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The completion of the 5,373,180-bp genome sequence of the marine psychrophilic bacterium Colwellia psychrerythraea 34H, a model for the study of life in permanently cold environments, reveals capabilities important to carbon and nutrient cycling, bioremediation, production of secondary metabolites, and cold-adapted enzymes. From a genomic perspective, cold adaptation is suggested in several broad categories involving changes to the cell membrane fluidity, uptake and synthesis of compounds conferring cryotolerance, and strategies to overcome temperature-dependent barriers to carbon uptake. Modeling of three-dimensional protein homology from bacteria representing a range of optimal growth temperatures suggests changes to proteome composition that may enhance enzyme effectiveness at low temperatures. Comparative genome analyses suggest that the psychrophilic lifestyle is most likely conferred not by a unique set of genes but by a collection of synergistic changes in overall genome content and amino acid composition.

The γ-carbonic anhydrases (CAs, EC 4.2.1.1) present in the Antarctic marine bacteria Pseudoalteromonas haloplanktis and Colwellia psychrerythraea, herein referred to as PhaCA and CpsCA, respectively, were investigated for their activation with a panel of 24 amino acids and amines. Both bacteria are considered Antarctic models for the investigation of photosynthetic and metabolic pathways in organisms adapted to live in cold seawater. PhaCA was much more sensitive to activation by these compounds compared to the genetically related enzyme CpsCA. The most effective PhaCA activators were d-Phe, l-/d-DOPA, l-Tyr and 2-pyridyl-methylamine, with the activation constant KA values of 0.72–3.27 µM. d-His, l-Trp, d-Tyr, histamine, dopamine, serotonin anddicarboxylic amino acids were also effective activators of PhaCA, with KA values of 6.48–9.85 µM. CpsCA was activated by d-Phe, d-DOPA, l-Trp, l-/d-Tyr, 4-amino-l-Phe, histamine, 2-pyridyl-methylamine and l-/d-Glu with KA values of 11.2–24.4 µM. The most effective CpsCA activator was l-DOPA (KA of 4.79 µM). Given that modulators of CAs from Antarctic bacteria have not been identified and investigated in detail for their metabolic roles to date, this research sheds some light on these poorly understood processes.

Glutathione reductases catalyze the reduction of oxidized glutathione (glutathione disulfide, GSSG) using NADPH as the substrate to produce reduced glutathione (GSH), which is an important antioxidant molecule that helps maintain the proper reducing environment of the cell. A recombinant form of glutathione reductase from Colwellia psychrerythraea , a marine psychrophilic bacterium, has been biochemically characterized to determine its molecular and enzymatic properties. C. psychrerythraea glutathione reductase was shown to be a homodimer with a molecular weight of 48.7 kDa using SDS-PAGE, MALDI-TOF mass spectrometry and gel filtration. The C. psychrerythraea glutathione reductase sequence shows significant homology to that of Escherichia coli glutathione reductase (66 % identity), and it possesses the FAD and NADPH binding motifs, as well as absorption spectrum features which are characteristic of flavoenzymes such as glutathione reductase. The psychrophilic C. psychrerythraea glutathione reductase exhibits higher k cat and k cat / K m at lower temperatures (4 °C) compared to mesophilic Baker’s yeast glutathione reductase. However, C. psychrerythraea glutathione reductase was able to complement an E. coli glutathione reductase deletion strain in oxidative stress growth assays, demonstrating the functionality of C. psychrerythraea glutathione reductase over a broad temperature range, which suggests its potential utility as an antioxidant enzyme in heterologous systems.

Background Colwellia psychrerythraea 34H is a psychrophilic bacterium able to produce docosahexaenoic acid (DHA). Polyketide synthase pathway is assumed to be responsible for DHA production in marine bacteria. Results Five pfa genes from strain 34H were confirmed to be responsible for DHA formation by heterogeneous expression in Escherichia coli . The complexity of fatty acid profile of this strain was revealed by GC and GC–MS. Treatment of cells with cerulenin resulted in significantly reduced level of C16 monounsaturated fatty acid (C16:1 Δ9t , C16:1 Δ7 ). In contrast, the amount of saturated fatty acids (C10:0, C12:0, C14:0), hydroxyl fatty acids (3-OH C10:0 and 3-OH C12:0), as well as C20:4ω3, C20:5ω3 and C22:6ω3 were increased. RNA sequencing (RNA-Seq) revealed the altered gene expression pattern when C. psychrerythraea cells were treated with cerulenin. Genes involved in polyketide synthase pathway and fatty acid biosynthesis pathway were not obviously affected by cerulenin treatment. In contrast, several genes involved in fatty acid degradation or β-oxidation pathway were dramatically reduced at the transcriptional level. Conclusions Genes responsible for DHA formation in C. psychrerythraea was first cloned and characterized. We revealed the complexity of fatty acid profile in this DHA-producing strain. Cerulenin could substantially change the fatty acid composition by affecting the fatty acid degradation at transcriptional level. Acyl-CoA dehydrogenase gene family involved in the first step of β-oxidation pathway may be important to the selectivity of degraded fatty acids. In addition, inhibition of FabB protein by cerulenin may lead to the accumulation of malonyl-CoA, which is the substrate for DHA formation.

In the two cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea ( Cm IDH and Cp IDH, respectively), the combined substitutions of amino acid residues between the Leu693, Leu724 and Phe735 residues of Cm IDH and the corresponding Phe693, Gln724 and Leu735 residues of Cp IDH were introduced by site-directed mutagenesis. A double mutant of Cm IDH substituted its Leu724 and Phe735 residues by the corresponding ones of Cp IDH, Cm L724Q/F735L, and the triple mutant of Cp IDH, Cp F693L/Q724L/L735F, showed the most decrease and increase of activity, respectively, of each wild-type and its all mutated enzymes. In the case of Cm IDH, the substitutions of these three amino acid residues resulted in the decrease of catalytic activity and thermostability for activity, but the combined substitutions of amino acid residues did not necessarily exert additive effects on these properties. On the other hand, similar substitutions in Cp IDH had quite opposite effects to Cm IDH, and the effects of the combined substitutions were additive. All multiple mutants of Cm IDH and Cp IDH showed lower and higher catalytic efficiency ( k cat / K m ) values than the respective wild-type enzymes. Single and multiple mutations of the substituted amino acid residues in the Cm IDH and Cp IDH led to the increase and decrease of sensitivity to tryptic digestion, indicating that the stability of protein structure was decreased and increased by the mutations, respectively.

We investigated the protein folding mechanism of the GroEL system of a psychrophilic bacterium, Colwellia psychrerythraea 34H. The amount of mRNA of the groESL operon of C. psychrerythraea was increased about 6-fold after a temperature upshift from 8 to 18 °C for 30 min, suggesting that this temperature causes heat stress in this bacterium. A σ 32 -type promoter was found upstream of the groESL , suggesting that the C. psychrerythraea groESL is regulated by the σ 32 system, like the groESL in E. coli . The maximum ATPase and CTPase activities of Cp GroEL were observed at 45 and 35 °C, respectively, which are much higher than the growth temperatures of C. psychrerythraea . We found that the refolding activity of the Cp GroEL system in the presence of ATP is lower than that in the presence of CTP. This suggests that ATP is not the optimum energy source of the Cp GroEL system. Analyses for the interaction of Cp GroEL– Cp GroES revealed that CTP could weaken this interaction, resulting in effective refolding function of the Cp GroEL system. From these findings, we consider that the Cp GroEL system possesses an energy-saving mechanism for avoiding excess consumption of ATP to ensure growth in a low-temperature environment.

Two structurally different monomeric and dimeric types of isocitrate dehydrogenase (IDH; EC 1.1.1.42) isozymes were confirmed to exist in a psychrophilic bacterium, Colwellia psychrerythraea, by Western blot analysis and the genes encoding them were cloned and sequenced. Open reading frames of the genes (icd-M and icd-D) encoding the monomeric and dimeric IDHs of this bacterium, IDH-M and IDH-D, were 2,232 and 1,251 bp in length and corresponded to polypeptides composed of 743 and 416 amino acids, respectively. The deduced amino acid sequences of the IDH-M and IDH-D showed high homology with those of monomeric and dimeric IDHs from other bacteria, respectively. Although the two genes were located in tandem, icd-M then icd-D, on the chromosomal DNA, a Northern blot analysis and primer extension experiment revealed that they are transcribed independent of each other. The expression of the monomeric and dimeric IDH isozyme genes in C. maris, a psychrophilic bacterium of the same genus as C. psychrerythraea, is known to be induced by low temperature and acetate, respectively, but no such induction in the expression of the C. psychrerythraea icd-M and icd-D genes was detected. IDH-M and IDH-D overexpressed in Escherichia coli were purified and characterized. In C. psychrerythraea, the IDH-M isozyme is cold-active whereas IDH-D is mesophilic, which is similar to C. maris that contains both cold-adapted and mesophilic isozymes of IDH. Experiments with chimeric enzymes between the cold-adapted monomeric IDHs of C. psychrerythraea and C. maris (IDH-M and ICD-II, respectively) suggested that the C-terminal region of the C. maris IDH-II is involved in its catalytic activity.

The gene (icl) encoding cold-adapted isocitrate lyase (ICL) of a psychrophilic bacterium, Colwellia psychrerythraea, was cloned and sequenced. Open reading frame of the gene was 1,587 bp in length and corresponded to a polypeptide composed of 528 amino acids. The deduced amino acid sequence showed high homology with that of cold-adapted ICL from other psychrophilic bacterium, C. maris (88% identity), but the sequential homology with that of the Escherichia coli ICL was low (28% identity). Primer extension analysis revealed that transcriptional start site for the C. psychrerythraea icl gene was guanine, located at 87 bases upstream of translational initiation codon. The expression of this gene in the cells of an E. coli mutant defective in ICL was induced by not only low temperature but also acetate. However, cis-acting elements for cold-inducible expression known in the several other bacterial genes were absent in the promoter region of the C. psychrerythraea icl gene. The substitution of Ala214 for Ser in the C. psychrerythraea ICL introduced by point mutation resulted in the increased thermostability and lowering of the specific activity at low temperature, indicating that Ala214 is important for psychrophilic properties of this enzyme.

Isocitrate lyase (ICL) from Colwellia psychrerythraea, a psychrophilic bacterium, was purified and characterized. The subunit molecular mass was 64 kDa, which is larger than that of other bacterial ICLs. The optimal temperature for its activity was 25 degrees C, the value of K(m) for the substrate ( DL-isocitrate) was minimum at 15 degrees C, and the catalytic efficiency ( k(cat)/ K(m)) value was maximum at 20 degrees C. Furthermore, the enzyme was remarkably thermolabile and completely inactivated by incubation for 2 min at 30 degrees C. These features indicate that ICL from this bacterium is a typical cold-adapted enzyme. A partial amino acid sequence of the C. psychrerythraea ICL was very similar to that of the closely related psychrophile Colwellia maris. Expression of the gene encoding the C. psychrerythraea ICL was found to be induced by low temperatures and by acetate in the medium. The cold adaptation of the catalytic properties of ICL and the stimulated expression of its gene at low temperatures strongly suggest that this enzyme is important for the growth of this bacterium in a cold environment.