Bacterial Cold Shock Proteins Research Articles

An internal loop region is responsible for inherent target specificity of bacterial Cold-shock proteins.

Cold shock proteins (Csps), of around 70 amino acids, share a protein fold for the cold shock domain (CSD) that contains RNA binding motifs, RNP1 and RNP2, and constitute one family of bacterial RNA-binding proteins. Despite similar amino acid composition, Csps have been shown to individually possess inherent specific functions. Here we identify the molecular differences in Csps that allow selective recognition of RNA targets. Using chimeras and mutants of Escherichia coli CspD and CspA, we demonstrate that Lys43-Ala44 in an internal loop of CspD and the N-terminal portion with Lys4 of CspA are important for determining their target specificities. Pull-down assays suggest these distinct specificities reflect differences in the ability to act on the target RNAs rather than differences in binding to the RNA targets. A phylogenetic tree constructed from 1,573 Csps reveals that the Csps containing Lys-Ala in the loop form a monophyletic clade, and the members in this clade are shown to have target specificities similar to E. coli CspD. The phylogenetic tree also finds a small cluster of Csps containing Lys-Glu in the loop, and these exhibit different specificity than E. coli CspD. Examination of this difference suggests a role of the loop of CspD type proteins in recognition of specific targets. Additionally, each identified type of Csp shows a different distribution pattern among bacteria. Our findings provide a basis for subclassification of Csps based on target RNA specificity, which will be useful for understanding of the functional specialization of Csps.

Bacterial Cold Shock Proteins as a Tool in Adaption to Stress

Bacterial Cold Shock Proteins as a Tool in Adaption to Stress

Bacterial Cold Shock Proteins as a Factor of Adaptation to Stresses

Bacteria have evolved a number of mechanisms to cope with stresses and adapt to changing environmental conditions. A family of bacterial proteins containing a functional cold shock domain are highly conserved nucleic acid-binding proteins that modulate transcription and post-transcriptional events in bacteria. For many bacteria, these proteins have been shown to regulate the expression of various genes involved in virulence and resistance of bacteria to stresses. The review discusses the new data on the mechanisms of action and the roles of cold shock proteins in the regulation of expression in intracellular bacterial pathogens.

Evidence for Elicitation of an Oxidative Burst in Vitis vinifera by Xylella fastidiosa Cold Shock Protein Peptide csp20

Plant response to pathogen challenge involves recognition by specific receptors of conserved molecular patterns associated with pathogenic organisms. Understanding a wider range of pathogen detection pathways in different plant species is valuable for identification and development of pathogen-resistant plant material. Xylella fastidiosa is an important bacterial pathogen of several agricultural crops including table and wine grapes. Although some pathogen recognition pathways have been identified for X. fastidiosa in host plants, there are likely several other molecular interactions that could be exploited to increase plant resistance to this pathogen. This study identified a conserved bacterial cold shock protein component (Xfcsp20) as an elicitor of reactive oxygen species production by Vitis vinifera plant tissue, an indication of pathogen recognition response. Characterization of Xfcsp20 as an elicitor of an oxidative burst in grapevine hosts of X. fastidiosa will be useful to continued work in the areas of bacterial-plant interactions and plant resistance. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

DgCspC gene overexpression improves cotton yield and tolerance to drought and salt stress comparison with wild-type plants.

Drought and high salinity are key limiting factors for cotton quality and yield. Therefore, research is increasingly focused on mining effective genes to improve the stress resistance of cotton. Few studies have demonstrated that bacterial Cold shock proteins (Csps) overexpression can enhance plants stress tolerance. Here, we first identified and cloned a gene DgCspC encoding 88 amino acids (aa) with an open reading frame (ORF) of 264 base pairs (bp) from a Deinococcus gobiensis I-0 with high resistance to strong radiation, drought, and high temperature. In this study, heterologous expression of DgCspC promoted cotton growth, as exhibited by larger leaf size and higher plant height than the wild-type plants. Moreover, transgenic cotton lines showed higher tolerance to drought and salts stresses than wild-type plants, as revealed by susceptibility phenotype and physiological indexes. Furthermore, the enhanced stresses tolerance was attributed to high capacity of cellular osmotic regulation and ROS scavenging resulted from DgCspC expression modulating relative genes upregulated to cause proline and betaine accumulation. Meanwhile, photosynthetic efficiency and yield were significantly higher in the transgenic cotton than in the wild-type control under field conditions. This study provides a newly effective gene resource to cultivate new cotton varieties with high stresses resistance and yield.

Overexpression of RNA-binding bacterial chaperones in rice leads to stay-green phenotype, improved yield and tolerance to salt and drought stresses.

Genes encoding bacterial cold shock proteins A (CspA, 213 bp) and B (CspB, 216 bp) were isolated from Escherichia coli strain K12, which showed 100% homology with gene sequences isolated from other bacterial species. In silico domain, analysis showed eukaryotic conserved cold shock domain (CSD) and ribonuclease-binding domain (RBD) indicating that they bind to RNA and are involved in temperature stress tolerance. Overexpression of these two genes in E. coli resulted in higher growth in presence of 200 mM NaCl and 300 mM mannitol. Western blot confirmed the translational products of the two genes. Seedlings of indica rice were transformed with Agrobacterium tumefaciens containing pCAMBIA1301 CspA and CspB genes. Transgene integration was confirmed by β-glucuronidase (GUS) histochemical assay, polymerase chain reaction (PCR) amplification, and gene copy number by Southern blotting. Chlorophyll, proline, Na+ , and K+ contents were higher in transgenics exposed to 150 mM NaCl and drought (imposed by withholding water) stresses during floral initiation stage. Catalase (CAT), superoxide dismutase (SOD), and guaiacol peroxidase (GPX) activities increased, while malondialdehyde (MDA) content was low in transgenics. Transgenics displayed increased root, shoot, and panicle lengths, root dry mass, and a distinct stay-green (SGR) phenotype. Higher transcript levels of CspA, CspB, SGR, chlorophyllase, isopentenyl adenine transferase 1 (IPT1), 9-cis-epoxycarotenoid dioxygenase (NCED), SOD, and sirtuin 1 (SIRT1) genes were observed in transgenics compared to wild type plants (WT) under multiple stresses. Present work indicates that bacterial chaperone proteins are capable of imparting SGR phenotype, salt and drought stress tolerance alongside grain improvement.

Cold-Shock Domains-Abundance, Structure, Properties, and Nucleic-Acid Binding.

Simple SummaryProteins are composed of compact domains, often of known three-dimensional structure, and natively unstructured polypeptide regions. The abundant cold-shock domain is among the set of canonical nucleic acid-binding domains and conserved from bacteria to man. Proteins containing cold-shock domains serve a large variety of biological functions, which are mostly linked to DNA or RNA binding. These functions include the regulation of transcription, RNA splicing, translation, stability and sequestration. Cold-shock domains have a simple architecture with a conserved surface ideally suited to bind single-stranded nucleic acids. Because the binding is mostly by non-specific molecular interactions which do not involve the sugar-phosphate backbone, cold-shock domains are not strictly sequence-specific and do not discriminate reliably between DNA and RNA. Many, but not all functions of cold shock-domain proteins in health and disease can be understood based of the physical and structural properties of their cold-shock domains.The cold-shock domain has a deceptively simple architecture but supports a complex biology. It is conserved from bacteria to man and has representatives in all kingdoms of life. Bacterial cold-shock proteins consist of a single cold-shock domain and some, but not all are induced by cold shock. Cold-shock domains in human proteins are often associated with natively unfolded protein segments and more rarely with other folded domains. Cold-shock proteins and domains share a five-stranded all-antiparallel β-barrel structure and a conserved surface that binds single-stranded nucleic acids, predominantly by stacking interactions between nucleobases and aromatic protein sidechains. This conserved binding mode explains the cold-shock domains’ ability to associate with both DNA and RNA strands and their limited sequence selectivity. The promiscuous DNA and RNA binding provides a rationale for the ability of cold-shock domain-containing proteins to function in transcription regulation and DNA-damage repair as well as in regulating splicing, translation, mRNA stability and RNA sequestration.

In silico identification and evaluation of Bacillus subtilis cold shock protein B (cspB)-like plant RNA chaperones

Cold shock domain (CSD) proteins with nucleic acid binding properties are well conserved from bacteria to higher organisms. In bacteria, the cold shock proteins (CSPs) are single domain RNA chaperones, whereas in animals and plants, CSDs are accompanied by additional domains with roles in transcription regulation. Bacterial CSPs (Escherischia coli-cspA and Bacilus subtilis-cspB) have successfully imparted drought tolerance in transgenic plants; however, these cannot be deployed in food crops due to their low public acceptance of transgenics with bacterial genes. Therefore, this study aimed to identify CSPB-like proteins from plants that can be used for developing drought tolerant transgenic crops. Twelve single domain plant CSPs presenting >40% sequence identity with CSPB were identified. All 12 plant CSPs were modeled by homology modeling and refined by molecular dynamics simulation for 10 ns. Selected plant CSPs and CSPB exhibited high structural similarity (Tm-score: 0.63–0.86). Structure based phylogenetic analysis revealed that Triticum aestivum-csp1 and Aegilops tauschii-cspE are structurally closer to CSPB compared to their orthologs and paralogs. Molecular docking with three RNA molecules (5U, UC3U, and C2UC) indicates that Ricinus communis-csd1 and T. aestivum-csp1 have a binding pattern and docking scores similar to those of CSPB. Furthermore, MD simulations for 20 ns and analysis of RMSD, RMSF, Rg as well as the number of hydrogen bonds in all the three complexes revealed that plant CSP-RNA complexes behave in a similar manner to that of the CSPB-RNA complex, making them highly potential candidate genes for developing drought tolerance in transgenic plants. Communicated by Ramaswamy H. Sarma

Interplay of cold shock protein E with an uncharacterized protein, YciF, lowers porin expression and enhances bile resistance in Salmonella Typhimurium

Bacterial cold shock proteins (CSPs) function as RNA chaperones. To assess CSP's roles in the intracellular human pathogen Salmonella Typhimurium, we analyzed their expression in varied stress conditions. We found that cold shock protein E (cspE or STM14_0732) is up-regulated during bile salt-induced stress and that an S. Typhimurium strain lacking cspE (ΔcspE) displays dose-dependent sensitivity to bile salts, specifically to deoxycholate. We also found that an uncharacterized gene, yciF (STM14_2092), is up-regulated in response to bile stress in WT but not in the ΔcspE strain. Complementation with WT CspE, but not with a F30V CspE variant, abrogated the bile sensitivity of ΔcspE as did multicopy overexpression of yciF. Northern blotting experiments with rifampicin disclosed that the regulation of yciF expression is, most likely, due to the RNA-stabilizing activity of CspE. Importantly, electrophoretic mobility shift assays indicated that purified CspE, but not the F30V variant, directly binds yciF mRNA. We also observed that the extra-cytoplasmic stress-response (ESR) pathway is augmented in the bile-treated ΔcspE strain, as judged by induction of RpoE regulon genes (rpoE, degP, and rybB) and downstream ESR genes (hfq, rne, and PNPase). Moreover, the transcript levels of the porin genes, ompD, ompF, and ompC, were higher in bile salts-stressed ΔcspE and correlated with higher intracellular accumulation of the fluorescent DNA stain bisBenzimide H 33258, indicating greater cell permeability. In conclusion, our study has identified YciF, a CspE target involved in the regulation of porins and in countering bile stress in S. Typhimurium.

YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis.

The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.

Software for top-down proteomic identification of a plasmid-borne factor (and other proteins) from genomically sequenced pathogenic bacteria using MALDI-TOF-TOF-MS/MS and post-source decay

A plasmid-borne factor (hypothetical protein) was identified in three Shiga toxin-producing Escherichia coli O113:H21 strains using antibiotic-induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS), post-source decay (PSD) and top-down proteomic analysis. The plasmid-encoded factor was identified from three pairs of complementary fragment ions (b/y) which were also used to obtain a more accurate calculation of the mass of the protein than that possible from MS analysis. A software application tool was developed to rapidly search amino acid sequences from whole genome sequencing-derived data to match the observed protein mass and fragment ions on the assumption that the fragment ions are the result of polypeptide backbone cleavage on the C-terminal side of aspartic acid and glutamic acid residues, i.e. aspartic acid effect. Manual inspection and top-down proteomic analysis confirmed the correctness of the identification of this protein using this new application tool. Although the function of the plasmid-borne factor is not known, its antibiotic-induced expression may suggest a role in antimicrobial stress. This approach was also used to identify three highly conserved bacterial cold-shock proteins: CspC, CspE, CsbD from one of the STEC strains. In addition to polypeptide backbone cleavage, metastable CsbD showed multiple small dissociative losses perhaps suggesting ammonia loss from the four arginine residues in its sequence. This phenomenon had been previously observed for metastable ubiquitin which also has four arginine residues.

Contribution of the cold shock protein CspA to virulence in Xanthomonas oryzae pv. oryzae.

Summary Xanthomonas oryzae pv. oryzae (Xoo) causes a damaging bacterial leaf blight disease in rice. Cold shock proteins (Csps) are highly conserved nucleic acid‐binding proteins present in various bacterial genera, but relatively little is known about their functions in Xanthomonas. Herein, we identified four Csps (CspA–CspD) in the Xoo PXO99A strain. Deletion of cspA decreased cold adaptation and a few known pathogenic factors, including bacterial pathogenicity, biofilm formation and polysaccharide production. Furthermore, we performed transcriptomic and chromosome immunoprecipitation (ChIP) experiments to identify direct targets of CspA and to determine its DNA‐binding sequence. Integrative data analysis revealed that CspA directly regulates two genes, PXO_RS11830 and PXO_RS01060, by binding to a conserved CCAAT sequence in the promoter region. We generated single‐deletion mutants of each gene and the results indicate that both are responsible for Xanthomonas pathogenicity. In addition, quantitative real‐time polymerase chain reaction and western blotting showed that CspA suppressed the expression of its direct targets. In summary, our study clarifies the characteristics of Csps in Xanthomonas and greatly advances our understanding of the mechanisms underlying the contribution of CspA to bacterial virulence.

Применение бактериальных белков холодового шока в биотехнологии

Применение бактериальных белков холодового шока в биотехнологии

RNA-Binding Domain is Necessary for PprM Function in Response to the Extreme Environmental Stress in Deinococcus radiodurans.

Deinococcus radiodurans was considered as one of the most radiation-resistant organisms on Earth because of its strong resistance to the damaging factors of both DNA and protein, including ionizing radiation, ultraviolet radiation, oxidants, and desiccation. PprM, as a bacterial cold shock protein homolog, was involved in the radiation resistance and oxidative stress response of D. radiodurans, but its potential mechanisms are poorly expounded. In this study, we found that PprM was highly conserved with the RNA-binding domain in Deinococcus genus through performing phylogenic analysis. Moreover, the paper presents the analysis on the tolerance of environmental stresses both in the wild-type and the pprM/pprM RBD mutant strains, demonstrating that pprM and RNA-binding domain disruptant strain were with higher sensitivity than the wild-type strain to cold stress, mitomycin C, UV radiation, and hydrogen peroxide. In the following step, the recombinant PprM was purified, with the finding that PprM was bound to the 5'-untranslated region of its own mRNA by gel mobility shift assay in vitro. With all these findings taken into consideration, it was suggested that PprM act as a cold shock protein and its RNA-binding domain may be involved in reaction to the extreme environmental stress in D. radiodurans.

Author Correction: The pattern-recognition receptor CORE of Solanaceae detects bacterial cold-shock protein.

In the version of this Article originally published, Fig. 6b, which is composed of individual pictures of six plants, inadvertently and erroneously displayed the same image of one Col-0 wt plant twice. This has been corrected so that Fig. 6bnow shows two different representative plants for the Col-0 wt control.

Improved drought tolerance in wheat plants overexpressing a synthetic bacterial cold shock protein gene SeCspA

Cold shock proteins (CSPs) enhance acclimatization of bacteria to adverse environmental circumstances. The Escherichia coli CSP genes CspA and CspB were modified to plant-preferred codon sequences and named as SeCspA and SeCspB. Overexpression of exogenous SeCspA and SeCspB in transgenic Arabidopsis lines increased germination rates, survival rates, and increased primary root length compared to control plants under drought and salt stress. Investigation of several stress-related parameters in SeCspA and SeCspB transgenic wheat lines indicated that these lines possessed stress tolerance characteristics, including lower malondialdehyde (MDA) content, lower water loss rates, lower relative Na+ content, and higher chlorophyll content and proline content than the control wheat plants under drought and salt stresses. RNA-seq and qRT-PCR expression analysis showed that overexpression of SeCsp could enhance the expression of stress-responsive genes. The field experiments showed that the SeCspA transgenic wheat lines had great increases in the 1000-grain weight and grain yield compared to the control genotype under drought stress conditions. Significant differences in the stress indices revealed that the SeCspA transgenic wheat lines possessed significant and stable improvements in drought tolerance over the control plants. No such improvement was observed for the SeCspB transgenic lines under field conditions. Our results indicated that SeCspA conferred drought tolerance and improved physiological traits in wheat plants.

RNA binding and chaperone activity of the E. coli cold-shock protein CspA.

Ensuring the correct folding of RNA molecules in the cell is of major importance for a large variety of biological functions. Therefore, chaperone proteins that assist RNA in adopting their functionally active states are abundant in all living organisms. An important feature of RNA chaperone proteins is that they do not require an external energy source to perform their activity, and that they interact transiently and non-specifically with their RNA targets. So far, little is known about the mechanistic details of the RNA chaperone activity of these proteins. Prominent examples of RNA chaperones are bacterial cold shock proteins (Csp) that have been reported to bind single-stranded RNA and DNA. Here, we have used advanced NMR spectroscopy techniques to investigate at atomic resolution the RNA-melting activity of CspA, the major cold shock protein of Escherichia coli, upon binding to different RNA hairpins. Real-time NMR provides detailed information on the folding kinetics and folding pathways. Finally, comparison of wild-type CspA with single-point mutants and small peptides yields insights into the complementary roles of aromatic and positively charged amino-acid side chains for the RNA chaperone activity of the protein.

Terahertz Frequency Spectroscopy to Determine Cold Shock Protein Stability upon Solvation and Evaporation - A Molecular Dynamics Study.

Infrared (IR) and Terahertz (THz) spectroscopy simulations were carried out using CHARMM35b2 to determine protein stability. The stabilities of three bacterial cold shock proteins (Csps) originating from mesophiles, thermophiles and hyper- thermophiles respectively were investigated in this study. The three different Csps were investigated by Normal-Mode analysis and Molecular Dynamics simulation of THz spectra using the Hessian matrix for solvated systems, interpreted in the harmonic approximation at optimum near-melting temperatures of each homologue, by incorporating differences in the hydrous and anhydrous states of the Csps. The results show slight variations in the large scale protein motion. However, the IR spectra of Csps observed at the low frequency saddle surface region, clearly distinguishes the thermophilic and mesophilic proteins based on their stability. Further studies on protein stability employing low-frequency collective modes have the potential to reveal functionally important conformational changes that are biologically significant.

Predicted Cold Shock Proteins from the Extremophilic Bacterium Deinococcus maricopensis and Related Deinococcus Species.

While many studies have examined the mechanisms by which extremophilic Deinococci survive exposure to ionizing radiation, very few publications have characterized the cold shock adaptations of this group, despite many species being found in persistent cold environments and environments prone to significant daily temperature fluctuations. Bacterial cold shock proteins (Csps) are a family of conserved, RNA chaperone proteins that commonly play a role in cold temperature adaptation, including a downward shift in temperature (i.e., cold shock). The primary aim of this study was to test whether a representative, desert-dwelling Deinococcus, Deinococcus maricopensis, encodes Csps as part of its genome. Bioinformatic approaches were used to identify a Csp from D. maricopensis LB-34. The Csp, termed Dm-Csp1, contains sequence features of Csps including a conserved cold shock domain and nucleic acid binding motifs. A tertiary model of Dm-Csp1 revealed an anticipated Csp structure containing five anti-parallel beta-strands, and ligand prediction experiments identified N-terminally located residues capable of binding single-stranded nucleic acids. Putative Csps were identified from 100% of (27 of 27) Deinococci species for which genome information is available; and the Deinococci-encoded Csps identified contain a C-terminally located region that appears to be limited to members of the class Deinococci.

The pattern-recognition receptor CORE of Solanaceae detects bacterial cold-shock protein.

Plants and animals recognize microbial invaders by detecting microbe-associated molecular patterns (MAMPs) by cell surface receptors. Many plant species of the Solanaceae family detect the highly conserved nucleic acid binding motif RNP-1 of bacterial cold-shock proteins (CSPs), represented by the peptide csp22, as a MAMP. Here, we exploited the natural variation in csp22 perception observed between cultivated tomato (Solanum lycopersicum) and Solanum pennellii to map and identify the leucine-rich repeat (LRR) receptor kinase CORE (cold shock protein receptor) of tomato as the specific, high-affinity receptor site for csp22. Corroborating its function as a genuine receptor, heterologous expression of CORE in Arabidopsis thaliana conferred full sensitivity to csp22 and, importantly, it also rendered these plants more resistant to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our study also confirms the biotechnological potential of enhancing plant immunity by interspecies transfer of highly effective pattern-recognition receptors such as CORE to different plant families.