A Cold-Shock Protein from the South Pole-Dwelling Soil Bacterium Arthrobacter sp. Confers Cold Tolerance to Rice.

Low temperature is a critical environmental factor restricting the physiology of organisms across kingdoms. In prokaryotes, cold shock induces the expression of various genes and proteins involved in cellular processes. Here, a cold-shock protein (ArCspA) from the South Pole-dwelling soil bacterium Arthrobacter sp. A2-5 was introduced into rice, a monocot model plant species. Four-week-old 35S:ArCspA transgenic rice plants grown in a cold chamber at 4 °C survived for 6 days. Cold stress significantly decreased the chlorophyll content in WT plants after 4 days compared with that in 35S:ArCspA transgenic plants. RNA-seq analysis was performed on WT and 35S:ArCspA transgenic rice with/without cold stress. GO terms such as "response to stress (GO:0006950)", "response to cold (GO:0009409)", and "response to heat (GO:0009408)" were significantly enriched among the upregulated genes in the 35S:ArCspA transgenic rice under normal conditions, even without cold-stress treatment. The expression of five cold stress-related genes, Rab16B (Os11g0454200), Rab21 (Os11g0454300), LEA22 (Os01g0702500), ABI5 (Os01 g0859300), and MAPK5 (Os03g0285800), was significantly upregulated in the transgenic rice compared with the WT rice. These results indicate that the ArCspA gene might be involved in the induction of cold-responsive genes and provide cold tolerance.

Transcriptome profiling of fully open flowers in a frost-tolerant almond genotype in response to freezing stress.

Spring frost is a major limiting abiotic stress for the cultivation of almonds [Prunus dulcis (Mill.)] in Mediterranean areas or the Middle East. Spring frost, in particular, damages almond fully open flowers, resulting to significant reduction in yield. Little is known about the genetic factors expressed after frost stress in Prunus spp. as well as in almond fully open flowers. Here, we provide the molecular signature of pistils of fully open flowers from a frost-tolerant almond genotype. The level of frost tolerance in this genotype was determined for all three flowering stages and was confirmed by comparing it to two other cultivars using several physiological analyses. Afterwards, comprehensive expression profiling of genes expressed in fully open flowers was performed after being exposed to frost temperatures (during post-thaw period). Clean reads, 27,104,070 and 32,730,772, were obtained for non-frost-treated and frost-treated (FT) libraries, respectively. A total of 62.24 Mb was assembled, generating 50,896 unigenes and 66,906 transcripts. Therefore, 863 upregulated genes and 555 downregulated genes were identified in the FT library. Functional annotation showed that most of the upregulated genes were related to various biological processes involved in responding to abiotic stress. For the first time, a highly expressed cold-shock protein was identified in the reproductive organ of fruit trees. The expression of six genes was validated by RT-PCR. As the first comprehensive analysis of open flowers in a frost-tolerant almond genotype, this study represents a key step toward the molecular breeding of fruit tree species for frost tolerance.

Comparative Proteomics Enables Identification of Nonannotated Cold Shock Proteins in E. coli.

Recent advances in mass spectrometry-based proteomics have revealed translation of previously nonannotated microproteins from thousands of small open reading frames (smORFs) in prokaryotic and eukaryotic genomes. Facile methods to determine cellular functions of these newly discovered microproteins are now needed. Here, we couple semiquantitative comparative proteomics with whole-genome database searching to identify two nonannotated, homologous cold shock-regulated microproteins in Escherichia coli K12 substr. MG1655, as well as two additional constitutively expressed microproteins. We apply molecular genetic approaches to confirm expression of these cold shock proteins (YmcF and YnfQ) at reduced temperatures and identify the noncanonical ATT start codons that initiate their translation. These proteins are conserved in related Gram-negative bacteria and are predicted to be structured, which, in combination with their cold shock upregulation, suggests that they are likely to have biological roles in the cell. These results reveal that previously unknown factors are involved in the response of E. coli to lowered temperatures and suggest that further nonannotated, stress-regulated E. coli microproteins may remain to be found. More broadly, comparative proteomics may enable discovery of regulated, and therefore potentially functional, products of smORF translation across many different organisms and conditions.

Purification and Characterization of a Novel Cold Shock Protein-Like Bacteriocin Synthesized by Bacillus thuringiensis.

Bacillus thuringiensis (Bt), one of the most successful biopesticides, may expand its potential by producing bacteriocins (thuricins). The aim of this study was to investigate the antimicrobial potential of a novel Bt bacteriocin, thuricin BtCspB, produced by Bt BRC-ZYR2. The results showed that this bacteriocin has a high similarity with cold-shock protein B (CspB). BtCspB lost its activity after proteinase K treatment; however it was active at 60 °C for 30 min and was stable in the pH range 5-7. The partial loss of activity after the treatments of lipase II and catalase were likely due to the change in BtCspB structure and the partial degradation of BtCspB, respectively. The loss of activity at high temperatures and the activity variation at different pHs were not due to degradation or large conformational change. BtCspB did not inhibit four probiotics. It was only active against B. cereus strains 0938 and ATCC 10987 with MIC values of 3.125 µg/mL and 0.781 µg/mL, and MBC values of 12.5 µg/mL and 6.25 µg/mL, respectively. Taken together, these results provide new insights into a novel cold shock protein-like bacteriocin, BtCspB, which displayed promise for its use in food preservation and treatment of B. cereus-associated diseases.

Expression, purification and characterization of cold shock protein A of Corynebacterium pseudotuberculosis.

The gram-positive bacterium Corynebacterium pseudotuberculosis is the causative agent of different diseases that cause dramatically reduced yields of wool and milk, and results in weight loss, carcass condemnation and also death mainly in sheep, equids, cattle and goats and therefore globally results in considerable economical loss. Cold shock proteins are conserved in many bacteria and eukaryotic cells and they help to restore normal cell functions after cold shock in which some appear to have specific functions at normal growth temperature as well. Cold shock protein A from C. pseudotuberculosis was expressed in Escherichia coli and purified. The thermal unfolding/refolding process characterized by circular dichroism, differential scanning calorimetry and NMR spectroscopy techniques indicated that the refolding process was almost completely reversible.

Isolation of two strong poly (U) binding proteins from moderate halophile Halomonas eurihalina and their identification as cold shock proteins.

Cold shock proteins (Csp) are known to be expressed in response to sudden decrease in temperature. They are thought to be involved in a number of cellular processes viz., RNA chaperone activity, translation, transcription, nucleoid condensation. During our studies on ribosomal protein S1 in moderate halophile Halomonas eurihalina, we observed the presence of two strong poly (U) binding proteins in abundance in cell extracts from cells grown under normal growth conditions. The proteins can be isolated in a single step using Poly (U) cellulose chromatography. The proteins were identified as major cold shock proteins belonging to Csp A family by MALDI-TOF and bioinformatic analysis. Csp 12 kDa was found in both exponential and stationary phases whereas Csp 8 kDa is found only in exponential phase.