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To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage 1,2 . In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery 3-6 . Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon-EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation., (© 2024. The Author(s).)

( R )-3-Hydroxybutyrate dehydrogenase (HBDH) catalyzes the NADH-dependent reduction of 3-oxocarboxylates to ( R )-3-hydroxycarboxylates. The active sites of a pair of cold- and warm-adapted HBDHs are identical except for a single residue, yet kinetics evaluated at -5, 0, and 5 °C show a much higher steady-state rate constant ( k cat ) for the cold-adapted than for the warm-adapted HBDH. Intriguingly, single-turnover rate constants ( k STO ) are strikingly similar between the two orthologues. Psychrophilic HBDH primary deuterium kinetic isotope effects on k cat ( D k cat ) and k STO ( D k STO ) decrease at lower temperatures, suggesting more efficient hydride transfer relative to other steps as the temperature decreases. However, mesophilic HBDH D k cat and D k STO are generally temperature-independent. The D k STO data allowed calculation of intrinsic primary deuterium kinetic isotope effects. Intrinsic isotope effects of 4.2 and 3.9 for cold- and warm-adapted HBDH, respectively, at 5 °C, supported by quantum mechanics/molecular mechanics calculations, point to a late transition state for both orthologues. Conversely, intrinsic isotope effects of 5.7 and 3.1 for cold- and warm-adapted HBDH, respectively, at -5 °C indicate the transition state becomes nearly symmetric for the psychrophilic enzyme, but more asymmetric for the mesophilic enzyme. His-to-Asn and Asn-to-His mutations in the psychrophilic and mesophilic HBDH active sites, respectively, swap the single active-site position where these orthologues diverge. At 5 °C, the His-to-Asn mutation in psychrophilic HBDH decreases D k cat to 3.1, suggesting a decrease in transition-state symmetry, while the His-to-Asn mutation in mesophilic HBDH increases D k cat to 4.4, indicating an increase in transition-state symmetry. Hence, temperature adaptation and a single divergent active-site residue may influence transition-state geometry in HBDHs.

Psychrobacter submarinus KMM 225 T is a Gram-negative bacterium isolated from a sea-water sample collected at a depth of 300 m in the Northwest Pacific Ocean. Here we report the structure of the capsular polysaccharide from P. submarinus KMM 225 T and its effect on the viability and colony formation of cancer cells. The glycopolymer was purified by ultracentrifugation and chromatography methods, and the structure was elucidated using NMR spectroscopy and composition analyses. The following structure of the acidic capsular polysaccharide, containing 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido]-d-glucose [d-QuipNAc4N(S-Hb)] and 4,6-O-[(S)-1-carboxyethylidene]-2-acetamido-2-deoxy-d-glucose [d-GlcpNAc4,6(S-Pyr)] was established: The capsular polysaccharide slightly reduced the viability but effectively suppressed the colony formation of different types of cancer cells, of which the most pronounced inhibition was shown for the human chronic myelogenous leukemia K-562 cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Although Psychrobacter strain M9-54-1 had been previously isolated from the microbiota of holothurians and shown to degrade quorum sensing (QS) signal molecules C6 and C10-homoserine lactone (HSL), little was known about the gene responsible for this activity. In this study, we determined the whole genome sequence of this strain and found that the full 16S rRNA sequence shares 99.78-99.66% identity with Psychrobacter pulmonis CECT 5989 T and P. faecalis ISO-46 T . M9-54-1, evaluated using the agar well diffusion assay method, showed high quorum quenching (QQ) activity against a wide range of synthetic N -acylhomoserine lactone (AHLs) at 4, 15, and 28 °C. High-performance liquid chromatography-mass-spectrometry (HPLC-MS) confirmed that QQ activity was due to an AHL-acylase. The gene encoding for QQ activity in strain M9-54-1 was identified from its genome sequence whose gene product was named AhaP. Purified AhaP degraded substituted and unsubstituted AHLs from C4- to C14-HSL. Furthermore, heterologous expression of ahaP in the opportunistic pathogen Pseudomonas aeruginosa PAO1 reduced the expression of the QS-controlled gene lecA , encoding for a cytotoxic galactophilic lectin and swarming motility protein. Strain M9-54-1 also reduced brine shrimp mortality caused by Vibrio coralliilyticus VibC-Oc-193, showing potential as a biocontrol agent in aquaculture.

The RidA subfamily proteins catalyze the deamination reaction of enamine/imine intermediates, which are metabolites of amino acids such as threonine and serine. Numerous structural and functional studies have been conducted on RidA isolated from mesophiles and thermophiles. However, little is known about the structure of the RidA proteins isolated from psychrophiles. In the present study, we elucidated the crystal structure of RidA from the Antarctic bacterium Psychrobacter sp. PAMC 21119 (Pp-RidA) at 1.6 Å resolution to identify the structural properties contributing to cold-adaptability. Although the overall structure of Pp-RidA is similar to those of its homologues, it exhibits specific structural arrangements of a loop positioned near the active site, which is assumed to play a role in covering the active site of catalysis. In addition, the surface electrostatic potential of Pp-RidA suggested that it exhibits stronger electrostatic distribution relative to its homologues. Our results provide novel insights into the key determinants of cold-adaptability., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.

Substrate and reaction promiscuity is a remarkable property of some enzymes and facilitates the adaptation to new metabolic demands in the evolutionary process. Substrate promiscuity is also a basis for protein engineering for biocatalysis. However, molecular principles of enzyme promiscuity are not well understood. Even for the widely studied PLP-dependent transaminases of class III, the reliable prediction of the biocatalytically important amine transaminase activity is still difficult if the desired activity is unrelated to the natural activity. Here, we show that 7,8-diaminopelargonic acid transaminase (synthase), previously considered to be highly specific, is able to convert (S)-(-)-1-phenylethylamine and a number of aldehydes and diketones. We were able to characterize the (S)-amine transaminase activity of 7,8-diaminopelargonic acid transaminase from Psychrobacter cryohalolentis (Pcryo361) and analyzed the three-dimensional structure of the enzyme. New substrate specificity for α-diketones was observed, though only a weak activity towards pyruvate was found. We examined the organization of the active site and binding modes of S-adenosyl-L-methionine and (S)-(-)-1-phenylethylamine using X-ray analysis and molecular docking. We suggest that the Pcryo361 affinity towards (S)-(-)-1-phenylethylamine arises from the recognition of the hydrophobic parts of the specific substrates, S-adenosyl-L-methionine and 7-keto-8-aminopelargonic acid, and from the flexibility of the active site. Our results support the observation that the conversion of amines is a promiscuous activity of many transaminases of class III and is independent from their natural function. The analysis of amine transaminase activity from among various transaminases will help to make the sequence-function prediction for biocatalysis more reliable.

Psychrobacter arcticus 273-4 is a Gram-negative bacterium isolated from a 20,000-to-30,000-year-old continuously frozen permafrost in the Kolyma region in Siberia. The survival strategies adopted to live at subzero temperatures include all the outer membrane molecules. A strategic involvement in the well-known enhancement of cellular membrane fluidity is attributable to the lipopolysaccharides (LPSs). These molecules covering about the 75% of cellular surface contribute to cold adaptation through structural modifications in their portions. In this work, we elucidated the exact structure of lipid A moiety obtained from the lipopolysaccharide of P. arcticus grown at 4 °C, to mimic the response to the real environment temperatures. The lipid A was obtained from the LPS by mild acid hydrolysis. The lipid A and its partially deacylated derivatives were exhaustively characterized by chemical analysis and by means of ESI Q-Orbitrap mass spectrometry. Moreover, biological assays indicated that P. arcticus 273-4 lipid A may behave as a weak TLR4 agonist.

Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisG S ) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg 2+ -dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N 1 -(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, the first reaction of histidine biosynthesis. While HisG S is catalytically active on its own, its activity is allosterically enhanced by HisZ in the absence of histidine. In the presence of histidine, HisZ mediates allosteric inhibition of ATPPRT. Here, initial velocity patterns, isothermal titration calorimetry, and differential scanning fluorimetry establish a distinct kinetic mechanism for ATPPRT where PRPP is the first substrate to bind. AMP is an inhibitor of HisG S , but steady-state kinetics and 31 P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg 2+ by Mn 2+ enhances catalysis by HisG S but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an S N 2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn 2+ increasing HisG S reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisG S 's catalytic rate, but decreases the hetero-octamer's, indicating that chemistry and product release are rate-limiting for HisG S and ATPPRT, respectively. This is confirmed by pre-steady-state kinetics, with a burst in product formation observed with the hetero-octamer but not with HisG S . These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisG S , accelerating chemistry beyond the product release rate.

Marine environment represents a promising source of new, unconventional bioactive compounds with health-promoting abilities, which can be used as food supplements. The present study was carried out to evaluate the efficacy of marine Psychrobacter namhaensis SO89 on growth performance and immune response of Nile tilapia (Oreochromis niloticus). P. namhaensis were isolated from marine environments and phylogenetically identified by 16S rRNA gene sequences. The bacterial isolate was incorporated in Nile tilapia diets (30% crude protein) at three concentrations (0.0, 0.5 and 1.0%; w/w) (designated as T 0 , T 0.5 and T 1 , respectively), which were equivalent to 0.0, 2.8 × 10 7 and 5.6 × 10 7  CFU g -1 diet, respectively. The diets were fed to Nile tilapia fingerlings (4.58 ± 0.14 g average weight) at a daily rate of 3% of their live body weights (BW), 3 times a day for 50 days. The best growth rates and feed utilization efficiency were obtained at 0.5% P. namhaensis SO89 concentration. Hematocrit (Ht%), hemoglobin (Hb%), erythrocytes (RBC) and total leukocyte (WBCs) values were significantly higher in P. namhaensis SO89- fed groups than in the control group. Similarly, immunoglobulin M (IgM), alternative complement hemolysis (ACH50), phagocytic and lysozyme activities significantly increased following dietary P. namhaensis SO89 supplementation at 0.5% concentration compared to the control group. The expression of IL-4 and IL-12 genes was also significantly up-regulated in P. namhaensis SO89-treated groups up to 0.5% concentration. Increasing bacterial concentration to 1% resulted in a significant decrease in fish performance and immune response. The present results suggest that marine psychrotolerant (Psychrobacter namhaensis) can be considered as a novel feed additive in Nile tilapia feeds., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Microorganisms living in extreme environments represent a huge reservoir of novel antimicrobial compounds and possibly of novel chemical families. Antarctica is one of the most extraordinary places on Earth and exhibits many distinctive features. Antarctic microorganisms are well known producers of valuable secondary metabolites. Specifically, several Antarctic strains have been reported to inhibit opportunistic human pathogens strains belonging to Burkholderia cepacia complex (Bcc). Herein, we applied a biodiscovery pipeline for the identification of anti-Bcc compounds. Antarctic sub-sea sediments were collected from the Ross Sea, and used to isolate 25 microorganisms, which were phylogenetically affiliated to three bacterial genera (Psychrobacter, Arthrobacter, and Pseudomonas) via sequencing and analysis of 16S rRNA genes. They were then subjected to a primary cell-based screening to determine their bioactivity against Bcc strains. Positive isolates were used to produce crude extracts from microbial spent culture media, to perform the secondary screening. Strain Pseudomonas BNT1 was then selected for bioassay-guided purification employing SPE and HPLC. Finally, LC-MS and NMR structurally resolved the purified bioactive compounds. With this strategy, we achieved the isolation of three rhamnolipids, two of which were new, endowed with high (MIC < 1 μg/mL) and unreported antimicrobial activity against Bcc strains.

Psychrophilic microorganisms have successfully colonized all permanently cold environments from the deep sea to mountain and polar regions. The ability of an organism to survive and grow in cryoenviroments depends on a number of adaptive strategies aimed at maintaining vital cellular functions at subzero temperatures, which include the structural modifications of the membrane. To understand the role of the membrane in the adaptation, it is necessary to characterize the cell-wall components, such as the lipopolysaccharides, that represent the major constituent of the outer membrane. The aim of this study was to investigate the structure of the carbohydrate backbone of the lipooligosaccharide (LOS) isolated from the cold-adapted Psychrobacter arcticus 273-4. The strain, isolated from a 20,000-to-30,000-year-old continuously frozen permafrost in Siberia, was cultivated at 4 °C. The LOS was isolated from dry cells and analyzed by means of chemical methods. In particular, it was degraded either by mild acid hydrolysis or by hydrazinolysis and investigated in detail by (1)H and (13)C NMR spectroscopy and by ESI FT-ICR mass spectrometry. The oligosaccharide was characterized by the substitution of the heptose residue, usually linked to Kdo in the inner core, with a glucose, and for the unusual presence of N-acetylmuramic acid.

Dimerization of bacterial chaperone trigger factor (TF) is an inherent protein concentration based property which available biophysical characterization and crystal structures have kept debatable. We acquired small-angle X-ray scattering (SAXS) intensity data from different TF homologues from Escherichia coli (ECTF), Vibrio cholerae (VCTF), and Psychrobacter frigidicola (PFTF) while varying each protein concentration. We found that ECTF and VCTF adopt a compact dimeric shape at higher concentrations which did not resemble the "back-to-back" conformation reported earlier for ECTF from crystallography (PDB ID: 1W26 ). In contrast, PFTF remained monomeric throughout the concentration range 2-90 μM displaying a multimodal open extended conformation. OLIGOMER analysis showed that both the ECTF and VCTF remained completely monomeric at lower concentrations (2-11 μM), while, at higher concentrations (60-90 μM), they adopted a dimeric form. Interestingly, the equilibrium existed in the medium concentration range (>11 and <60 μM), which correlates with the physiological concentration (40-50 μM) of TF in cell cytoplasm. Additionally, circular dichroism data revealed that solution structures of ECTF and VCTF contain predominantly α-helical content, while PFTF contains 310-helical content.

Correlation between the chemical structure of lipid A from various Gram-negative bacteria and biological activity of their lipopolysaccharide (LPS) as an agonist of the innate immune receptor Toll-like receptor 4 was investigated. Purified LPS species were quantitatively evaluated by their ability to activate the production of tumor necrosis factor (TNF) by murine bone marrow-derived macrophages in vitro. Wild-type LPS from plague-causing bacteria Yersinia pestis was compared to LPS from mutant strains with defects in acyltransferase genes (lpxM, lpxP) responsible for the attachment of secondary fatty acid residues (12:0 and 16:1) to lipid A. Lipid A of Y. pestis double ΔlpxM/ΔlpxP mutant was found to have the chemical structure that was predicted based on the known functions of the respective acyltransferases. The structures of lipid A from two members of the ancient psychrotrophic bacteria of the genus Psychrobacter were established for the first time, and biological activity of LPS from these bacteria containing lipid A fatty acids with shorter acyl chains (C10-C12) than those in lipid A from LPS of Y. pestis or E. coli (C12-C16) was determined. The data revealed a correlation between the ability of LPS to activate TNF production by bone marrow-derived macrophages with the number and the length of acyl chains within lipid A.

Lipid A is the essential component of endotoxin (Gram-negative lipopolysaccharide), a potent immunostimulatory compound. As the outer surface of the outer membrane, the details of lipid A structure are crucial not only to bacterial pathogenesis but also to membrane integrity. This work characterizes the structure of lipid A in two psychrophiles, Psychromonas marina and Psychrobacter cryohalolentis, and also two mesophiles to which they are related using MALDI-TOF MS and fatty acid methyl ester (FAME) GC-MS. P. marina lipid A is strikingly similar to that of Escherichia coli in organization and total acyl size, but incorporates an unusual doubly unsaturated tetradecadienoyl acyl residue. P. cryohalolentis also shows structural organization similar to a closely related mesophile, Acinetobacter baumannii, however it has generally shorter acyl constituents and shows many acyl variants differing by single methylene (-CH2-) units, a characteristic it shares with the one previously reported psychrotolerant lipid A structure. This work is the first detailed structural characterization of lipid A from an obligate psychrophile and the second from a psychrotolerant species. It reveals distinctive structural features of psychrophilic lipid A in comparison to that of related mesophiles which suggest constitutive adaptations to maintain outer membrane fluidity in cold environments.

Herein we present the draft genomes of three Psychrobacter strains isolated from Antarctic sponges and able to inhibit the growth of bacteria belonging to the Burkholderia cepacia complex, responsible for infections of the respiratory system in patients affected by Cystic Fibrosis. The comparative analysis of the annotated genomes of these Psychrobacter strains highlighted their differences in terms of overall genomic content (e.g. shared gene sets) and allowed the identification of gene clusters hypothetically involved in the biosynthesis of antimicrobial compounds., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The spoilage potential of several Psychrobacter and Pseudoalteromonas species (Psychrobacter cibarius, Psychrobacter maritimus, Pseudoalteromonas elyakovii, Pseudoalteromonas paragorgicola and Pseudoalteromonas nigrifaciens) was determined and quantified based on the presence of volatile organic compounds (VOCs). Psychrobacter and Pseudoalteromonas species dominate the microbiota of cooked brown shrimp (Crangon crangon). Additionally, API ZYM analyses determined the species' enzymatic capacity to contribute to spoilage by degrading lipids, amino acids and proteins. The bacterial species used in this study were isolated from cooked brown shrimp during storage under different storage and processing conditions and were selected for analysis of their spoilage potential based on their difference in the (GTG)₅-rep profile, 16S rRNA and gyrB sequences and API ZYM profile. The isolates were inoculated as pure cultures on heat-sterilised shrimp. The inoculated samples were stored at 4 °C and the production of VOCs by the pure strains on the shrimp matrix was identified via gas chromatography coupled to mass spectrometry (GC-MS). VOC production was quantified daily by selected ion flow tube mass spectrometry (SIFT-MS) until the bacterial count exceeded 10⁸-10⁹ cfu/g. The sensory profile of Psychrobacter species revealed very low spoilage potential as measured by the production of VOCs, but these species may nevertheless contribute to spoilage. Based on the API ZYM results, Pseudoalteromonas as well as Psychrobacter species might enhance spoilage by breaking down lipids and hydrolysing amino acids and proteins. Pseudoalteromonas species, especially Psa. elyakovii and Psa. nigrifaciens, have a high spoilage potential and might be responsible for the off-odours produced during spoilage of brown shrimp. These isolates produced significant amounts of volatile compounds such as sulphides, acetone, ammonia, and ethanol, which are all involved in seafood spoilage., (© 2013 Elsevier B.V. All rights reserved.)

A novel constituent of bacterial polysaccharides, 2,3,4-triacetamido-2,3,4-trideoxy-L-arabinose, was found in the O-specific polysaccharide from the lipopolysaccharide of Psychrobacter cryohalolentis K5(T) and identified by 1D and 2D (1)H and (13)C NMR studies of the polysaccharide and a disaccharide obtained by solvolysis of the polysaccharide with triflic acid. The following structure of the branched polysaccharide was established by sugar analysis, triflic acid solvolysis, Smith degradation, and 2D NMR spectroscopy.

An acidic polysaccharide was obtained from Psychrobacter maritimus 3pS isolated from a Siberian cryopeg sample (Kolyma lowland). The following structure of the tetrasaccharide repeating unit of the polysaccharide was established by sugar analysis along with (1)H and (13)C NMR spectroscopy: →2)-α-L-Rhap-(1→4)-α-D-GalpNAcA-(1→3)-α-D-QuipNAc4NHb-(1→3)-β-D-QuipNAc4NHb-(1→ where D-GalNAcA indicates 2-acetamido-2-deoxy-D-galacturonic acid and d-QuiNAc4NHb indicates 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutanoyl]amino-D-glucose., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cold-adapted enzymes possess high specific activity at low and moderate temperatures with respect to their mesophilic and thermophilic homologs; it is accepted that they have a less rigid and more flexible structure in the region surrounding the active site. However, the low stability of such molecules could represent the main barrier for their application in some industrial bioprocesses. The aim of this article was to investigate the ability of the naturally occurring osmolytes to increase the thermal stability and the specific activity of the cold-active lipase from Psychrobacter sp. TA144 (PsyHSL), which belongs to the hormone-sensitive lipase group. The effect of trimethylamine N-oxide (TMAO), betaine, and L-proline addition on the activity and thermal stability of PsyHSL was investigated by means of biochemical and biophysical techniques. It turned out that in the presence of 3 M TMAO, the enzyme specific activity enhanced up to 250% at 50°C, while the addition of 1 M TMAO increased the thermostability fivefold at 45°C. Our experiments demonstrated that, even in the case of a psychrophilic enzyme, osmoprotectants, particularly TMAO, addition may be considered an efficient strategy to improve the protein thermal stability and specific activity at higher temperatures., (Copyright © 2012 American Institute of Chemical Engineers (AIChE).)

Psychrotrophic bacteria of the genus Psychrobacter have not been studied in respect to lipopolysaccharide structure. In this work, we determined the structure of the O-specific polysaccharide of the lipopolysaccharide of Psychrobacter muricolla 2pS(T) isolated from overcooled (-9°C) water brines within permafrost. The polysaccharide was found to be acidic due to the presence of an amide of 2-acetamido-2-deoxy-l-guluronic acid with glycine (l-GulNAcA6Gly), which has not been hitherto found in nature. The following structure of the disaccharide repeating unit of the polysaccharide was established using composition analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy: →4)-α-l-GulpNAcA6Gly-(1→3)-β-d-GlcpNAc-(1→, (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are significant pathogens in Cystic Fibrosis (CF) patients and are resistant to a plethora of antibiotics. In this context, microorganisms from Antarctica are interesting because they produce antimicrobial compounds inhibiting the growth of other bacteria. This is particularly true for bacteria isolated from Antarctic sponges. The aim of this work was to characterize a set of Antarctic bacteria for their ability to produce new natural drugs that could be exploited in the control of infections in CF patients by Bcc bacteria. Hence, 11 bacterial strains allocated to different genera (e.g., Pseudoalteromonas, Arthrobacter and Psychrobacter) were tested for their ability to inhibit the growth of 21 Bcc strains and some other human pathogens. All these bacteria completely inhibited the growth of most, if not all, Bcc strains, suggesting a highly specific activity toward Bcc strains. Experimental evidences showed that the antimicrobial compounds are small volatile organic compounds, and are constitutively produced via an unknown pathway. The microbial volatile profile was obtained by SPME-GC-MS within the m/z interval of 40-450. Solid phase micro extraction technique affords the possibility to extract the volatile compounds in head space with a minimal sample perturbation. Principal component analysis and successive cluster discriminant analysis was applied to evaluate the relationships among the volatile organic compounds with the aim of classifying the microorganisms by their volatile profile. These data highlight the potentiality of Antarctic bacteria as novel sources of antibacterial substances to face Bcc infections in CF patients., (Copyright © 2011 John Wiley & Sons, Ltd.)

The cold-active lipase gene Lip-948, cloned from Antarctic psychrotrophic bacterium Psychrobacter sp. G, was ligated into plasmid pColdI. The recombinant plasmid pColdI+Lip-948 was then transformed into Escherichia coli BL21. SDS-PAGE analysis showed that there was substantive expression of lipase LIP-948 in E. coli with a yield of about 39% of total protein, most of which was present in the inclusion body. The soluble protein LIP-948 only consisted of 1.7% of total LIP-948 with a specific activity of 66.51U/mg. Co-expression of molecular chaperones with the pColdI+Lip-948 were also carried out. The results showed that co-expression of different chaperones led to an increase or decrease in the formation of soluble LIP-948 in varying degrees. Co-expression of pColdI+Lip-948 with chaperone pTf16 and pGro7 decreased the amount of soluble LIP-948, while the soluble expression was enhanced when pColdI+Lip-948 was co-expressed with "chaperone team" plasmids (pKJE7, pG-Tf2, pG-KJE8), respectively. LIP-948 was most efficiently expressed in soluble form when it was co-expressed with pG-KJE8, which was up to 19.8% of intracellular soluble proteins and with a specific activity of 108.77U/mg. The soluble LIP-948 was purified with amylase affinity chromatography and its enzymatic characters were studied. The optimal temperature and pH of LIP-948 was 35°C and 8, respectively. The activity of LIP-948 dropped dramatically after incubation at 50°C for 15min and was enhanced by Sr(2+), Ca(2+). It preferentially hydrolyzed 4-nitrophenyl esters with the shorter carbon chain., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In our search for bioactive metabolites from a marine sponge-associated bacterium Psychrobacter sp., a new bile acid derivative (1), which was assumed to be an artifact, were isolated along with six known (2-7) compounds by bioactivity-guided fractionation. Elucidation of the structure of the new compound was done using a combination of NMR ((1)H, (13)C, HMBC, HSQC, and COSY) and MS spectroscopy. Compound 1 exhibited moderate suppressive effects on both NO and IL-6 production at a concentration of 200 microM (87.3 microg/mL) without significant cytotoxicity against cells. Compounds 2-5 and 7 showed selective inhibitory activity against several human pathogenic bacterial strains at the low concentration of 30 microg/well. In a cytotoxicity evaluation, only compound 7 showed mild cytotoxicity against five human solid tumor cell lines (A-549, SK-OV-3, SK-MEL-2, XF-498, and HCT-15) with ED(50) values in the range of 11-14 microg/mL.

Psychrobacter 273-4 was isolated from a 20,000-40,000-year-old Siberian permafrost core, which is characterized by low temperature, low water activity, and high salinity. To explore how 273-4 survives in the permafrost environment, proteins in four 273-4 samples cultured at 4 and 22 degrees C in media with and without 5% sodium chloride were profiled and comparatively studied using 2-D HPLC and MS. The method used herein involved fractionation via a pH gradient using chromatofocusing followed by nonporous silica (NPS) RP-HPLC and on-line electrospray mass mapping. It was observed that 33 proteins were involved in the adaptation to low temperature in the cells grown in the nonsaline media while there were only 14 proteins involved in the saline media. There were 45 proteins observed differentially expressed in response to salt at 22 degrees C while there were 22 proteins at 4 degrees C. In addition, 5% NaCl and 4 degrees C showed a combination effect on protein expression. A total of 56 proteins involved in the adaptation to low temperature and salt were identified using MS and database searching. The differentially expressed proteins were classified into different functional categories where the response of the regulation system to stress appears to be very elaborate. The evidence shows that the adaptation of 273-4 is based primarily on the control of translation and transcription, the synthesis of proteins (chaperones) to facilitate RNA and protein folding, and the regulation of metabolic pathways.

Twelve strains of psychrophilic bacteria were isolated from cyanobacterial mat samples collected from various water bodies in the McMurdo Dry Valley region of Antarctica. All the isolates were Gram-negative, non-motile, coccoid, psychrophilic, halotolerant bacteria and had C(16 : 1)omega7c, C(17 : 1)omega8c and C(18 : 1)omega9c as the major fatty acids, ubiquinone-8 as the respiratory quinone and DNA G+C content of 41-46 mol%. Based on these characteristics, the isolates were assigned to the genus Psychrobacter. Based on their SDS-PAGE profiles, the 12 isolates could be categorized into three groups. Six isolates of Group I were identified as representing strains of Psychrobacter okhotskensis. However, using detailed phenotypic and chemotaxonomic characteristics and phylogenetic analysis based on their 16S rRNA gene sequences, strain CMS 39(T), the only strain from Group II, and strain CMS 56(T), a representative strain of Group III, were different from each other and from all recognized species of Psychrobacter. Therefore, it is proposed to classify CMS 39(T) (=DSM 15337(T)=MTCC 4208(T)) and CMS 56(T) (=DSM 15339(T)=MTCC 4386(T)) as representing the type strains of novel species of Psychrobacter, for which the names Psychrobacter vallis sp. nov. and Psychrobacter aquaticus sp. nov., respectively, are proposed.

Two Gram-negative, non-motile, non-spore-forming, moderately halophilic strains, JG-100T and JG-102, were isolated from squid jeotgal, a traditional Korean fermented seafood. The two strains grew optimally at 30 degrees C and in the presence of 2-3 % (v/w) NaCl. Strains JG-100T and JG-102 were characterized chemotaxonomically; they both had ubiquinone-8 as the predominant respiratory lipoquinone and C(18 : 1)omega9c as the major fatty acid. Their DNA G+C content was 44 mol%. Strains JG-100T and JG-102 showed 1 bp difference in their 16S rRNA gene sequences and a mean DNA-DNA relatedness level of 88 %. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains JG-100T and JG-102 form a distinct phylogenetic lineage within the cluster comprising Psychrobacter species. The 16S rRNA gene sequences of strains JG-100T and JG-102 had similarity levels of 95.2-98.4 % to sequences of the type strains of recognized Psychrobacter species. Levels of DNA-DNA relatedness between strains JG-100T and JG-102 and the type strains of some phylogenetically related Psychrobacter species were 6-24 %. On the basis of phenotypic and phylogenetic data and genomic distinctiveness, strains JG-100T and JG-102 should be placed in the genus Psychrobacter as a novel species, for which the name Psychrobacter alimentarius sp. nov. (type strain, JG-100T=KCTC 12186T=DSM 16065T) is proposed.

Nine psychrotolerant bacteria were isolated from fast ice in the middle of Geologie Archipelago, Adelie Land, Antarctica and were categorized into two groups, based on their SDS-PAGE profiles. Representatives from each of the two groups, namely strains DD 48T and SJ 14T exhibited phenotypic and chemotaxonomic characteristics confirming to the genus Psychrobacter. The 16S rRNA gene sequence indicated that the two isolates are closely related to each other and to the already reported fifteen species of Psychrobacter. Detailed studies on the phenotypic characteristics, chemotaxonomic properties and phylogenetic analysis of strains DD 48T and SJ 14T indicated that they are distinctly different from each other and the reported species of Psychrobacter. At the DNA-DNA hybridisation level, the two species exhibit less than 70% similarity. Thus, strains DD 48T and SJ 14T are identified as new species of the genus Psychrobacter for which the names Psychrobacter salsus sp. nov. and Psychrobacter adeliensis sp. nov. respectively are proposed.