Psychroflexus Torquis Research Articles

Psychroflexus maritimus sp. nov., isolated from coastal sediment.

A Gram-stain-negative, strictly aerobic, non-flagellated, gliding, rod-shaped bacterium, designated C1T was isolated from the coastal sediment of Xiaoshi Island, Weihai, China. Phylogenetic analysis of the 16S rRNA gene and the genome sequence of the newly isolated strain revealed that it belonged to the genus Psychroflexus within the family Flavobacteriaceae. The 16S rRNA gene sequence similarities between strain C1T and the type strains of Psychroflexus members ranged from 91.6 to 94.9%. The AAI, POCP, ANI and dDDH values between strain C1T and Psychroflexus torquis ATCC 700755T were 67.0%, 53.1%, 70.5% and 20.2%, respectively. The DNA G+C content was 34.0mol%. Strain C1T grows optimally at 28-30℃, at pH 7.5-8.0 and with 3.0-5.0% (w/v) NaCl, and its colonies were orange-colored, convex and circular on the MA plate. Positive for hydrolysis of Tween 20 and catalase activities. The dominant respiratory quinone was menaquinone-6, and the major fatty acids were iso-C17:0 3-OH and iso-C15:0. The polar lipids of strain C1T consisted of phosphatidylethanolamine and three unidentified lipids. On the basis of phylogenetic, phenotypic and chemotaxonomic data, it is considered that strain C1T represents a novel species within the genus Psychroflexus, for which, the name Psychroflexus maritimus sp. nov., is proposed. The type strain is C1T (= MCCC 1H00415T = KCTC 72796T).

Identification and characterization of the psychrophilic bacterium CidnaK gene in the Antarctic Chlamydomoas sp. ICE-L under freezing conditions

Heat shock protein DnaK (Hsp70) can prevent irreversible protein denaturation by chaperoning the unfolded polypeptides under extremely cold environment, and enhance cold and freezing tolerance of microorganism. From an Antarctic sea-ice green alga Chlamydomonas sp. ICE-L, eight Hsp70 genes were identified and divided into seven subfamilies: four in the cytoplasmic subfamilies, two in the chloroplast subgroups, and one in the mitochondria or endoplasmic reticulum subfamilies. Phylogenetic analyses showed that except CidnaK, each of the other Cihsp70 genes had their homologs in Chlamydomonas reinhardtii and Volvox carteri. CiDnak is a cytoplasmic protein with highly homologous to DnaK proteins from prokaryotes, and it had 100% amino acid sequence identities with the DnaK of Psychroflexus torquis ATCC 700755, a psychrophilic bacterium isolated from Antarctic sea ice. The transcription of CidnaK was significantly induced upon freezing stress in ICE-L, eminently higher than that of other Cihsp70 genes. In addition, the transformed C. reinhardtii 137c with CidnaK gene showed much higher rates of survival and growth than the wild type under freezing or low temperature conditions. Our results suggested that CidnaK was likely originated from the symbiotic bacterium in the sea-ice brine, and is an important candidate freezing inducible gene that confers low temperature tolerance to Chlamydomonas cells, and plays a crucial role in ICE-L adapting the frigid Antarctic environments.

In situ light responses of the proteorhodopsin-bearing Antarctic sea-ice bacterium, Psychroflexus torques.

Proteorhodopsin (PR) is a wide-spread protein found in many marine prokaryotes. PR allows for the potential conversion of solar energy to ATP, possibly assisting in cellular growth and survival during periods of high environmental stress. PR utilises either blue or green light through a single amino acid substitution. We incubated the PR-bearing bacterium Psychroflexus torquis 50 cm deep within Antarctic sea ice for 13 days, exposing cultures to diurnal fluctuations in light and temperature. Enhanced growth occurred most prominently in cultures incubated under irradiance levels of ∼50 μmol photons m-2 s-1, suggesting PR provides a strong selective advantage. In addition, cultures grown under blue light yielded over 5.5 times more live cells per photon compared to green-light incubations. Because P. torquis expresses an apparently 'green-shifted' PR gene variant, this finding infers that the spectral tuning of PR is more complex than previously thought. This study supports the theory that PR provides additional energy to bacteria under sub-optimal conditions, and raises several points of interest to be addressed by future research.

Proteomic Insight into Functional Changes of Proteorhodopsin-Containing Bacterial Species Psychroflexus torquis under Different Illumination and Salinity Levels.

The extremely psychrophilic proteorhodopsin-containing bacterial species Psychroflexus torquis is considered to be a model sea-ice microorganism, which has adapted to an epiphytic lifestyle. So far, not much is known about proteorhodopsin-based phototrophy and associated life strategies of sea ice bacteria, although it has been previously shown that P. torquis can gain growth advantage from light using a proteorhodopsin proton pump, the activity of which is influenced by environmental salinity. The comprehensive quantitative proteomic study performed here indicated that P. torquis responds to changing salinity and illumination conditions. Proteins in the electron-transfer chain were down-regulated at a suboptimal salinity level, TonB-dependent transporters increased in abundance under supra-optimal salinity and decreased under suboptimal salinity. In addition, several anaplerotic CO2 fixation proteins and three putative light sensing proteins that contain PAS and GAF domains became more abundant under illumination. Furthermore, central metabolic pathways (TCA and glycolysis) were also induced by both salinity stress and illumination. The data suggest that P. torquis responded to changes in both light energy and salinity to modulate membrane and central metabolic proteins that are involved in energy production as well as nutrient uptake and gliding motility processes that would be especially advantageous during the polar summer ice algal bloom.

Expression of cold-adapted β-1,3-xylanase as a fusion protein with a ProS2 tag and purification using immobilized metal affinity chromatography with a high concentration of ArgHCl.

Cold-adapted β-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes.

Characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria Desulfotalea psychrophila, Flavobacterium psychrophilum, Psychrobacter arcticus, Psychrobacter cryohalolentis, Psychromonas ingrahamii, Psychroflexus torquis, and Photobacterium profundum.

BackgroundSingle-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination and repair in Bacteria, Archaea and Eukarya. In recent years, there has been an increasing interest in SSBs, since they find numerous applications in diverse molecular biology and analytical methods.ResultsWe report the characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria Desulfotalea psychrophila (DpsSSB), Flavobacterium psychrophilum (FpsSSB), Psychrobacter arcticus (ParSSB), Psychrobacter cryohalolentis (PcrSSB), Psychromonas ingrahamii (PinSSB), Photobacterium profundum (PprSSB), and Psychroflexus torquis (PtoSSB). The proteins show a high differential within the molecular mass of their monomers and the length of their amino acid sequences. The high level of identity and similarity in respect to the EcoSSB is related to the OB-fold and some of the last amino acid residues. They are functional as homotetramers, with each monomer encoding one single stranded DNA binding domain (OB-fold). The fluorescence titrations indicated that the length of the ssDNA-binding site size is approximately 30 ± 2 nucleotides for the PinSSB, 31 ± 2 nucleotides for the DpsSSB, and 32 ± 2 nucleotides for the ParSSB, PcrSSB, PprSSB and PtoSSB. They also demonstrated that it is salt independent. However, when the ionic strength was changed from low salt to high, binding-mode transition was observed for the FpsSSB, at 31 ± 2 nucleotides and 45 ± 2 nucleotides, respectively. As expected, the SSB proteins under study cause duplex DNA destabilization. The greatest decrease in duplex DNA melting temperature was observed in the presence of the PtoSSB 17°C. The SSBs in question possess relatively high thermostability for proteins derived from cold-adapted bacteria.ConclusionThe results showed that SSB proteins from psychrophilic microorganisms are typical bacterial SSBs and possess relatively high thermostability, offering an attractive alternative to other thermostable SSBs in molecular biology applications.

Extensive Gene Acquisition in the Extremely Psychrophilic Bacterial Species Psychroflexus torquis and the Link to Sea-Ice Ecosystem Specialism

Sea ice is a highly dynamic and productive environment that includes a diverse array of psychrophilic prokaryotic and eukaryotic taxa distinct from the underlying water column. Because sea ice has only been extensive on Earth since the mid-Eocene, it has been hypothesized that bacteria highly adapted to inhabit sea ice have traits that have been acquired through horizontal gene transfer (HGT). Here we compared the genomes of the psychrophilic bacterium Psychroflexus torquis ATCC 700755T, associated with both Antarctic and Arctic sea ice, and its closely related nonpsychrophilic sister species, P. gondwanensis ACAM 44T. Results show that HGT has occurred much more extensively in P. torquis in comparison to P. gondwanensis. Genetic features that can be linked to the psychrophilic and sea ice-specific lifestyle of P. torquis include genes for exopolysaccharide (EPS) and polyunsaturated fatty acid (PUFA) biosynthesis, numerous specific modes of nutrient acquisition, and proteins putatively associated with ice-binding, light-sensing (bacteriophytochromes), and programmed cell death (metacaspases). Proteomic analysis showed that several genes associated with these traits are highly translated, especially those involved with EPS and PUFA production. Because most of the genes relating to the ability of P. torquis to dwell in sea-ice ecosystems occur on genomic islands that are absent in closely related P. gondwanensis, its adaptation to the sea-ice environment appears driven mainly by HGT. The genomic islands are rich in pseudogenes, insertional elements, and addiction modules, suggesting that gene acquisition is being followed by a process of genome reduction potentially indicative of evolving ecosystem specialism.

Light-stimulated growth of proteorhodopsin-bearing sea-ice psychrophile Psychroflexus torquis is salinity dependent

Proteorhodopsins (PRs) are commonly found in marine prokaryotes and allow microbes to use light as an energy source. In recent studies, it was reported that PR stimulates growth and survival under nutrient-limited conditions. In this study, we tested the effect of nutrient and salinity stress on the extremely psychrophilic sea-ice bacterial species Psychroflexus torquis, which possesses PR. We demonstrated for the first time that light-stimulated growth occurs under conditions of salinity stress rather than nutrient limitation and that elevated salinity is related to increased growth yields, PR levels and associated proton-pumping activity. PR abundance in P. torquis also is post-transcriptionally regulated by both light and salinity and thus could represent an adaptation to its sea-ice habitat. Our findings extend the existing paradigm that light provides an energy source for marine prokaryotes under stress conditions other than nutrient limitation.

Psychroflexus tropicus sp. nov., an obligately halophilic Cytophaga-Flavobacterium-Bacteroides group bacterium from an Hawaiian hypersaline lake.

A Gram-negative bacterium designated LA1(T) was isolated from water collected in hypersaline Lake Laysan on Laysan Island in the Northwestern Hawaiian Islands. Cells occurred singly as fine rods to short filaments. Growth in 50 % strength marine broth occurred optimally when the medium contained 7.5-10 % (w/v) NaCl. The major fatty acids in LA1(T) grown at 15 and 30 degrees C were 12-methyl tetradecanoic acid and 13-methyl tetradecanoic acid, respectively. The nucleotide sequence of the 16S rRNA gene showed that LA1(T) belonged in the Cytophaga-Flavobacterium-Bacteroides (CFB) group in the domain Bacteria. The closest described neighbour in terms of 16S rRNA gene sequence identity was Psychroflexus torquis ACAM 623(T) (94.4 % over 1423 bases), an obligate psychrophile from Antarctic sea-ice. The G+C content of 35.0 mol% was consistent with this affiliation. Phenotypic and genotypic analyses, including DNA hybridization, indicated that LA1(T) could be assigned to the genus Psychroflexus but, based on significant differences, including growth at 43 degrees C, it constitutes a novel species, Psychroflexus tropicus sp. nov., for which LA1(T) (=ATCC BAA-734(T)=DSM 15496(T)) is the type strain.

Distribution of prokaryotic genetic diversity in athalassohaline lakes of the Atacama Desert, Northern Chile

Athalassohaline lakes are inland saline aquatic environments with ionic proportions quite different from the dissolved salts in seawater. Prokaryotes inhabiting athalassohaline environments are poorly known and very few of such places have been surveyed for microbial diversity studies around the world. We analyzed the planktonic bacterial and archaeal assemblages inhabiting several of these evaporitic basins in a remote and vast area in northern Chile by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene fragments. Most systems were springs and athalassohaline ponds in different saltflats of the Atacama Desert region, including Salar de Llamará (in the Central Depression), Salar de Atacama (in the Pre-Andean Depression) and Salar de Ascotán (in the Altiplano). Overall, we analyzed more than 25 samples from 19 different environments with strong gradients of altitude, qualitative ionic compositions and UV influence. Between 4 and 25 well-defined DGGE bands were detected for Bacteria in each sample, whereas Archaea ranged between 1 and 5. Predominant DGGE bands (defined by intensity and frequency of appearance) were excised from the gel and sequenced. Bacterial assemblages were dominated by the Cytophaga–Flavobacterium–Bacteroides (CFB) phylum and a few Proteobacteria. There was a tendency for increasing contribution of CFB with higher salinities and altitude. Thus, CFB accounted for the major fraction of band intensity in the Ascotán samples and for lower percentages in Atacama and Llamará. When the distribution of particular CFB sequences was examined, there were several relatives of Psychroflexus torquis substituting each other as salinity changed in Ascotán. Another set of CFB sequences, very distantly related to Cytophaga marinovorus, was abundant in both Llamará and Atacama at salinities lower than 7%. Archaeal assemblages were dominated by uncultured haloarchaea distantly related to cultured strains mostly obtained from thalassohaline environments. Most of the archaeal sequences did not have a close match with environmental 16S rRNA genes deposited in the database either. Therefore, athalassohaline environments are excellent sources of new microorganisms different from their counterparts in thalassohaline sites and useful tools to relate microbial genetic diversity and environmental characteristics such as changes in salinity (both qualitative and quantitative) and altitude.

Distribution of two triamines, spermidine and homospermidine, and an aromatic amine, 2-phenylethylamine, within the phylum Bacteroidetes

Cellular polyamines of the newly additional 19 species belonging to the class Bacteroides of the phylum Bacteroidetes were analyzed by HPLC to display polyamine distribution as a chemotaxonomic marker within the total 41 species. Three profiles, the presence of spermidine, the presence of homospermidine and the absence of both triamines, corresponded to their phylogenetical positions within the four families of the class. The occurrence of an aromatic amine, 2-phenylethylamine, extracted into cellular polyamine fraction, was also determined within the 121 species distributed within the phylum. This aromatic amine was found in Cellulophaga lytica, Cytophaga latercula, Tenacibaculum amylolyticum, Tenacibaculum martimum, Tenacibaculum mesophilum and Psychroflexus torquis belonging to the family Flavobacteriaceae of the class Flavobacteria, and Flexibacter flexilis and Microscilla marina belonging to the family Flexibacteraceae of the class Sphingobacteria.

Large production of an aromatic amine, 2-phenylethylamine, in a psychrophilic marine bacterium, Psychroflexus torquis.

During the analysis of the cellular polyamines by high-performance liquid chromatography and gas chromatography within the Cytophaga-Flavobacterium Bacteroides phylum, an usual amine corresponding to 2-phenylethylamine was detected in Psychroflexus torqu s belonging to the family Flavobacteriaceae. Homospermidine, agmatine and 2-phenylethylamine were detected as the major cellular polyamine components of the organism. A part of 2-phenylethylamine produced in the cells was secreted into the medium whereas agmatine and homospermidine were scarcely secreted. i

Psychroflexus torquis gen. nov., sp. nov. a psychrophilic species from Antarctic sea ice, and reclassification of Flavobacterium gondwanense (Dobson et al. 1993) as Psychroflexus gondwanense gen. nov., comb. nov.

A group of sea-ice-derived psychrophilic bacterial strains possessing the unusual ability to synthesize the polyunsaturated fatty acids eicosapentaenoic acid (20:5 omega 3) and arachidonic acid (20:4 omega 6) belong to the Family Flavobacteriaceae (Flexibacter-Bacteroides-Flavobacterium phylum), according to 16S rRNA sequence analysis. Surprisingly, the isolates were also found to cluster closely to the moderately halophilic and psychrotrophic species [Flavobacterium] gondwanense (sequence similarity 97.8-98.1%). The whole-cell fatty acid profiles of this group and [Flavobacterium] gondwanense were very similar and distinct from other related flavobacteria. The sea ice strains and [Flavobacterium] gondwanense differed substantially in terms of ecophysiology, possibly representing divergent adaptations to sympagic and planktonic marine habitats, respectively. Evidence based on phylogeny and fatty acid profiles supports the conclusion that the taxa are close relatives distinct from other bacterial groups. It is thus proposed that the sea ice strains represent a novel taxon designated Psychroflexus torquis gen. nov., sp. nov. (type strain ACAM 623T) while [Flavobacterium] gondwanense becomes Psychroflexus gondwanense gen. nov., comb. nov.