Complete genome sequence of Psychrobacter cibarius AOSW16051, a trimeric autotransporter adhesin synthesizing bacterium isolated from the Baltic Sea.

Bacteria of the genus Psychrobacter are widely distributed in the global low-temperature marine environment and have been studied for their effects on the settlement and metamorphosis of marine invertebrates. Psychrobacter cibarius AOSW16051 was isolated from the surface water samples of the Baltic Sea on the edge of the Arctic Ocean. Here, we present the complete genome of strain AOSW16051, which consists of a circular chromosome composed of 3,425,040 nucleotides with 42.98% G + C content and a circular plasmid composed of 5846 nucleotides with 38.66% G + C content. The genes predicted in this strain showed its strong outer membrane system, type VI secretion system and adhesion system. Trimeric autotransporter adhesins (TAAs) has been identified in the genome of P. cibarius AOSW16051, which has a variety of biological functions in interacting with host cells. However, there are no reports on TAAs in marine bacteria and aquatic pathogenic bacteria. By analyzing the genomic data, we can gain valuable insights to enhance our understanding of the physiological characteristics of P. cibarius, as well as the biological functions of TAAs and their role in triggering metamorphosis of invertebrate larvae.

A new family of bacterial ribosome hibernation factors.

To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. 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 discovery3-6. 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.

Psychrobacter species enrichment as potential microplastic degrader and the putative biodegradation mechanism in Shenzhen Bay sediment, China.

Microplastic (MP) pollution has emerged as a pressing environmental concern due to its ubiquity and longevity. Biodegradation of MPs has garnered significant attention in combatting global MP contamination. This study focused on MPs within sediments near the sewage outlet of Shenzhen Bay. The objective was to elucidate the microbial communities in sediments with varying MPs, particularly those with high MP loads, and to identify microorganisms associated with MP degradation. The results revealed varying MP abundance, ranging from 211 to 4140 items kg-1 dry weight (d. w.), with the highest concentration observed near the outfall. Metagenomic analysis confirmed the enrichment of Psychrobacter species in sediments with high MP content. Psychrobacter accounted for ∼16.71% of the total bacterial community and 41.71% of hydrocarbon degrading bacteria at the S3 site, exhibiting a higher abundance than at other sampling sites. Psychrobacter contributed significantly to bacterial function at S3, as evidenced by the Kyoto Encyclopedia of Genes and Genomes pathway and enzyme analysis. Notably, 28 enzymes involved in MP biodegradation were identified, predominantly comprising oxidoreductases, hydrolases, transferases, ligases, lyases, and isomerases. We propose a putative mechanism for MP biodegradation, involving the breakdown of long-chain plastic polymers and subsequent oxidation of short-chain oligomers, ultimately leading to thorough mineralization.

Heterologous protein production using Psychrobacter sp. PAMC 21119 analyzed with a green fluorescent protein-based reporter system.

Insolubility and low expression are typical bottlenecks in the production of proteins for studying their function and structure using X-ray crystallography or nuclear magnetic resonance spectroscopy. Cold-active enzymes from polar microorganisms have unique structural features that render them unstable and thermolabile, and are responsible for decreased protein yield in heterologous expression systems. To address these challenges, we developed a heterologous protein expression system using a psychrophilic organism, Psychrobacter sp. PAMC 21119, as a protein expression host with its own promoter. We screened 11 promoters and evaluated their strength using quantitative real-time polymerase chain reaction and a reporter system harboring the SfGFP gene. The highest expression was achieved using promoters RH96_RS13655 (P21119_20930) and RH96_RS15090 (P21119_23410), regardless of the temperature used. The p20930 strain exhibited a maximum expression level 19.6-fold higher than that of its control at 20 °C and produced approximately 0.5 mg of protein per gram of dry cell weight. To our knowledge, this is the first report of a low-temperature recombinant protein expression system developed using Psychrobacter sp. that can be used to express various difficult-to-express and cold-active proteins.

Phylogenomics-based reclassifications in the genus Psychrobacter including emended descriptions of Psychrobacter pacificensis, Psychrobacter proteolyticus and Psychrobacter submarinus.

The taxonomic status of 43 Psychrobacter species was examined based upon the genome sequences of their type strains. Three groups of type strains were found to be conspecific, Psychrobacter salsus Shivaji et al. (Syst Appl Microbiol 27:628-635, 2004. 10.1078/0723202042369956) and Psychrobacter submarinus Romanenko et al. (Int J Syst Evol Microbiol 52:1291-1297, 2002. 10.1099/00207713-52-4-1291); Psychrobacter oceani Matsuyama et al. (Int J Syst Evol Microbiol 65:1450-1455, 2015. 10.1099/ijs.0.000118) and Psychrobacter pacificensis Maruyama et al. (Int J Syst Evol Microbiol 50:835-846, 2000. 10.1099/00207713-50-2-835); and Psychrobacter proteolyticus Denner et al. (Syst Appl Microbiol 24:44-53, 2001. 10.1078/0723-2020-00006), Psychrobacter marincola Romanenko et al. (Int J Syst Evol Microbiol 52:1291-1297, 2002. 10.1099/00207713-52-4-1291) and Psychrobacter adeliensis Shivaji et al. (Syst Appl Microbiol 27:628-635, 2004. 10.1078/0723202042369956). For all three groups, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values are > 97.69% and > 80.2%, respectively. This conclusion is supported by similarities in morphology, growth properties, and fatty acid compositions. Based on this evidence, we propose the reclassification of Psychrobacter salsus Shivaji et al. (Syst Appl Microbiol 27:628-635, 2004. 10.1078/0723202042369956) as a later heterotypic synonym of Psychrobacter submarinus Romanenko et al. (Int J Syst Evol Microbiol 52:1291-1297, 2002. 10.1099/00207713-52-4-1291); Psychrobacter oceani Matsuyama et al. (Int J Syst Evol Microbiol 65:1450-1455, 2015. 10.1099/ijs.0.000118) as a later heterotypic synonym of Psychrobacter pacificensis Maruyama et al. (Int J Syst Evol Microbiol 50:835-846, 2000. 10.1099/00207713-50-2-835), and Psychrobacter marincola Romanenko et al. (Int J Syst Evol Microbiol 52:1291-1297, 2002. 10.1099/00207713-52-4-1291) and Psychrobacter adeliensis Shivaji et al. (Syst Appl Microbiol 27:628-635, 2004. 10.1078/0723202042369956) as later heterotypic synonyms of Psychrobacter proteolyticus Denner et al. (Syst Appl Microbiol 24:44-53, 2001. 10.1078/0723-2020-00006).

Psychrobacter Phage Encoding an Antibiotics Resistance Gene Represents a Novel Caudoviral Family.

Psychrobacter is an important bacterial genus that is widespread in Antarctic and marine environments. However, to date, only two complete Psychrobacter phage sequences have been deposited in the NCBI database. Here, the novel Psychrobacter phage vB_PmaS_Y8A, infecting Psychrobacter HM08A, was isolated from sewage in the Qingdao area, China. The morphology of vB_PmaS_Y8A was characterized by transmission electron microscopy, revealing an icosahedral head and long tail. The genomic sequence of vB_PmaS_Y8A is linear, double-stranded DNA with a length of 40,226 bp and 44.1% G+C content, and encodes 69 putative open reading frames. Two auxiliary metabolic genes (AMGs) were identified, encoding phosphoadenosine phosphosulfate reductase and MarR protein. The first AMG uses thioredoxin as an electron donor for the reduction of phosphoadenosine phosphosulfate to phosphoadenosine phosphate. MarR regulates multiple antibiotic resistance mechanisms in Escherichia coli and is rarely found in viruses. No tRNA genes were identified and no lysogeny-related feature genes were detected. However, many similar open reading frames (ORFs) were found in the host genome, which may indicate that Y8A also has a lysogenic stage. Phylogenetic analysis based on the amino acid sequences of whole genomes and comparative genomic analysis indicate that vB_PmaS_Y8A contains a novel genomic architecture similar only to that of Psychrobacter phage pOW20-A, although at a low similarity. vB_PmaS_Y8A represents a new family-level virus cluster with 22 metagenomic assembled viral genomes, here named Minviridae. IMPORTANCE Although Psychrobacter is a well-known and important bacterial genus that is widespread in Antarctic and marine environments, genetic characterization of its phages is still rare. This study describes a novel Psychrobacter phage containing an uncharacterized antibiotic resistance gene and representing a new virus family, Minviridae. The characterization provided here will bolster current understanding of genomes, diversity, evolution, and phage-host interactions in Psychrobacter populations.

Structure and functions of a multireplicon genome of Antarctic Psychrobacter sp. ANT_H3: characterization of the genetic modules suitable for the construction of the plasmid-vectors for cold-active bacteria.

Among Psychrobacter spp., there are several multireplicon strains, carrying more than two plasmids. Psychrobacter sp. ANT_H3 carries as many as 11 extrachromosomal replicons, which is the highest number in Psychrobacter spp. Plasmids of this strain were subjected to detailed genomic analysis, which enables an insight into the structure and functioning of this multireplicon genome. The replication and conjugal transfer modules of ANT_H3 plasmids were analyzed functionally to discover their potential for being used as building blocks for the construction of novel plasmid-vectors for cold-active bacteria. It was shown that two plasmids have a narrow host range as they were not able to replicate in species other than Psychrobacter, while remaining plasmids had a wider host range and were functional in various Alpha- and Gammaproteobacteria. Moreover, it was confirmed that mobilization modules of seven plasmids were functional, i.e., could be mobilized for conjugal transfer by the RK2 conjugation system. Auxiliary genes were also distinguished in ANT_H3 plasmids, including these encoding putative DNA-protecting protein DprA, multidrug efflux SMR transporter of EmrE family, glycine cleavage system T protein, MscS small-conductance mechanosensitive channel protein, and two type II restriction-modification systems. Finally, all genome-retrieved plasmids of Psychrobacter spp. were subjected to complex genome- and proteome-based comparative analyses showing that Antarctic replicons are significantly different from plasmids from other locations.

Detection and description of a novel Psychrobacter glacincola infection in some Red Sea marine fishes in Hurghada, Egypt.

An important food-producing sector in Egypt is aquaculture and fisheries; however, several pathogenic microorganisms lead to high mortalities and significant economic losses. The occurrence of Psychrobacter glacincola infection among 180 wild marine fishes collected from the Red sea at Hurghada, Egypt were investigated in the present study. The disease prevalence rate was 6.7%. The recovered isolates were subjected to biochemical and molecular identification. The study also investigated pathogenicity and the antibiogram profile of the recovered isolates. The clinical examination of the infected fish revealed various signs that included lethargy and sluggish movement, hemorrhages and ulcers on the body and the operculum, scale loss, and fin congestion and rot, especially at the tail fin. Furthermore, during postmortem examination, congestion of the liver, spleen, and kidney was observed. Interestingly, 12 isolates were recovered and were homogenous bacteriologically and biochemically. The phylogenetic analysis based on 16S rRNA gene confirmed that MRB62 identified strain was closely related the genus Psychrobacter and identified as P. glacincola and was pathogenic to Rhabdosargus haffara fish, causing 23.3% mortality combined with reporting a series of clinical signs similar to that found in naturally infected fishes. The present study also showed that P. glacincola isolates were sensitive to all antibiotics used for sensitivity testing. Our findings add to the body of knowledge regarding the occurrence of pathogenic P. glacincola infection in Egyptian marine fishes and its potential effects on fish. Future large-scale surveys exploring this bacterium among other freshwater and marine fishes in Egypt would be helpful for the implementation of effective strategies for the prevention and control of this infection are warranted.

A Plasmid-Borne Gene Cluster Flanked by Two Restriction-Modification Systems Enables an Arctic Strain of Psychrobacter sp. to Decompose SDS.

The cold-adapted Psychrobacter sp. strain DAB_AL62B, isolated from ornithogenic deposits on the Arctic island of Spitsbergen, harbors a 34.5 kb plasmid, pP62BP1, which carries a genetic SLF module predicted to enable the host bacterium to metabolize alkyl sulfates including sodium dodecyl sulfate (SDS), a common anionic surfactant. In this work, we experimentally confirmed that the pP62BP1-harboring strain is capable of SDS degradation. The slfCHSL genes were shown to form an operon whose main promoter, PslfC, is negatively regulated by the product of the slfR gene in the absence of potential substrates. We showed that lauryl aldehyde acts as an inducer of the operon. The analysis of the draft genome sequence of the DAB_AL62B strain revealed that the crucial enzyme of the SDS degradation pathway-an alkyl sulfatase-is encoded only within the plasmid. The SLF module is flanked by two restriction-modification systems, which were shown to exhibit the same sequence specificity. We hypothesize that the maintenance of pP62BP1 may be dependent on this unique genetic organization.

Molecular cloning, characterization, and homology modeling of serine hydroxymethyltransferase from psychrophilic bacterium Psychrobacter sp.

Serine hydroxymethyltransferase (SHMT) plays a significant role in the synthesis of l-serine, purine, and thymidylate, which could be extensively applied in the treatment of cancers and the development of antibiotics. In this study, cloned from Psychrobacter sp. ANT206, a novel cold-adapted SHMT gene (psshmt, 1257 bp) encoding a protein of 418 amino acids was expressed in Escherichia coli. The homology modeling result revealed that PsSHMT owned fewer Proline (Pro) residues and hydrogen bonds compared with its homologs from mesophilic E. coli and thermophilic Geobacillus stearothermophilus. In addition, the molecular weight of the purified recombinant PsSHMT (rPsSHMT) was identified to be 45 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, approximately. The enzymatic characteristics of the cold-adapted rPsSHMT displayed that its optimum temperature and pH were 30°C and 7.5, respectively, and its enzymatic activity could be inhibited by Cu2+ , significantly. rPsSHMT also showed a high kcat value and low ΔG at low temperatures. Furthermore, arginine (Arg) could affect the activity of rPsSHMT and be vital to its active sites. The results of this study reflected that these characteristics of the cold-adapted rPsSHMT made it a remarkable candidate that could be utilized in multiple industrial fields under low temperatures.

Functional and molecular characterization of a cold-active lipase from Psychrobacter celer PU3 with potential antibiofilm property.

The lipase gene from Psychrobacter celer PU3 was cloned into pET-28a(+) expression vector and overexpressed in E. coli BL21 (DE3) pLysS cells. The purified Psychrobacter celer lipase (PCL) was characterized as an alkaline active enzyme and has a molecular mass of around 30 kDa. The PCL was active even at a low temperature and the optimum range was observed between 10 and 40 °C temperatures. MALDI-TOF and phylogenetic analysis ensured that Psychrobacter celer PU3 lipase (PCL) was closely related to P. aureginosa lipase (PAL). MD simulation results suggest that temperature change did not affect the overall structure of PCL, but it might altered the temperature-dependent PCL functional changes. R1 (129-135 AA) and R2 (187-191 AA) regions could be important for temperature-dependent PCL function and they fluctuated much at 35 °C temperature. PMSF completely inhibited PCL lipase activity and it demonstrates the presence of serine residues in the active site of PCL. PCL is moderately halophilic and most of the tested organic solvents found to be inhibiting the lipase activity except the solvents ethanol and methanol. PCL activity was increased with surfactants (SDS and CTAB) and bleaching agents (hydrogen peroxide). The effect of different metal ions on PCL resulted that only mercuric chloride was found as the enhancer of the lipase activity. Antibiofilm property of PCL was evaluated against pathogenic Vibrio parahaemolyticus isolated from the diseased shrimp and MIC value was 500 U. PCL significantly altered the morphology and biofilm density of V. parahaemolyticus and the same was observed through scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) imaging. RT-PCR analysis revealed that the mRNA expression level of biofilm, colony morphology and major toxin-related (aphA, luxS, opaR, tolC, toxR) genes of V. parahaemolyticus were significantly downregulated with PCL treatment.

Glutaredoxin Interacts with GR and AhpC to Enhance Low-Temperature Tolerance of Antarctic Psychrophile Psychrobacter sp. ANT206.

Glutaredoxin (Grx) is an important oxidoreductase to maintain the redox homoeostasis of cells. In our previous study, cold-adapted Grx from Psychrobacter sp. ANT206 (PsGrx) has been characterized. Here, we constructed an in-frame deletion mutant of psgrx (Δpsgrx). Mutant Δpsgrx was more sensitive to low temperature, demonstrating that psgrx was conducive to the growth of ANT206. Mutant Δpsgrx also had more malondialdehyde (MDA) and protein carbonylation content, suggesting that PsGrx could play a part in the regulation of tolerance against low temperature. A yeast two-hybrid system was adopted to screen interacting proteins of 26 components. Furthermore, two target proteins, glutathione reductase (GR) and alkyl hydroperoxide reductase subunit C (AhpC), were regulated by PsGrx under low temperature, and the interactions were confirmed via bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP). Moreover, PsGrx could enhance GR activity. trxR expression in Δpsgrx, Δahpc, and ANT206 were illustrated 3.7, 2.4, and 10-fold more than mutant Δpsgrx Δahpc, indicating that PsGrx might increase the expression of trxR by interacting with AhpC. In conclusion, PsGrx may participate in glutathione metabolism and ROS-scavenging by regulating GR and AhpC to protect the growth of ANT206. These findings preliminarily suggest the role of PsGrx in the regulation of oxidative stress, which could improve the low-temperature tolerance of ANT206.

Genome analysis of a new biosurfactants source: The Antarctic bacterium Psychrobacter sp. TAE2020.

Biosurfactants are considered a possible green alternative to chemical surfactants for countless commercial products including detergents and cleaners, personal care products, cosmetics, pharmaceuticals and therapeutics, food additives, emulsifiers, and dispersants for bioremediation. Organisms from extreme environments are well-adapted to the harsh conditions and represent an exciting avenue of discovery of naturally occurring biosurfactants. In this study, we report the genome analysis of Psychrobacter sp. TAE2020, an aerobic Æ´-proteobacterium isolated from an Antarctic coastal seawater sample collected in the vicinity of the French Antarctic station Dumont d'Urville, Terre Adelie (66°40' S; 140° 01' E) which has been shown to produce biosurfactants. Biochemical assays indicate that Psychrobacter sp. TAE2020 can produce one or more excellent emulsifiers and a biosurfactant which is able to reduce the surface tension of a Gut medium. Next generation sequencing and genome mining allowed the identification of a plethora of biosynthetic gene clusters possibly involved in the production of emulsifying agents, just waiting to be isolated and characterized. This study paves the way for a more thorough investigation into the potential biotechnological applications of this new Antarctic strain.

Increased Synthesis of a Magnesium Transporter MgtA During Recombinant Autotransporter Expression in Escherichia coli.

Overproduction of the membrane proteins in Escherichia coli cells is a common approach to obtain sufficient material for their functional and structural studies. However, the efficiency of this process can be limited by toxic effects which decrease the viability of the host and lead to low yield of the product. During the expression of the esterase autotransporter AT877 from Psychrobacter cryohalolentis K5T, we observed significant growth inhibition of the C41(DE3) cells in comparison with the same cells producing other recombinant proteins. Induction of AT877 synthesis also resulted in the elevated expression of a magnesium transporter MgtA and decreased ATP content of the cells. To characterize the response to overexpression of the autotransporter in bacterial cells, we performed a comparative analysis of their proteomic profile by mass spectrometry. According to the obtained data, E. coli cells which synthesize AT877 experience complex stress condition presumably associated with secretion apparatus overloading and improper localization of the recombinant protein. Several response pathways were shown to be activated by AT877 overproduction including Cpx, PhoP/PhoQ, Psp, and σE The obtained results open new opportunities for optimization of the recombinant membrane protein expression in E. coli for structural studies and biotechnological applications.

NADP+ -dependent isocitrate dehydrogenase isozymes from a psychrotrophic bacterium, Psychrobacter sp. strain 13A.

The genes encoding dimeric and monomeric isocitrate dehydrogenase (IDH) isozymes from a psychrotrophic bacterium, strain 13A (13AIDH-D and 13AIDH-M, respectively), were cloned and sequenced. The deduced amino acid sequences of these two IDHs showed high degrees of identity with those of bacteria of genus Psychrobacter. Analysis of the 16S ribosomal RNA gene of the strain 13A revealed that this bacterium is classified to genus Psychrobacter. The optimum temperatures for activities of 13AIDH-D and 13AIDH-M were 55°C and 45°C, respectively, indicating that they are mesophilic. On the contrary, 13AIDH-D maintained 90% of its maximum activity after incubation for 10 min at 50°C, while the 13AIDH-M activity was completely lost under the same condition. In addition, 13AIDH-D showed much higher specific activity than 13AIDH-M. From northern and western blot analyses, the 13AIDH-D gene was found to be not transcribed under the growth conditions tested in this study. However, the catalytic ability of the mesophilic 13AIDH-M was concluded to be enough to sustain the growth of strain 13A at low temperatures. Therefore, a novel pattern of the contribution of IDH isozymes in cold-living bacteria to their growth at low temperatures was confirmed in strain 13A.

A novel ATP dependent dimethylsulfoniopropionate lyase in bacteria that releases dimethyl sulfide and acryloyl-CoA.

Dimethylsulfoniopropionate (DMSP) is an abundant and ubiquitous organosulfur molecule in marine environments with important roles in global sulfur and nutrient cycling. Diverse DMSP lyases in some algae, bacteria, and fungi cleave DMSP to yield gaseous dimethyl sulfide (DMS), an infochemical with important roles in atmospheric chemistry. Here, we identified a novel ATP-dependent DMSP lyase, DddX. DddX belongs to the acyl-CoA synthetase superfamily and is distinct from the eight other known DMSP lyases. DddX catalyses the conversion of DMSP to DMS via a two-step reaction: the ligation of DMSP with CoA to form the intermediate DMSP-CoA, which is then cleaved to DMS and acryloyl-CoA. The novel catalytic mechanism was elucidated by structural and biochemical analyses. DddX is found in several Alphaproteobacteria, Gammaproteobacteria, and Firmicutes, suggesting that this new DMSP lyase may play an overlooked role in DMSP/DMS cycles.

The global sulfur cycle is a collection of geological and biological processes that circulate sulfur-containing compounds through the oceans, rocks and atmosphere. Sulfur itself is essential for life and important for plant growth, hence its widespread use in fertilizers. Marine organisms such as bacteria, algae and phytoplankton produce one particular sulfur compound, called dimethylsulfoniopropionate, or DMSP, in massive amounts. DMSP made in the oceans gets readily converted into a gas called dimethyl sulfide (DMS), which is the largest natural source of sulfur entering the atmosphere. In the air, DMS is converted to sulfate and other by-products that can act as cloud condensation nuclei, which, as the name suggests, are involved in cloud formation. In this way, DMS can influence weather and climate, so it is often referred to as 'climate-active' gas. At least eight enzymes are known to cleave DMSP into DMS gas with a few by-products. These enzymes are found in algae, bacteria and fungi, and are referred to as lyases, for the way they breakdown their target compounds (DMSP, in this case). Recently, researchers have identified some bacteria that produce DMS from DMSP without using known DMSP lyases. This suggests there are other, unidentified enzymes that act on DMSP in nature, and likely contribute to global sulfur cycling. Li, Wang et al. set out to uncover new enzymes responsible for converting the DMSP that marine bacteria produce into gaseous DMS. One new enzyme called DddX was identified and found to belong to a superfamily of enzymes quite separate to other known DMSP lyases. Li, Wang et al. also showed how DddX drives the conversion of DMSP to DMS in a two-step reaction, and that the enzyme is found across several classes of bacteria. Further experiments to characterise the protein structure of DddX also revealed the molecular mechanism for its catalytic action. This study offers important insights into how marine bacteria generate the climatically important gas DMS from DMSP, leading to a better understanding of the global sulfur cycle. It gives microbial ecologists a more comprehensive perspective of these environmental processes, and provides biochemists with data on a family of enzymes not previously known to act on sulfur-containing compounds.

Characterization of two enzymes from Psychrobacter cryohalolentis that are required for the biosynthesis of an unusual diacetamido-d-sugar.

Psychrobacter cryohalolentis strain K5T is a Gram-negative organism first isolated in 2006. It has a complex O-antigen that contains, in addition to l-rhamnose and d-galactose, two diacetamido- and a triacetamido-sugar. The biochemical pathways for the production of these unusual sugars are presently unknown. Utilizing the published genome sequence of the organism, we hypothesized that the genes 0612, 0638, and 0637 encode for a 4,6-dehydratase, an aminotransferase, and an N-acetyltransferase, respectively, which would be required for the biosynthesis of one of the diacetamido-sugars, 2,4-diacetamido-2,4,6-trideoxy-d-glucose, starting from UDP-N-acetylglucosamine. Here we present functional and structural data on the proteins encoded by the 0638 and 0637 genes. The kinetic properties of these enzymes were investigated by a discontinuous HPLC assay. An X-ray crystallographic structure of 0638, determined in its external aldimine form to 1.3 Å resolution, demonstrated the manner in which the UDP ligand is positioned into the active site. It is strikingly different from that previously observed for PglE from Campylobacter jejuni, which functions on the same substrate. Four X-ray crystallographic structures were also determined for 0637 in various complexed states at resolutions between 1.3 and 1.55 Å. Remarkably, a tetrahedral intermediate mimicking the presumed transition state was trapped in one of the complexes. The data presented herein confirm the hypothesized functions of these enzymes and provide new insight into an unusual sugar biosynthetic pathway in Gram-negative bacteria. We also describe an efficient method for acetyl-CoA synthesis that allowed us to overcome its prohibitive cost for this analysis.

The elucidation of the biodegradation of nitrobenzene and p-nitrophenol of nitroreductase from Antarctic psychrophile Psychrobacter sp. ANT206 under low temperature.

Psychrobacter is one important typical strain in the Antarctic environment. In our previous study, Psychrobacter sp. ANT206 from Antarctica with novel cold-adapted nitroreductase (PsNTR) could biodegrade nitrobenzene and p-nitrophenol in low temperature environment. In this study, the in-frame deletion mutant of psntr (Δpsntr-ANT206) that displayed well genetic stability and kanamycin resistance stability was constructed using allelic replacement method. Additionally, Δpsntr-ANT206 was more sensitive to nitrobenzene and p-nitrophenol in the comparison of heat and hyperosmolarity, suggesting that psntr gene participated in the regulation of the tolerance against nitro-aromatic compounds (NACs). Further analysis was conducted by integrated gas chromatography-mass spectrometry (GC-MS), and several metabolites were identified. Among them, ethylbenzene, L-Alanine, citric acid, aniline, 4-aminophenol and other metabolites were different between the wild-type strain and Δpsntr-ANT206 under nitrobenzene and p-nitrophenol stress at different time periods under low temperature, respectively. These data could increase the knowledge of the construction of deletion mutant strains and biodegradation mechanism of NACs of typical strains Psychrobacter from Antarctica, which would also provide the basis of the molecular technique on the regulation of bioremediation of the contaminants under low temperature in the future.

AhaP, A Quorum Quenching Acylase from Psychrobacter sp. M9-54-1 That Attenuates Pseudomonas aeruginosa and Vibrio coralliilyticus Virulence.

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 5989T and P. faecalis ISO-46T. 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.

HRM and 16S rRNA gene sequencing reveal the cultivable microbiota of the European sea bass during ice storage.

The total cultivable microbiota of the ice-stored European sea bass (Dicentrarchus labrax), the most important commercial fish species of the Mediterranean aquaculture, was determined using 16S rRNA gene sequence analysis. High Resolution Melting (HRM) curve profiles and sequencing analysis (V3-V4 region of the 16S rRNA gene) were used respectively for the differentiation and identification of the collected isolates from six time intervals (day 0, 4, 8, 12, 14 and 16) while fish were stored in ice. HRM analysis differentiated the unknown microbiota in ten groups (208 isolates) and in two single isolates based on their HRM curve profiles. The isolates with HRM profiles which were >91% similar within each group were found to belong to the same species using sequencing analysis. Thus, the ten groups consist of representatives of Psychrobacter glacincola, Ps. alimentarius, Ps. cryohalolentis, Ps. maritimus, Ps. fozii, Pseudomonas sp., Paeniglutamicibacter sp., Carnobacterium sp., Leucobacter aridicolis and Bacillus thuringiensis. Based on this approach, Ps. cryohalolentis was found to be the most dominant phylotype at the beginning of fish shelf-life compared to other species. The abundance of this bacterium decreased throughout storage, while Ps. glacincola increased and dominated at the time of the sensory minimum acceptability (day 14) and rejection (day 16). To conclude, HRM could be used for the rapid determination of sea bass microbiota, using the representatives of each group as reference bacterial strains, in order for scientists to solve rapidly stakeholders problems related with microbial quality or safety of fish.