Genome sequence of a novel member of the genus Psychrobacter isolated from Antarctic soil

Psychrobacter spp. have shown characteristics indicating remarkable capabilities at subzero temperatures that identify them as potential model organisms for the study of low-temperature adaptations. Here we present the draft genome sequence of Psychrobacter sp. PAMC 21119, which was isolated from permafrost soil of Antarctica; this information could provide insight into adaptation and evolution strategies under extreme environmental conditions.     

Mechanisms of subzero growth in the cryophile Planococcus halocryophilus determined through proteomic analysis

The eurypsychrophilic bacterium Planococcus halocryophilus is capable of growth down to ?15°C, making it ideal for studying adaptations to subzero growth. To increase our understanding of the mechanisms and pathways important for subzero growth, we performed proteomics on P. halocryophilus grown at 23°C, 23°C with 12% w/v NaCl and ?10°C with 12% w/v NaCl. Many proteins with increased abundances at ?10°C versus 23°C also increased at 23C‐salt versus 23°C, indicating a closely tied relationship between salt and cold stress adaptation. Processes which displayed the largest changes in protein abundance were peptidoglycan and fatty acid (FA) synthesis, translation processes, methylglyoxal metabolism, DNA repair and recombination, and protein and nucleotide turnover. We identified intriguing targets for further research at ?10°C, including PlsX and KASII (FA metabolism), DD‐transpeptidase and MurB (peptidoglycan synthesis), glyoxalase family proteins (reactive electrophile response) and ribosome modifying enzymes (translation turnover). PemK/MazF may have a crucial role in translational reprogramming under cold conditions. At ?10°C P. halocryophilus induces stress responses, uses resources efficiently, and carefully controls its growth and metabolism to maximize subzero survival. The present study identifies several mechanisms involved in subzero growth and enhances our understanding of cold adaptation.     

Proteomics of Colwellia psychrerythraea at subzero temperatures – a life with limited movement,flexible membranes and vital DNA repair

The mechanisms that allow psychrophilic bacteria to remain metabolically active at subzero temperatures result from form and function of their proteins. We present first proteomic evidence of physiological changes of the marine psychrophile Colwellia psychrerythraea 34H (Cp34H) after exposure to subzero temperatures (?1, and ?10°C in ice) through 8 weeks. Protein abundance was compared between different treatments to understand the effects of temperature and time, independently and jointly, within cells transitioning to, and being maintained in ice. Parallel [3H]‐leucine and [3H]–thymidine incubations indicated active protein and DNA synthesis to ?10°C. Mass spectrometry‐based proteomics identified 1763 proteins across four experimental treatments. Proteins involved in osmolyte regulation and polymer secretion were found constitutively present across all treatments, suggesting that they are required for metabolic success below 0°C. Differentially abundant protein groups indicated a reallocation of resources from DNA binding to DNA repair and from motility to chemo‐taxis and sensing. Changes to iron and nitrogen metabolism, cellular membrane structures, and protein synthesis and folding were also revealed. By elucidating vital strategies during life in ice, this study provides novel insight into the extensive molecular adaptations that occur in cold‐adapted marine organisms to sustain cellular function in their habitat.     

Gilthead sea bream liver proteome altered at low temperatures by oxidative stress

Gilthead sea bream exposed to the cold show multiple physiological alterations, particularly in liver. A typical cold‐stress response was reproduced in gilthead sea bream acclimated to 20°C (Warm group) when the water temperature was lowered to 8°C (Cold group). After 10 days, thiobarbituric acid reactive substances in the liver had increased by 50%, and nitric oxide had increased twofold. This indicates that lipid peroxidation and oxidative stress had occurred. Protein profiles of liver from fish in warm and cold environments were obtained by 2‐DE. Quantification of differential expression by matching spots showed that a total of 57 proteins were altered significantly. Many proteins were downregulated following cold exposure, including actin, the most abundant protein in the proteome; enzymes of amino acid metabolism; and enzymes with antioxidant capacity, such as betaine‐homocysteine‐methyl transferase, glutathione‐S‐transferase and catalase. Some proteins associated with protective action were upregulated at low temperatures, including peroxiredoxin, thioredoxin and lysozyme; as well as enzymes such as aldehyde dehydrogenase and adenosin‐methionine synthetase. However, the upregulation of proteases, proteasome activator protein and trypsinogen‐like protein indicated an increase in proteolysis. Increases in elongation factor‐1α, the GAPDH oxidative form, tubulin and Raf‐kinase inhibitor protein indicated oxidative stress and the induction of apoptosis. These data indicate that cold exposure induced oxidative damage in hepatocytes.     

Proteomics of life at low temperatures: trigger factor is the primary chaperone in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

The proteomes expressed at 4°C and 18°C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis have been compared using two‐dimensional differential in‐gel electrophoresis, showing that translation, protein folding, membrane integrity and anti‐oxidant activities are upregulated at 4°C. This proteomic analysis revealed that the trigger factor is the main upregulated protein at low temperature. The trigger factor is the first molecular chaperone interacting with virtually all newly synthesized polypeptides on the ribosome and also possesses a peptidyl‐prolyl cis‐trans isomerase activity. This suggests that protein folding at low temperatures is a rate‐limiting step for bacterial growth in cold environments. It is proposed that the psychrophilic trigger factor rescues the chaperone function as both DnaK and GroEL (the major bacterial chaperones but also heat‐shock proteins) are downregulated at 4°C. The recombinant psychrophilic trigger factor is a monomer that displays unusually low conformational stability with a Tm value of 33°C, suggesting that the essential chaperone function requires considerable flexibility and dynamics to compensate for the reduction of molecular motions at freezing temperatures. Its chaperone activity is strongly temperature‐dependent and requires near‐zero temperature to stably bind a model‐unfolded polypeptide.     

Cold tolerance of Pseudomonas sp. 30-3 isolated from oil-contaminated soil, Antarctica

Pseudomonas sp. 30-3 was enriched from oil-contaminated soil from Wright Valley, Antarctica using JP8 jet fuel as sole carbon source. This isolate exhibited tolerance to temperatures ranging from 0°C to 35°C when cultured in laboratory medium. In a freeze-thaw study, an 89% survival was observed when Pseudomonas sp. 30-3 was exposed to 4°C prior to freezing. PCR amplification of a 248-bp DNA fragment in Pseudomonas sp. 30-3 using capB-gene specific primers showed a 98% amino acid sequence homology with CapB of Pseudomonas fragi and 62% homology with CspA of Escherichia coli. Radiolabeling of total cellular proteins exhibited elevated expression of an 8-kDa protein at 4°C, which suggests that the CapB in Pseudomonas sp. 30-3 may play a pivotal role in survival and tolerance at cold and subzero temperatures. Tolerance to cold temperatures and the ability to degrade hydrocarbons by Pseudomonas sp. 30-3 provide support for the application of bioremediation for petroleum hydrocarbons in Antarctic soils.     

Proteomic analysis of <Emphasis Type="Italic">Psychrobacter</Emphasis><Emphasis Type="Italic"> cryohalolentis</Emphasis> K5 during growth at subzero temperatures

It is crucial to examine the physiological processes of psychrophiles at temperatures below 4°C, particularly to facilitate extrapolation of laboratory results to in situ activity. Using two dimensional electrophoresis, we examined patterns of protein abundance during growth at 16, 4, and −4°C of the eurypsychrophile Psychrobacter cryohalolentis K5 and report the first identification of cold inducible proteins (CIPs) present during growth at subzero temperatures. Growth temperature substantially reprogrammed the proteome; the relative abundance of 303 of the 618 protein spots detected (∼31% of the proteins at each growth temperature) varied significantly with temperature. Five CIPs were detected specifically at −4°C; their identities (AtpF, EF-Ts, TolC, Pcryo_1988, and FecA) suggested specific stress on energy production, protein synthesis, and transport during growth at subzero temperatures. The need for continual relief of low-temperature stress on these cellular processes was confirmed via identification of 22 additional CIPs whose abundance increased during growth at −4°C (relative to higher temperatures). Our data suggested that iron may be limiting during growth at subzero temperatures and that a cold-adapted allele was employed at −4°C for transport of iron. In summary, these data suggest that low-temperature stresses continue to intensify as growth temperatures decrease to −4°C.     

Proteomic Adaptation of Australian Epidemic Bordetella pertussis

Bordetella pertussis causes whooping cough. The predominant strains in Australia changed to single nucleotide polymorphism (SNP) cluster I (pertussis toxin promoter allele ptxP3/pertactin gene allele prn2) from cluster II (non‐ptxP3/non‐prn2). Cluster I was mostly responsible for the 2008–2012 Australian epidemic and was found to have higher fitness compared to cluster II using an in vivo mouse competition assay, regardless of host's immunization status. This study aimed to identify proteomic differences that explain higher fitness in cluster I using isobaric tags for relative and absolute quantification (iTRAQ), and high‐resolution multiple reaction monitoring (MRM‐hr). A few key differences in the whole cell and secretome were identified between the cluster I and II strains tested. In the whole cell, nine proteins were upregulated (>1.2 fold change, q < 0.05) and three were downregulated (<0.8 fold change, q < 0.05) in cluster I. One downregulated protein was BP1569, a TLR2 agonist for Th1 immunity. In the secretome, 12 proteins were upregulated and 1 was downregulated which was Bsp22, a type III secretion system (T3SS) protein. Furthermore, there was a trend of downregulation in three T3SS effectors and other virulence factors. Three proteins were upregulated in both whole cell and supernatant: BP0200, molybdate ABC transporter (ModB), and tracheal colonization factor A (TcfA). Important expression differences in lipoprotein, T3SS, and transport proteins between the cluster I and II strains were identified. These differences may affect immune evasion, virulence and metabolism, and play a role in increased fitness of cluster I.     

Analysis of cotton (Gossypium hirsutum) root proteomes during a compatible interaction with the black root rot fungus Thielaviopsis basicola

A proteomic approach was used to uncover the inducible molecular defense mechanism of cotton root occurring during the compatible interaction with Thielaviopsis basicola. Microscopic observation of cotton root inoculated with a suspension of conidia showed that this necrotrophic hemibiotroph fungus interacts with the plant and completes its life cycle in our experimental system. 2‐DE analysis of root extracts taken after 1, 3, 5, and 7 days postinoculation and cluster analysis of the protein expression levels showed four major profiles (constant, upregulated, one slightly downregulated, and one dramatically downregulated). Spots significantly (p<0.05) upregulated were analyzed by LC‐MS/MS and identified using MASCOT MS/MS ion search software and associated databases. These proteins included defense and stress related proteins, such as pathogenesis‐related proteins and proteins likely to be involved in the oxidative burst, sugar, and nitrogen metabolism as well as amino acid and isoprenoid synthesis. While many of the identified proteins are common components of the defense response of most plants, a proteasome subunit and a protein reported to be induced only in cotton root following Meloidogyne incognita infection were also identified.     

Proteome profiling of aging in mouse models: Differential expression of proteins involved in metabolism,transport, and stress response in kidney

Aging is a time‐dependent complex biological phenomenon observed in various organs and organelles of all living organisms. To understand the molecular mechanism of age‐associated functional loss in aging kidneys, we have analyzed the expression of proteins in the kidneys of young (19–22 wk) and old (24 months) C57/BL6 male mice using 2‐DE followed by LC‐MS/MS. We found that expression levels of 49 proteins were upregulated (p ≤ 0.05), while that of only ten proteins were downregulated (p ≤ 0.05) due to aging. The proteins identified belong to three broad functional categories: (i) metabolism (e.g., aldehyde dehydrogenase family, ATP synthase β‐subunit, malate dehydrogenase, NADH dehydrogenase (ubiquinone), hydroxy acid oxidase 2), (ii) transport (e.g., transferrin), and (iii) chaperone/stress response (e.g., Ig‐binding protein, low density lipoprotein receptor‐related protein associated protein 1, selenium‐binding proteins (SBPs)). Some proteins with unknown functions were also identified as being differentially expressed. ATP synthase β subunit, transferrin, fumarate hydratase, SBPs, and albumin are present in multiple forms, possibly arising due to proteolysis or PTMs. The above functional categories suggest specific mechanisms and pathways for age‐related kidney degeneration.     

Identification of kaempferol‐regulated proteins in rat calvarial osteoblasts during mineralization by proteomics

Kaempferol, a flavonoid, promotes osteoblast mineralization in vitro and bone formation in vivo; however, its mechanism of action is yet unknown. We adopted proteomic approach to identify the differential effect of kaempferol on rat primary calvarial osteoblasts during mineralization. The primary rat calvarial osteoblasts were treated with kaempferol (5.0 μM) for 9 days under mineralizing condition that resulted in significant increase in alkaline phosphatase activity and mineralization of the cells. Further, 2‐D analysis of the kaempferol‐treated osteoblast lysates revealed 18 differentially expressed proteins (nine upregulated and nine downregulated) on the basis of >/<2.0‐fold as cut‐off (p<0.01) that were then identified by MALDI‐TOF MS. These included cytoskeletal proteins, intracellular signaling protein, chaperone, extracellular matrix protein, and proteins involved in glycolysis and cell–matrix interactions. Proteomics data were confirmed by Western blotting and quantitative real‐time PCR by randomly selecting two upregulated and two downregulated proteins. Western blot analysis confirmed upregulation of HSP‐70 and cytokeratin‐14 levels, and downregulation of aldose reductase and caldesmon expression. We further demonstrated that kaempferol treatment inhibits aldose reductase activity in osteoblasts indicating an altered cellular metabolism by decelerating polyol pathway that was associated with the kaempferol‐induced osteoblast mineralization. In conclusion, this is a first comprehensive study on the differential regulation of proteins by kaempferol in primary osteoblast, which would further help to elucidate the role of the identified proteins in the process of osteoblast mineralization.     

Proteomic profiling of eggs from a hybrid abalone and its parental lines: Haliotis discus hannai Ino and Haliotis gigantea

Proteomic analysis was performed on the eggs of hybrid abalone and their corresponding parental lines. A total of 915 ± 19 stained protein spots were detected from Haliotis discus hannai♀ × H. discus hannai♂ (DD), 935 ± 16 from H. gigantea♀ × H. gigantea♂ (GG) and 923 ± 13 from H. gigantea♀ × H. discus hannai♂ (GD). The spots from DD and GD were clustered together. The distance between DD and GG was maximal by hierarchical cluster analysis. A total of 112 protein gel spots were identified; of these, 59 were abalone proteins. The proteins were involved in major biological processes including energy metabolism, proliferation, apoptosis, signal transduction, immunity, lipid metabolism, electron carrier proteins, protein biosynthesis and decomposition, and cytoskeletal structure. Three of 20 differential expression protein spots involved in energy metabolism exhibited as upregulated in GD, 13 spots exhibited additivity, and four spots exhibited as downregulated in the offspring. Eleven protein spots were expressed at the highest level in DD. The proteins involved in stress responses included superoxide dismutase, peroxiredoxin 6, thioredoxin peroxidase and glutathione‐S‐transferase. Two of seven differential expression protein spots involved in response to stress exhibited as upregulated in GD, three exhibited additivity, and two exhibited as downregulated. These results might suggest that proteomic approaches are suitable for the analysis of hybrids and the functional prediction of abalone hybridization.     

Proteomic analysis of cold stress-responsive proteins in <Emphasis Type="Italic">Thellungiella</Emphasis> rosette leaves

Low temperature is one of the most severe environmental factors that impair plant growth and agricultural production. To investigate how Thellungiella halophila, an Arabidopsis-like extremophile, adapts to cold stress, a comparative proteomic approach based on two-dimensional electrophoresis was adopted to identify proteins that changed in abundance in Thellungiella rosette leaves during short term (6 h, 2 and 5 days) and long term (24 days) exposure to cold stress. Sixty-six protein spots exhibited significant change at least at one time point and maximal cold stress induced-proteome change was found in long-term cold stress group while the minimal change was found in 6-h cold treatment group. Fifty protein spots were identified by mass spectrometry analysis. The identified proteins mainly participate in photosynthesis, RNA metabolism, defense response, energy pathway, protein synthesis, folding and degradation, cell wall and cytoskeleton and signal transduction. These proteins might work cooperatively to establish a new homeostasis under cold stress. Nearly half of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology suggesting that the cold stress tolerance of T. halophila is achieved, at least partly, by regulation of chloroplast function. All protein spots involved in RNA metabolism, defense response, protein synthesis, folding and degradation were found to be upregulated markedly by cold treatment, indicating enhanced RNA metabolism, defense and protein metabolism may play crucial roles in cold tolerance mechanism in T. halophila.     

Growth dynamics and transcriptional responses of a Red Sea Prochlorococcus strain to varying temperatures

Prochlorococcus play a crucial role in the ocean's biogeochemical cycling, but it remains controversial how they will respond to global warming. Here we assessed the response to temperature (22–30°C) of the growth dynamics and gene expression profiles of a Red Sea Prochlorococcus strain (RSP50) in a non-axenic culture. Both the specific growth rate (0.55–0.80 day⁻¹) and cell size (0.04–0.07 μm³) of Prochlorococcus increased significantly with temperature. The primary production released extracellularly ranged from 20% to 34%, with humic-like fluorescent compounds increasing up to fivefold as Prochlorococcus reached its maximum abundance. At 30°C, genes involved in carbon fixation such as CsoS2 and CsoS3 and photosynthetic electron transport including PTOX were downregulated, suggesting a cellular homeostasis and energy saving mechanism response. In contrast, PTOX was found upregulated at 22°C and 24°C. Similar results were found for transaldolase, related to carbon metabolism, and citrate synthase, an important enzyme in the TCA cycle. Our data suggest that in spite of the currently warm temperatures of the Red Sea, Prochlorococcus can modulate its gene expression profiles to permit growth at temperatures lower than its optimum temperature (28°C) but is unable to cope with temperatures exceeding 30°C.     

Differential Proteomics in Response to Low Temperature Diazotrophy of Himalayan Psychrophilic Nitrogen Fixing Pseudomonas migulae S10724 Strain

Himalayas are considered as a reservoir of diversified and dynamic gene pool. This study describes the response of a Himalayan psychrophilic diazotroph to low temperature diazotrophy. Seven cold adaptive N₂ fixing bacteria were isolated and identified as Bacillus sp., Arthrobacter sp., Rhodococcus sp., Pseudomonas sp., etc. In order to examine the physiological response to low temperature diazotrophy, differential proteomic analysis of Pseudomonas migulae S10724 strain was carried out using two dimensional electrophoresis and MALDI–TOF–MS. Functional assessment of 66 differentially expressed proteins revealed several mechanisms thought to be involved in low temperature adaptation and nitrogen fixation, including general stress adaptation, protein and nucleic acid synthesis, energy metabolism, cell growth/maintenance, etc. Major fraction of the upregulated proteins was stress proteins, while majority of the downregulated proteins were related to cell division. Furthermore, MALDI–TOF–MS-based identification of randomly selected peptides encountered two exclusively expressed proteins: NifU family SUF system FeS assembly protein and membrane protein, suppressor for copper-sensitivity B precursor which might have a crucial role at low temperature nitrogen fixation. To the best of our knowledge, this is the first report of the isolation and differential proteomic analysis of psychrophilic diazotroph from Himalayan high altitude rhizospheric soil.     

Proteomics insights into the Burkholderia cenocepacia phosphorus stress response

The Burkholderia cepacia complex is a group of Burkholderia species that are opportunistic pathogens causing high mortality rates in patients with cystic fibrosis. An environmental stress often encountered by these soil-dwelling and pathogenic bacteria is phosphorus limitation, an essential element for cellular processes. Here, we describe cellular and extracellular proteins differentially regulated between phosphate-deplete (0 mM, no added phosphate) and phosphate-replete (1 mM) growth conditions using a comparative proteomics (LC–MS/MS) approach. We observed a total of 128 and 65 unique proteins were downregulated and upregulated respectively, in the B. cenocepacia proteome. Of those downregulated proteins, many have functions in amino acid transport/metabolism. We have identified 24 upregulated proteins that are directly/indirectly involved in inorganic phosphate or organic phosphorus acquisition. Also, proteins involved in virulence and antimicrobial resistance were differentially regulated, suggesting B. cenocepacia experiences a dramatic shift in metabolism under these stress conditions. Overall, this study provides a baseline for further research into the biology of Burkholderia in response to phosphorus stress.