Life at Low Temperatures

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS). It causes disease primarily in fresh water-reared salmonids, but other fish species can also be affected. A diverse array of clinical conditions is associated with BCWD, including tail rot (peduncle disease), necrotic myositis, and cephalic osteochondritis. Degradation of connective and muscular tissues by extracellular proteases is common to all of these presentations. There are no effective vaccines to prevent BCWD or RTFS, and antibiotics are often used to prevent and control disease. To identify virulence factors that might permit development of an efficacious vaccine, cDNA suppression subtractive hybridization (SSH) was used to identify cold-regulated genes in a virulent strain of F. psychrophilum. Genes predicted to encode a two-component system sensor histidine kinase (LytS), an ATP-dependent RNA helicase, a multidrug ABC transporter permease/ATPase, an outer membrane protein/protective antigen OMA87, an M43 cytophagalysin zinc-dependent metalloprotease, a hypothetical protein, and four housekeeping genes were upregulated at 8°C versus the level of expression at 20°C. Because no F. psychrophilum gene was known to be suitable as an internal standard in reverse transcription-quantitative real-time PCR (RT-qPCR) experiments, the expression stability of nine commonly used reference genes was evaluated at 8°C and 20°C. Expression of the 16S rRNA was equivalent at both temperatures, and this gene was used in RT-qPCR experiments to verify the SSH findings. With the exception of the ATCC 49513 strain, similar patterns of gene expression were obtained with 11 other representative strains of F. psychrophilum.Bacterial cold water disease (BCWD), caused by Flavobacterium psychrophilum, occurs at low water temperatures and can cause economic losses in the aquaculture industry as a result of direct mortality or vertebral deformities that decrease the market value of fish that survive the infection (31, 35). A number of putative virulence factors of this bacterium have been identified, including extracellular proteases involved in degradation of extracellular matrix components such as elastin, fibrinogen, type IV collagen, actin, and myosin (9,37,47,48). The best studied of these, psychrophilic metalloproteases Fpp1 and Fpp2, are thought to be involved in destruction of host tissues; however, their roles have not yet been demonstrated in vivo (47,48). In a recent study, Sudheesh et al. (53) used two-dimensional acrylamide gel electrophoresis and Western blot analysis to compare a virulent strain and a nonvirulent strain of F. psychrophilum and found a thermolysin that was unique to the virulent strain. A role for the iron uptake-associated gene exbD2 has also been demonstrated. Strains lacking this gene have decreased virulence and confer a high level of protection in rainbow trout fry when they are used for vaccination (4). Other putative virulence factors of F. psychrophilum include a lipo...

“…GyrA has previously been identified to be a cold-inducible protein in prokaryotes (44). The gyrA and gyrB genes were upregulated 28.36-and 3.48-fold at 8°C, respectively, in this study.…”

Section: Discussionsupporting

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Identification of Cold-Temperature-Regulated Genes in Flavobacterium psychrophilum

Hesami

Metcalf

Lumsden

et al. 2011

“…Microorganisms that sustain metabolic processes at cold temperatures produce cold acclimation and cold shock proteins involved in DNA replication (GyrA, RecA, and DnaA) (77)(78)(79) (79,(85)(86)(87), pyruvate metabolism (AceE and AceF) (88,89), and unsaturated fatty acid metabolism (fatty acid desaturases; DnaJ) (1,(90)(91)(92). Genes encoding such proteins were identified as containing cold adaptation features within the LH metagenome and originated mostly from Proteobacteria, Bacteroidetes, and Cyanobacteria (see Table S6 in the supplemental material).…”

Section: Resultsmentioning

confidence: 99%

Defining the Functional Potential and Active Community Members of a Sediment Microbial Community in a High-Arctic Hypersaline Subzero Spring

Lay

Mykytczuk

Yergeau

et al. 2013

bThe Lost Hammer (LH) Spring is the coldest and saltiest terrestrial spring discovered to date and is characterized by perennial discharges at subzero temperatures (؊5°C), hypersalinity (salinity, 24%), and reducing (Ϸ؊165 mV), microoxic, and oligotrophic conditions. It is rich in sulfates (10.0%, wt/wt), dissolved H 2 S/sulfides (up to 25 ppm), ammonia (Ϸ381 M), and methane (11.1 g day ؊1 ). To determine its total functional and genetic potential and to identify its active microbial components, we performed metagenomic analyses of the LH Spring outlet microbial community and pyrosequencing analyses of the cDNA of its 16S rRNA genes. Reads related to Cyanobacteria (19.7%), Bacteroidetes (13.3%), and Proteobacteria (6.6%) represented the dominant phyla identified among the classified sequences. Reconstruction of the enzyme pathways responsible for bacterial nitrification/denitrification/ammonification and sulfate reduction appeared nearly complete in the metagenomic data set. In the cDNA profile of the LH Spring active community, ammonia oxidizers (Thaumarchaeota), denitrifiers (Pseudomonas spp.), sulfate reducers (Desulfobulbus spp.), and other sulfur oxidizers (Thermoprotei) were present, highlighting their involvement in nitrogen and sulfur cycling. Stress response genes for adapting to cold, osmotic stress, and oxidative stress were also abundant in the metagenome. Comparison of the composition of the functional community of the LH Spring to metagenomes from other saline/ subzero environments revealed a close association between the LH Spring and another Canadian high-Arctic permafrost environment, particularly in genes related to sulfur metabolism and dormancy. Overall, this study provides insights into the metabolic potential and the active microbial populations that exist in this hypersaline cryoenvironment and contributes to our understanding of microbial ecology in extreme environments. Cryoenvironments are defined as permanently subzero or frozen environments, such as permafrost, glaciers, ice sheets, multiyear sea ice, high-elevation Antarctic dry valleys, and some cold saline springs (1-6). Microorganisms inhabiting cryoenvironments must face the challenges of subzero temperatures, low water activity, and, often, high solute concentrations to sustain their viability. The cold saline springs on Axel Heiberg Island (AHI) in the Canadian high Arctic discharge through 500 to 600 m of thick permafrost, maintain a liquid state at subzero temperatures, and offer a unique opportunity to assess microbial adaptations to extremes of both high salinity and subzero temperatures (3, 4, 7-9). These springs occur in an area with an average annual air temperature of Ϫ15°C, reaching below Ϫ40°C during the winter months, and probably originate from subpermafrost groundwater flow through carboniferous evaporites in areas of diapiric uplift on AHI (10, 11). Other Arctic cold springs, on Ellesmere Island in the Canadian high Arctic and on the Norwegian highArctic Svalbard archipelago, have been reported (12-14), although t...

“…All of the polar metagenomes contained genes encoding cold shock proteins, which are a common feature of prokaryotes growing in low temperatures (47). RNA chaperones (cold shock proteins CspA, CspB, CspE, CspI, and CspG) are essential for proper protein folding, especially at low temperatures, guiding nascent polypeptides into functional three-dimensional configurations (44).…”

Section: Fig 3 Statistical Analyses Of Metabolic Profiles For the Arcmentioning

confidence: 99%

Metagenomic Analysis of Stress Genes in Microbial Mat Communities from Antarctica and the High Arctic

Varin

Lovejoy

Jungblut

et al. 2012

163

107

Polar and alpine microbial communities experience a variety of environmental stresses, including perennial cold and freezing; however, knowledge of genomic responses to such conditions is still rudimentary. We analyzed the metagenomes of cyanobacterial mats from Arctic and Antarctic ice shelves, using high-throughput pyrosequencing to test the hypotheses that consortia from these extreme polar habitats were similar in terms of major phyla and subphyla and consequently in their potential responses to environmental stresses. Statistical comparisons of the protein-coding genes showed similarities between the mats from the two poles, with the majority of genes derived from Proteobacteria and Cyanobacteria; however, the relative proportions differed, with cyanobacterial genes more prevalent in the Antarctic mat metagenome. Other differences included a higher representation of Actinobacteria and Alphaproteobacteria in the Arctic metagenomes, which may reflect the greater access to diasporas from both adjacent ice-free lands and the open ocean. Genes coding for functional responses to environmental stress (exopolysaccharides, cold shock proteins, and membrane modifications) were found in all of the metagenomes. However, in keeping with the greater exposure of the Arctic to long-range pollutants, sequences assigned to copper homeostasis genes were statistically (30%) more abundant in the Arctic samples. In contrast, more reads matching the sigma B genes were identified in the Antarctic mat, likely reflecting the more severe osmotic stress during freeze-up of the Antarctic ponds. This study underscores the presence of diverse mechanisms of adaptation to cold and other stresses in polar mats, consistent with the proportional representation of major bacterial groups. Microbial mats dominated by cyanobacteria are commonly found in extreme environments, such as geothermal springs, hypersaline basins, ultraoligotrophic ponds, and hot and cold desert soils (10,14). Cyanobacterial mats are also a dominant feature of polar lake, pond, and river ecosystems, with some of the most luxuriant communities growing on the thick ice shelves that float on Arctic and Antarctic seas (55). The stresses encountered by organisms on polar ice shelves include sparse nutrients, freezethaw cycles, bright sunlight exposure during summer, prolonged darkness during winter, salinity fluctuations, desiccation, and persistent low temperatures (29, 56). Extreme cold is an overarching stress in the polar regions because it drastically modifies the physical-chemical environment of living cells, with effects on biochemical reaction rates, substrate transport, membrane fluidity, and conformation of macromolecules, such as DNA and proteins (45, 61). Once the freezing point is crossed, there are additional physical and chemical stresses imposed by ice crystal formation, water loss, and increasing solute concentrations.Although polar ice shelf mats are visually dominated by cyanobacteria, other microorganisms, including Bacteria, Archaea, and protists, live wit...