Recombinant Psychrobacter arcticus Protein CrcB homolog (crcB)

What is Psychrobacter arcticus and what are its primary characteristics?

Psychrobacter arcticus is a Gram-negative, non-motile, non-pigmented, oxidase-positive coccobacillus isolated from Siberian permafrost. It belongs to the Gammaproteobacteria class and was characterized as a novel species in 2006. The bacterium demonstrates remarkable cold adaptation, capable of growth at temperatures ranging from -10°C to 30°C and tolerating salinities of 0 to 1.7 M NaCl. This psychrophilic organism has been isolated from permafrost core samples and characterized through extensive gene sequencing studies, including both 16S rRNA and gyrB gene analysis, with the latter providing more reliable phylogenetic markers for taxonomic classification within the Psychrobacter genus. The type strain designation is 273-4 (DSM 17307 = VKM B-2377), which is now widely used in research settings for studying cold adaptation mechanisms.

What is the CrcB homolog protein in Psychrobacter arcticus and what is its genetic context?

The CrcB homolog protein in Psychrobacter arcticus is encoded by the crcB gene (Ordered Locus Name: Psyc_0215). It is a full-length protein consisting of 123 amino acids with the sequence: MQWLAIGLGAAFGACLRGWLARFNPLHHWIPLGTLGANVLGGLLIGLALVWFERMGSGLSPNIRLFVITGFLGGLTTFSTFSAEVFTFIHHGRLLAALGLVGLHVGMTLLATALGFYCFKLVL. This protein is part of a larger family of CrcB-like proteins found across bacterial species. In P. arcticus, the CrcB homolog is likely involved in membrane-associated functions based on its predicted structure and amino acid composition, which suggests it contains transmembrane regions. The protein has been assigned the UniProt accession number Q4FV70 and has been recombinantly produced for research purposes.

What growth conditions are optimal for Psychrobacter arcticus in laboratory settings?

Psychrobacter arcticus grows optimally in laboratory settings under specific conditions that reflect its adaptation to cold environments. For biofilm formation studies, it forms biofilms at temperatures between 4°C and 22°C when acetate is provided as the sole carbon source and in the presence of 1-7% sea salt. The organism can also colonize substrates such as quartz sand under these conditions. For general cultivation, researchers have developed defined media containing carbon sources such as lactate (20 mM) or pyruvate (100 mM), with nitrogen sources including glutamate (5 mM) or ammonium chloride (5 mM). Additional components include K₂HPO₄ (1 mM), Wolin's vitamins, MOPS buffer, and trace minerals. Various buffers have been tested for supporting growth, including HEPES, PIPES, MOPSO, MOPS, and phosphate buffers at pH 7.0. When transitioning from marine broth to defined media, a serial culturing approach with a 1% inoculum is recommended for optimal adaptation.

How does the genome of Psychrobacter arcticus reveal its adaptations to cold environments?

The 2.65-Mb genome of Psychrobacter arcticus 273-4 reveals multiple sophisticated adaptations enabling survival and growth at temperatures as low as -10°C. Analysis of the complete genome, which contains 2,147 putative proteins distributed relatively evenly between forward (53.5%) and reverse (46.5%) strands with an average CDS length of 994 bp, indicates three primary cold adaptation strategies. First, P. arcticus exhibits modifications in membrane composition to maintain fluidity at low temperatures. Second, it synthesizes specialized cold shock proteins that facilitate cellular processes under cold stress. Third, its metabolism is adapted to utilize acetate as an energy source, which is significant in permafrost environments. The genome shows an average GC content of 42.8%, with four rRNA operons (one on the positive strand, three on the negative strand).

Comparative genomic analysis revealed perhaps the most striking adaptation: significant alterations in amino acid usage across the proteome. Specifically, P. arcticus shows reduced utilization of acidic amino acids, proline, and arginine. This pattern is consistent with increased protein flexibility at low temperatures, which is essential for maintaining enzymatic activity when thermal energy is limited. Importantly, these amino acid adaptations are not randomly distributed but occur more frequently in gene categories essential for cell growth and reproduction, strongly suggesting directed evolutionary adaptation to subzero growth conditions. These genomic adaptations likely evolved in response to the consistent long-term freezing temperatures (-10°C to -12°C) of the Kolyma permafrost soil from which this strain was isolated.

What is known about the role of the cat1 gene in biofilm formation by Psychrobacter arcticus?

Biofilm formation is a critical survival strategy for Psychrobacter arcticus in its natural permafrost habitat. Through transposon mutagenesis studies, researchers have identified a remarkably large gene (20.1-kbp) that plays a crucial role in this process. This gene, designated cat1 (cold attachment gene 1), encodes a protein of 6,715 amino acids (Psyc_1601) that appears to be specifically involved in biofilm formation. Mutants with disruptions in the cat1 gene demonstrate significantly reduced biofilm formation capabilities compared to wild-type strains, while exhibiting no impairment in planktonic growth characteristics. This finding indicates that the Cat1 protein has a specialized function in surface attachment and biofilm development rather than affecting general cellular metabolism or growth.

Genomic context analysis shows that cat1 is likely part of an operon with an upstream gene (Psyc_1602) encoding an outer membrane protein, with 64 bp separating these two genes. Located 379 bp downstream of cat1 is a separate operon predicted to encode an ABC transporter (comprising genes Psyc_1600, Psyc_1599, and Psyc_1598). Time-course studies using static microtiter plate assays have further characterized the role of cat1 in the biofilm formation process, confirming its importance in the attachment phase of biofilm development. This gene represents one of the largest coding sequences identified in bacterial genomes and appears to be specialized for facilitating attachment at low temperatures, hence its designation as "cold attachment gene 1."

How does amino acid composition in the Psychrobacter arcticus proteome differ from mesophilic bacteria, and what implications does this have for protein function?

Comparative genomic analysis of Psychrobacter arcticus reveals a distinctive pattern of amino acid usage that differentiates it from mesophilic bacteria. The P. arcticus proteome shows a statistically significant reduction in acidic amino acids (aspartic acid and glutamic acid) as well as proline and arginine residues. This adaptation has profound implications for protein function at low temperatures. The reduction in charged and rigid amino acids results in proteins with greater structural flexibility and reduced stability of secondary structures, which is advantageous in cold environments where thermal energy is limited and protein rigidity could impair function.

This differential amino acid usage is not randomly distributed throughout the proteome but shows a bias toward genes essential for cell growth and reproduction. This non-random distribution strongly suggests that these adaptations evolved specifically to enable growth at low temperatures rather than merely surviving cold stress in a dormant state. The amino acid composition changes likely contribute to maintaining critical enzyme kinetics and protein-protein interactions at temperatures where most mesophilic organisms' proteins would become too rigid to function effectively.

These proteomic adaptations, combined with the organism's ability to prevent intracellular water from freezing at temperatures as low as -10°C to -12°C (the in situ temperatures of its native permafrost environment), enable P. arcticus to maintain active metabolism and cellular processes under conditions that would be prohibitive for most bacteria. This represents a fascinating example of environmental adaptation at the molecular level with implications for protein engineering and biotechnological applications in cold environments.

What are the recommended procedures for handling recombinant Psychrobacter arcticus CrcB homolog protein in laboratory settings?

For optimal handling of recombinant Psychrobacter arcticus CrcB homolog protein in laboratory settings, specific storage and handling protocols should be followed. The recombinant protein is typically supplied in a Tris-based buffer containing 50% glycerol, which has been optimized for protein stability. When received, the protein should be stored at -20°C for regular storage, or at -80°C for extended preservation. It is critical to avoid repeated freeze-thaw cycles, as these can lead to protein denaturation and loss of structural integrity. For ongoing experiments, working aliquots should be prepared and stored at 4°C, but should not be kept for longer than one week to maintain protein quality.

When preparing the protein for functional assays or structural studies, researchers should consider the amino acid composition and potential membrane association of this protein. The hydrophobic nature of several regions in the sequence suggests potential membrane interaction, which may require specific buffer conditions for maintaining native conformation. For experimental reproducibility, it is advisable to document the specific tag used in the recombinant protein, as this may affect protein behavior in various assay systems. The tag type may vary depending on the production process, and this information should be confirmed with the supplier.

What genomic sequencing and annotation approaches were used for Psychrobacter arcticus, and how can these be applied to similar psychrophilic organisms?

The genome sequencing and annotation of Psychrobacter arcticus 273-4 employed a comprehensive methodology that can serve as a model for similar psychrophilic organisms. The genome was sequenced by the Joint Genome Institute using standard shotgun methods with Sanger sequencing technology. Coding sequences were identified through a combination of computational approaches, utilizing both Critica and Glimmer gene modeling software within the Oakridge National Laboratory Genome Analysis Pipeline.

The annotation process involved multiple verification steps, including confirmation of coding sequences by comparing amino acid translations with GenBank's non-redundant database using BLASTP, and manual identification of ribosomal binding sites using Artemis software. Transfer RNA genes were identified with tRNAScanSE, while ribosomal RNAs (16S and 23S) were located by comparing genome fragments against an rRNA database using BLASTN. Additional structural RNAs, including 5S rRNA, RnpB, transfer-messenger RNA, and signal recognition particle RNA, were identified using the Infernal search tool.

Functional assignments for each coding sequence were made through a hierarchical approach that considered multiple databases: KEGG, InterPro, TIGRFams, PFams, and Clusters of Orthologous Groups of Proteins (COGs). This multi-database approach allowed for comprehensive functional prediction based on sequence identity, alignment quality, and database ranking. Final annotation polishing was performed by the Integrated Microbial Genomes annotation group.

For researchers working with similar psychrophilic organisms, this multi-layered approach combining various gene prediction algorithms with manual curation represents a gold standard methodology. Special attention should be paid to genes potentially involved in cold adaptation by comparing amino acid compositions with mesophilic counterparts and identifying cold-specific gene families.

How can the CrcB homolog protein from Psychrobacter arcticus be used in comparative studies of cold adaptation mechanisms?

The CrcB homolog protein from Psychrobacter arcticus presents a valuable model for comparative studies of cold adaptation mechanisms at the molecular level. Researchers can utilize this protein to investigate how membrane-associated proteins adapt to function at low temperatures by comparing its structural features and amino acid composition with homologs from mesophilic and thermophilic organisms. Such comparative analyses should focus on identifying cold-adaptive features such as increased flexibility in key functional regions, altered charge distribution, and modified hydrophobic interactions.

For experimental approaches, recombinant expression of the CrcB homolog alongside its mesophilic counterparts, followed by comparative biochemical characterization at various temperatures (ranging from -10°C to 30°C), can reveal temperature-dependent functional differences. Techniques such as circular dichroism spectroscopy can determine structural stability differences, while fluorescence-based thermal shift assays can quantify temperature-dependent unfolding profiles. Activity assays performed across temperature gradients can establish the relationship between structural flexibility and functional capacity.

Additionally, researchers can employ site-directed mutagenesis to systematically replace amino acids unique to the P. arcticus CrcB homolog with those commonly found in mesophilic homologs, creating a series of variants with intermediate properties. This approach can help identify which specific residues are critical for cold adaptation. Such comparative studies contribute to our fundamental understanding of protein evolution in extreme environments and may inform protein engineering efforts for applications requiring low-temperature functionality.

What role might the CrcB homolog play in fluoride resistance, and how can this be experimentally verified?

Based on studies of CrcB homologs in other bacterial species, the Psychrobacter arcticus CrcB protein may play a significant role in fluoride resistance, functioning as part of a fluoride ion channel or transporter system that helps maintain ionic homeostasis under environmental stress conditions. To verify this hypothesis experimentally, researchers could employ several complementary approaches.

First, gene knockout or silencing studies would establish whether the crcB gene is essential for fluoride resistance in P. arcticus. By creating a crcB deletion mutant and comparing its growth in media containing various fluoride concentrations to wild-type bacteria, researchers can quantify the contribution of this protein to fluoride tolerance. Complementation studies, reintroducing the wild-type gene on a plasmid vector, would confirm that any observed phenotypic changes are specifically attributable to the absence of CrcB.

Second, fluoride transport assays using radioactive ¹⁸F or fluorescent probes could directly measure the protein's capacity to transport fluoride ions across membranes. These assays could be performed in whole cells, membrane vesicles, or reconstituted liposomes containing the purified recombinant CrcB protein. Comparing transport kinetics at different temperatures (e.g., 4°C, 15°C, and 22°C) would reveal how temperature affects the protein's function, potentially identifying cold-specific adaptations in its transport mechanism.

Third, structural studies using techniques such as X-ray crystallography or cryo-electron microscopy could determine whether the P. arcticus CrcB homolog possesses unique structural features compared to mesophilic homologs that might contribute to its function at low temperatures. Combined with molecular dynamics simulations, these structural insights could reveal how the protein maintains functional flexibility in cold environments.

What is the amino acid composition profile of CrcB homolog protein and how does it reflect cold adaptation?

The amino acid composition of the Psychrobacter arcticus CrcB homolog protein reflects specific adaptations to cold environments that are consistent with broader patterns observed throughout the P. arcticus proteome. Analysis of the 123-amino acid sequence reveals several key features that likely contribute to maintained functionality at low temperatures.

The amino acid sequence (MQWLAIGLGAAFGACLRGWLARFNPLHHWIPLGTLGANVLGGLLIGLALVWFERMGSGLSPNIRLFVITGFLGGLTTFSTFSAEVFTFIHHGRLLAALGLVGLHVGMTLLATALGFYCFKLVL) shows a notably high glycine content, which contributes to backbone flexibility without sacrificing stability. The reduced proportion of charged amino acids, particularly acidic residues, is consistent with the cold adaptation strategy observed across the P. arcticus proteome, likely preventing excessive rigidity at low temperatures. The high proportion of hydrophobic residues suggests strong membrane association, which may be particularly important for maintaining membrane integrity and function in cold environments.

What are the key growth parameters for Psychrobacter arcticus in defined laboratory media?

The following table summarizes the key growth parameters for Psychrobacter arcticus in defined laboratory media, which are essential for researchers planning experiments with this psychrophilic organism:

Growth conditions should be carefully controlled when working with P. arcticus, particularly when transitioning from complex media to defined media. A serial culturing approach with a 1% inoculum is recommended for optimal adaptation. Growth is typically considered negative if no increase in turbidity is observed after 5 days of incubation at 22°C or after 10 days at 4°C.