enzymes from psychrophiles has only recently received concerted research attention, in measure as a result of the EC-funded project COLDZYME. This research effort has been stimulated by the realization that such cold-active enzymes offer novel opportunities for biotechnological exploitation. Only very recently has the first cold-active enzyme, α-amylase, been crystallized, and this success was followed rapidly by others. This effort has facilitated a direct approach to solving the three-dimensional structure of cold-active enzymes to complement the gene homology modeling that had been performed previously. Recently studies have highlighted how different adaptations are used by different enzymes to achieve conformational flexibility at low temperatures, and how such adaptations are not necessarily the opposite of those that confer thermostability to proteins in thermophilic counterparts. This review also highlights initial successes in engineering genetically improved thermal stability in cold-active enzymes to give improved catalysts for low-temperature biotechnology. Received: July 11, 1999 / Accepted: December 27, 1999     

Ecological and biogeographic relationships of class Flavobacteria in the Southern Ocean

The abundance, spatial distribution and diversity of class Flavobacteria were investigated in the Southern Ocean euphotic zone across a latitudinal transect and in the ice pack off Eastern Antarctica. Surface seawater samples filter-fractionated into 0.8 mum particulate and 0.2 m planktonic fractions were investigated with different molecular techniques. The abundance of particle-associated Flavobacteria, ascertained with real-time PCR and DGGE band analysis using Flavobacteria-specific primers, was found to be significantly higher in Polar Front Zone (PFZ) and Antarctic Zone (AZ) water samples than in nutrient limited Temperate Zone (TZ) and Sub-Antarctic Zone (SAZ) waters. Abundance of particle-associated Flavobacteria correlated positively with seawater chlorophyll a and nutrient concentrations, suggesting that increased Flavobacteria abundance may relate to enhanced primary production in the PFZ and AZ. This is supported by comparison of DGGE profiles that demonstrated significant differences in the total Flavobacteria community structure and 16S rRNA gene diversity between samples from the PFZ and AZ and those from TZ and SAZ. Sequence analysis revealed a broad diversity amongst class Flavobacteria in the Southern Ocean with several Flavobacteria clades detected in PFZ and AZ waters not detected in TZ and SAZ waters that putatively represent psychrophilic taxa. Sequence data included a large, so far uncultivated, cosmopolitan phylogenetic clade ("DE cluster 2") that is distributed throughout the Southern Ocean.     

Molecular and functional properties of the psychrophilic elongation factor G from the Antarctic Eubacterium <Emphasis Type="Italic">Pseudoalteromonas haloplanktis</Emphasis> TAC 125

The molecular and functional properties of the elongation factor (EF) G from the psychrophilic Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) were studied. PhEF-G catalyzed protein synthesis in vitro that was inhibited by fusidic acid, an antibiotic specifically acting on EF-G. The EF interacted with GDP only in the presence of P. haloplanktis ribosome and fusidic acid with an affinity similar to that displayed by Escherichia coli EF-G. The psychrophilic translocase elicited a ribosome-dependent GTPase that was competitively inhibited by GDP, the slowly hydrolyzable GTP analog GppNHp, and the protein synthesis inhibitor ppGDP. The temperature dependence of the activity of PhEF-G reached its maximum at least 26°C beyond the growth temperature of P. haloplanktis (4–20°C). The heat inactivation profile of the ribosome-dependent GTPase of PhEF-G gave a temperature for half inactivation (46°C), significantly lower than that for half denaturation measured by either UV- (57°C) or fluorescence-melting (62°C). This finding was attributed to a different effect of the temperature on the catalytic domain with respect to that elicited on the other domains constituting the EF, thus confirming the differential molecular flexibility present in psychrophilic enzymes. A molecular model, based on the 3D coordinates of a thermophilic EF-G, showed differences only in connecting loops. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biochemical and phylogenetic analyses of psychrophilic isolates belonging to the Arthrobacter subgroup and description of Arthrobacter psychrolactophilus, sp. nov.

During our work on psychrophilic microorganisms we obtained a large collection of new isolates. In order to identify six of these, we examined their growth properties, cell wall compositions, and their 16S rRNA gene sequences. The results showed that all of the isolates are gram-positive, aerobic, contain lysine in their cell walls, and belong to the high mol% G+C Arthrobacter subgroup. Phylogenetic analysis of the 16S rRNA genes grouped five isolates obtained from a small geographical region into a monophyletic clade. Isolate B7 had a 16S rRNA sequence that was 94.3% similar to that of Arthrobacter polychromogenes and 94.4% similar to that of Arthrobacter oxydans. Primary characteristics that distinguish isolate B7 from the Arthrobacter type strain (Arthrobacter globiformis) and A. polychromogenes include lack of growth at 37 degrees C, growth at 0-5 degrees C, the ability to use lactose as a sole carbon source, and the absence of blue pigments. Because of these differences, isolate B7 was chosen as a type strain representing a new Arthrobacter species, Arthrobacter psychrolactophilus. The sixth isolate, LV7, differed from the other five because it did not have the rod/ coccus morphological cycle and was most closely related to Arthrobacter agilis.     

嗜冷弧菌2693菌株的天冬氨酸转氨基甲酰酶基因克隆及测序

对嗜冷弧菌２６９３菌株编码天冬氨酸氨甲酰基转移酶的基因进行了克隆,并对其核苷酸序列进行了分析。菌株２６９３的ＡＴＣａｓｅ由ｐｒｙＢＩ操纵子ｐｙｒＢ编码催化链,ｐｙｒＩ编码调节莲。催化及调节多肽链由一条单一的双顺反操纵子编码,在同一启动基因控制之下进行转录。