ppGpp is a dual-role regulator involved in balancing iron absorption and prodiginine biosynthesis in Pseudoalteromonas.

Iron is an essential element for microbial survival and secondary metabolism. However, excess iron availability and overloaded secondary metabolites can hinder microbial growth and survival. Microorganisms must tightly control iron homeostasis and secondary metabolism. Our previous studies have found that the stringent starvation protein A (SspA) positively regulates prodiginine biosynthesis by activating iron uptake in Pseudoalteromonas sp. strain R3. It is believed that the interaction between SspA and the small nucleotide ppGpp is important for iron to exert regulation functions. However, the roles of ppGpp in iron absorption and prodiginine biosynthesis, and the underlying relationship between ppGpp and SspA in strain R3 remain unclear. In this study, we found that ppGpp accumulation in strain R3 could be induced by limiting iron. In addition, ppGpp not only positively regulated iron uptake and prodiginine biosynthesis via increasing the SspA level but also directly repressed iron uptake and prodiginine biosynthesis independent of SspA, highlighting the finding that ppGpp can stabilize both iron levels and prodiginine production. Notably, the abolishment of ppGpp significantly increased prodiginine production, thus providing a theoretical basis for manipulating prodiginine production in the future. This dynamic ppGpp-mediated interaction between iron uptake and prodiginine biosynthesis has significant implications for understanding the roles of nutrient uptake and secondary metabolism for the survival of bacteria in unfavorable environments.

New investigation of encoding secondary metabolites gene by genome mining of a marine bacterium, Pseudoalteromonas viridis BBR56.

Pseudoalteromonas viridis strain BBR56 was isolated from seawater at Dutungan Island, South Sulawesi, Indonesia. Bacterial DNA was isolated using Promega Genomic DNA TM050. DNA purity and quantity were assessed using NanoDrop spectrophotometers and Qubit fluorometers. The DNA library and sequencing were prepared using Oxford Nanopore Technology GridION MinKNOW 20.06.9 with long read, direct, and comprehensive analysis. High accuracy base calling was assessed with Guppy version 4.0.11. Filtlong and NanoPlot were used for filtering and visualizing the FASTQ data. Flye (2.8.1) was used for de novo assembly analysis. Variant calls and consensus sequences were created using Medaka. The annotation of the genome was elaborated by DFAST. The assembled genome and annotation were tested using Busco and CheckM. Herein, we found that the highest similarity of the BBR56 isolate was 98.37% with the 16 S rRNA gene sequence of P. viridis G-1387. The genome size was 5.5 Mb and included chromosome 1 (4.2 Mbp) and chromosome 2 (1.3 Mbp), which encoded 61 pseudogenes, 4 noncoding RNAs, 113 tRNAs, 31 rRNAs, 4,505 coding DNA sequences, 4 clustered regularly interspaced short palindromic repeats, 4,444 coding genes, and a GC content of 49.5%. The sequence of the whole genome of P. viridis BBR56 was uploaded to GenBank under the accession numbers CP072425-CP072426, biosample number SAMN18435505, and bioproject number PRJNA716373. The sequence read archive (SRR14179986) was successfully obtained from NCBI for BBR56 raw sequencing reads. Digital DNA-DNA hybridization results showed that the genome of BBR56 had the potential to be a new species because no other bacterial genomes were similar to the sample. Biosynthetic gene clusters (BGCs) were assessed using BAGEL4 and the antiSMASH bacterial version. The genome harbored diverse BGCs, including genes that encoded polyketide synthase, nonribosomal peptide synthase, RiPP-like, NRP-metallophore, hydrogen cyanide, betalactone, thioamide-NRP, Lant class I, sactipeptide, and prodigiosin. Thus, BBR56 has considerable potential for further exploration regarding the use of its secondary metabolite products in the human and fisheries sectors.

The impact of interspecific competition on the genomic evolution of Phaeobacter inhibens and Pseudoalteromonas tunicata during biofilm growth.

Interspecific interactions in biofilms have been shown to cause the emergence of community-level properties. To understand the impact of interspecific competition on evolution, we deep-sequenced the dispersal population of mono- and co-culture biofilms of two antagonistic marine bacteria (Phaeobacter inhibens 2.10 and Pseudoalteromononas tunicata D2). Enhanced phenotypic and genomic diversification was observed in the P. tunicata D2 populations under both mono- and co-culture biofilms in comparison to P. inhibens 2.10. The genetic variation was exclusively due to single nucleotide variants and small deletions, and showed high variability between replicates, indicating their random emergence. Interspecific competition exerted an apparent strong positive selection on a subset of P. inhibens 2.10 genes (e.g., luxR, cobC, argH, and sinR) that could facilitate competition, while the P. tunicata D2 population was genetically constrained under competition conditions. In the absence of interspecific competition, the P. tunicata D2 replicate populations displayed high levels of mutations affecting the same genes involved in cell motility and biofilm formation. Our results show that interspecific biofilm competition has a complex impact on genomic diversification, which likely depends on the nature of the competing strains and their ability to generate genetic variants due to their genomic constraints.

Genome-based taxonomic analysis of the genus Pseudoalteromonas reveals heterotypic synonyms.

The Pseudoalteromonas genus comprises members that have been demonstrated to play significant ecological roles and produce enzymes, natural products, and activities that are beneficial to the environment and economy. A comprehensive evaluation of the genus revealed that the genomes of several Pseudoalteromonas species are highly similar to each other, exceeding species cutoff values. This evaluation involved determining and comparing the average nucleotide identity, in silico DNA-DNA hybridization, average amino acid identity, and the difference in G + C% between Pseudoalteromonas type strains with publicly available genomes. The genome of the Pseudoalteromonas elyakovii type strain was further assessed through additional sequencing and genomic comparisons to historical sequences. These findings suggest that six Pseudoalteromonas species, namely P. mariniglutinosa, P. donghaensis, P. maricaloris, P. elyakovii, P. profundi, and P. issachenkonii, should be reclassified as later heterotypic synonyms of the following validly published species: P. haloplanktis, P. lipolytica, P. flavipulchra, P. distincta, P. gelatinilytica, and P. tetraodonis. Furthermore, two names without valid standing, 'P. telluritireducens' and 'P. spiralis', should be associated with the validly published Pseudoalteromonas species P. agarivorans and P. tetraodonis, respectively.

Biosynthetic mechanism of the yellow pigments in the marine bacterium Pseudoalteromonas sp. strain T1lg65.

The Pseudoalteromonas genus marine bacteria have attracted increasing interest because of their abilities to produce bioactive metabolites. The pigmented Pseudoalteromonas group encodes more secondary metabolite biosynthetic gene clusters (BGCs) than the non-pigmented group. Here, we report a yellow pigmented bacterium Pseudoalteromonas sp. strain T1lg65, which was isolated from a mangrove forest sediment. We showed that the yellow pigments of T1lg65 belong to the group of lipopeptide alterochromides. Further genetic analyses of the alterochromide BGC revealed that the yellow pigments are biosynthesized by aryl-polyene synthases and nonribosomal peptide synthases. Within the gene cluster, altA encodes a tyrosine ammonia acid lyase, which catalyzes synthesis of the precursor 4-hydroxycinnamic acid (4-HCA) from tyrosine in the alterochromide biosynthetic pathway. In addition, altN, encoding a putative flavin-dependent halogenase, was proven to be responsible for the bromination of alterochromides based on gene deletion, molecular docking, and site mutagenesis analyses. In summary, the biosynthetic pathway, precursor synthesis, and bromination mechanism of the lipopeptide alterochromides were studied in-depth. Our results expand the knowledge on biosynthesis of Pseudoalteromonas pigments and could promote the development of active pigments in the future.IMPORTANCEThe marine bacteria Pseudoalteromonas spp. are important biological resources because they are producers of bioactive natural products, including antibiotics, pigments, enzymes, and antimicrobial peptides. One group of the microbial pigments, alterochromides, holds a great value for their novel lipopeptide structures and antimicrobial activities. Previous studies were limited to the structural characterization of alterochromides and genome mining for the alterochromide biosynthesis. This work focused on the biosynthetic mechanism for alterochromide production, especially revealing functions of two key genes within the gene cluster for the alterochromide biosynthesis. On the one hand, our study provides a target for metabolic engineering of the alterochromide biosynthesis; on the other hand, the 4-HCA synthase AltA and brominase AltN show potential in the biocatalyst industry.

ι-Carrageenan catabolism is initiated by key sulfatases in the marine bacterium Pseudoalteromonas haloplanktis LL1.

Marine bacteria contribute substantially to cycle macroalgae polysaccharides in marine environments. Carrageenans are the primary cell wall polysaccharides of red macroalgae. The carrageenan catabolism mechanism and pathways are still largely unclear. Pseudoalteromonas is a representative bacterial genus that can utilize carrageenan. We previously isolated the strain Pseudoalteromonas haloplanktis LL1 that could grow on ι-carrageenan but produce no ι-carrageenase. Here, through a combination of bioinformatic, biochemical, and genetic analyses, we determined that P. haloplanktis LL1 processed a desulfurization-depolymerization sequential pathway for ι-carrageenan utilization, which was initiated by key sulfatases PhSulf1 and PhSulf2. PhSulf2 acted as an endo/exo-G4S (4-O-sulfation-ß-D-galactopyranose) sulfatase, while PhSulf1 was identified as a novel endo-DA2S sulfatase that could function extracellularly. Because of the unique activity of PhSulf1 toward ι-carrageenan rather than oligosaccharides, P. haloplanktis LL1 was considered to have a distinct ι-carrageenan catabolic pathway compared to other known ι-carrageenan-degrading bacteria, which mainly employ multifunctional G4S sulfatases and exo-DA2S (2-O-sulfation-3,6-anhydro-α-D-galactopyranose) sulfatase for sulfate removal. Furthermore, we detected widespread occurrence of PhSulf1-encoding gene homologs in the global ocean, indicating the prevalence of such endo-acting DA2S sulfatases as well as the related ι-carrageenan catabolism pathway. This research provides valuable insights into the enzymatic processes involved in carrageenan catabolism within marine ecological systems.IMPORTANCECarrageenan is a type of linear sulfated polysaccharide that plays a significant role in forming cell walls of marine algae and is found extensively distributed throughout the world's oceans. To the best of our current knowledge, the ι-carrageenan catabolism in marine bacteria either follows the depolymerization-desulfurization sequential process initiated by ι-carrageenase or starts from the desulfurization step catalyzed by exo-acting sulfatases. In this study, we found that the marine bacterium Pseudoalteromonas haloplanktis LL1 processes a distinct pathway for ι-carrageenan catabolism employing a specific endo-acting DA2S-sulfatase PhSulf1 and a multifunctional G4S sulfatase PhSulf2. The unique PhSulf1 homologs appear to be widely present on a global scale, indicating the indispensable contribution of the marine bacteria containing the distinct ι-carrageenan catabolism pathway. Therefore, this study would significantly enrich our understanding of the molecular mechanisms underlying carrageenan utilization, providing valuable insights into the intricate roles of marine bacteria in polysaccharide cycling in marine environments.

Co-existence of two antibiotic-producing marine bacteria: Pseudoalteromonas piscicida reduce gene expression and production of the antibacterial compound, tropodithietic acid, in Phaeobacter sp.

Many bacteria co-exist and produce antibiotics, yet we know little about how they cope and occupy the same niche. The purpose of the present study was to determine if and how two potent antibiotic-producing marine bacteria influence the secondary metabolome of each other. We established an agar- and broth-based system allowing co-existence of a Phaeobacter species and Pseudoalteromonas piscicida that, respectively, produce tropodithietic acid (TDA) and bromoalterochromides (BACs). Co-culturing of Phaeobacter sp. strain A36a-5a on Marine Agar with P. piscicida strain B39bio caused a reduction of TDA production in the Phaeobacter colony. We constructed a transcriptional gene reporter fusion in the tdaC gene in the TDA biosynthetic pathway in Phaeobacter and demonstrated that the reduction of TDA by P. piscicida was due to the suppression of the TDA biosynthesis. A stable liquid co-cultivation system was developed, and the expression of tdaC in Phaeobacter was reduced eightfold lower (per cell) in the co-culture compared to the monoculture. Mass spectrometry imaging of co-cultured colonies revealed a reduction of TDA and indicated that BACs diffused into the Phaeobacter colony. BACs were purified from Pseudoalteromonas; however, when added as pure compounds or a mixture they did not influence TDA production. In co-culture, the metabolome was dominated by Pseudoalteromonas features indicating that production of other Phaeobacter compounds besides TDA was reduced. In conclusion, co-existence of two antibiotic-producing bacteria may be allowed by one causing reduction in the antagonistic potential of the other. The reduction (here of TDA) was not caused by degradation but by a yet uncharacterized mechanism allowing Pseudoalteromonas to reduce expression of the TDA biosynthetic pathway.IMPORTANCEThe drug potential of antimicrobial secondary metabolites has been the main driver of research into these compounds. However, in recent years, their natural role in microbial systems and microbiomes has become important to determine the assembly and development of microbiomes. Herein, we demonstrate that two potent antibiotic-producing bacteria can co-exist, and one mechanism allowing the co-existence is the specific reduction of antibiotic production in one bacterium by the other. Understanding the molecular mechanisms in complex interactions provides insights for applied uses, such as when developing TDA-producing bacteria for use as biocontrol in aquaculture.

Development of capillary electrophoresis methods for the detection of microbial metabolites on potential future spaceflight missions.

The search for chemical indicators of life is a fundamental component of potential future spaceflight missions to ocean worlds. Capillary electrophoresis (CE) is a useful separation method for the determination of the small organic molecules, such as amino acids and nucleobases, that could be used to help determine whether or not life is present in a sample collected during such missions. CE is under development for spaceflight applications using multiple detection systems, such as laser induced fluorescence (LIF) and mass spectrometry (MS). Here we report CE-based methods for separation and detection of major polar metabolites in cells, such as amino acids, nucleobases/sides, and oxidized and reduced glutathione using detectors that are less expensive alternatives to LIF and MS. Direct UV detection, indirect UV detection, and capacitvely coupled contactless conductivity detection (C4D) were tested with CE, and a combination of direct UV and C4D allowed the detection of the widest variety of metabolites. The optimized method was used to profile metabolites found in samples of Escherichia coli and Pseudoalteromonas haloplanktis and showed distinct differences between the species.

Low-temperature features of the psychrophilic chaperonin from Pseudoalteromonas haloplanktis.

Chaperonins from psychrophilic bacteria have been shown to exist as single-ring complexes. This deviation from the standard double-ring structure has been thought to be a beneficial adaptation to the cold environment. Here we show that Cpn60 from the psychrophile Pseudoalteromonas haloplanktis (Ph) maintains its double-ring structure also in the cold. A strongly reduced ATPase activity keeps the chaperonin in an energy-saving dormant state, until binding of client protein activates it. Ph Cpn60 in complex with co-chaperonin Ph Cpn10 efficiently assists in protein folding up to 55 °C. Moreover, we show that recombinant expression of Ph Cpn60 can provide its host Escherichia coli with improved viability under low temperature growth conditions. These properties of the Ph chaperonin may make it a valuable tool in the folding and stabilization of psychrophilic proteins.

Psychrosphaera algicola sp. nov. and Paraglaciecola algarum sp. nov., and reclassification of Pseudoalteromonas elyakovii, Pseudoalteromonas flavipulchra, and Pseudoalteromonas profundi as later heterotypic synonyms of P. distincta, P. maricaloris, and P. gelatinilytica.

Two Gram-negative, obligately aerobic, rod-shaped bacteria, strains G1-22T and G1-23T, were isolated from the phycosphere of a marine brown alga. Both strains exhibited catalase- and oxidase-positive activities. Strain G1-22T displayed optimal growth at 25 °C, pH 8.0, and 2.0-3.0% (w/v) NaCl, while strain G1-23T exhibited optimal growth at 25 °C, pH 8.0, and 4.0% NaCl. Ubiquinone-8 was identified as the sole isoprenoid quinone in both strains. As major fatty acids (> 5%), strain G1-22T contained C16 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C12 : 1 3-OH, and C10 : 0 3-OH, while strain G1-23T contained C16 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), and C14 : 0. Phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol were major polar lipids in both strains. Strains G1-22T and G1-23T had DNA G+C contents of 40.2 and 38.9 mol%, respectively. Phylogenetic analyses based on 16S rRNA and genome sequences revealed that strains G1-22T and G1-23T formed distinct phylogenetic lineages within the genera Psychrosphaera and Paraglaciecola, respectively. Strain G1-22T showed closest relatedness to Psychrosphaera ytuae MTZ26T with 97.8% 16S rRNA gene sequence similarity, 70.2% average nucleotide identity (ANI), and a 21.5% digital DNA-DNA hybridization (dDDH) value, while strain G1-23T was most closely related to Paraglaciecola aquimarina KCTC 32108T with 95.6% 16S rRNA gene sequence similarity, 74.6% ANI, and a 20.1% dDDH value. Based on phenotypic and molecular characteristics, strains G1-22T and G1-23T are proposed to represent two novel species, namely Psychrosphaera algicola sp. nov. (type strain G1-22T=KACC 22486T=JCM 34971T) and Paraglaciecola algarum sp. nov. (type strain G1-23T=KACC 22490T=JCM 34972T), respectively. Additionally, based on the comparison of whole genome sequences, it is proposed that Pseudoalteromonas elyakovii, Pseudoalteromonas flavipulchra, and Pseudoalteromonas profundi are reclassified as later heterotypic synonyms of Pseudoalteromonas distincta, Pseudoalteromonas maricaloris, and Pseudoalteromonas gelatinilytica, respectively.

Crown ether dopant to reduce ion suppression and improve detection in the liquid microjunction surface sampling probe.

RATIONALE: Sodium and potassium are required in agar media for the growth of some microorganisms (e.g., marine bacteria). However, alkali cations are a significant source of contamination for mass spectrometry causing ion suppression and adduct formation. Conventionally, salts can be removed before mass spectrometric analysis with appropriate and often lengthy sample preparation. The direct mass spectrometric sampling of bacterial colonies grown on agar media seeks to minimize or eliminate sample preparation to improve workflow. However, this may exacerbate ion suppression and contamination since these metal cations will degrade spectral quality and limit the rapid profiling of microbial metabolites. Different approaches are needed to sequester sodium and potassium ions to minimize unwanted background interferences. Herein, we use crown ethers (CEs) in combination with a liquid microjunction surface sampling probe (LMJ-SSP) to directly sample the surface of the bacterial colonies from two marine bacteria species (Pseudoalteromonas rubra DSM6842 and Pseudoalteromonas tunicata DSM 14096). CEs (e.g., 18-crown-6 or 15-crown-5) are added to the carrier solvent of the LMJ-SSP, the chemical noise is reduced, and spectra are easier to interpret. METHODS: The liquid microjunction formed at the tip of LMJ-SSP was used to directly touch bacterial colonies on agar. The carrier solvent was either methanol (100%) or methanol: H2O (50:49.9%) with or without 0.01% CEs. Information-theoretic measures are employed to investigate qualitative changes between spectra before and after adding CEs. RESULTS: Our work demonstrates the capability of CEs to reduce background interferences within the direct profiling of bacterial colonies from agar plates. The data obtained from both P. rubra DSM6842 and P. tunicata DSM 14096 show that CEs can be used to mitigate the salty background and improve compound detection. CONCLUSION: Our approach can be implemented in natural product discovery using LMJ-SSP to allow fast and accurate detection of interesting/novel compounds.

Whole-genome sequencing of Pseudoalteromonas piscicida 2515 revealed its antibacterial potency against Vibrio anguillarum: a preliminary invitro study.

Pseudoalteromonas piscicida 2515, isolated from Litopenaeus vannamei culture water, is a potential marine probiotic with broad anti-Vibrio properties. However, genomic information on P. piscicida 2515 is scarce. In this study, the general genomic characteristics and probiotic properties of the P. piscicida 2515 strain were analysed. In addition, we determined the antibacterial mechanism of this bacterial strain by scanning electron microscopy (SEM). The results indicated that the whole-genome sequence of P. piscicida 2515 contained one chromosome and one plasmid, including a total length of 5,541,406 bp with a G + C content of 43.24%, and 4679 protein-coding genes were predicted. Various adhesion-related genes, amino acid and vitamin metabolism and biosynthesis genes, and stress-responsive genes were found with genome mining tools. The presence of genes encoding chitin, bromocyclic peptides, lantibiotics, and sactipeptides showed the strong antibacterial activity of the P. piscicida 2515 strain. Moreover, in coculture with Vibrio anguillarum, P. piscicida 2515 displayed vesicle/pilus-like structures located on its surface that possibly participated in its bactericidal activity, representing an antibacterial mechanism. Additionally, 16 haemolytic genes and 3 antibiotic resistance genes, including tetracycline, fluoroquinolone, and carbapenem were annotated, but virulence genes encoding enterotoxin FM (entFM), cereulide (ces), and cytotoxin K were not detected. Further tests should be conducted to confirm the safety characteristics of P. piscicida 2515, including long-term toxicology tests, ecotoxicological assessment, and antibiotic resistance transfer risk assessment. Our results here revealed a new understanding of the probiotic properties and antibacterial mechanism of P. piscicida 2515, in addition to theoretical information for its application in aquaculture.

Marine biofilm formation on flax fibre reinforced biocomposites.

Artificial reefs represent useful tools to revitalize coastal and ocean ecosystems. Their formulation determines the biofilm formation which is the prerequisite for the colonization process by marine micro- and macroorganisms. In comparison with concrete, biobased polymers offer improved characteristics, including architecture, formulation, rugosity and recycling. This article aims to explore a new scale of artificial reef made of biocomposites reinforced with a high flax fibre (Linum utilatissimum) content (30%). Cellular adhesion and resulting biofilm formation were assessed using two marine microorganisms: Pseudoalteromonas sp. 3J6 and Cylindrotheca closterium. The influence of flax fibre leachates and plastic monomers on the growth of those marine microorganisms were also evaluated. Results indicated that the introduction of flax fibres inside the polymer matrix modified its physicochemical properties thus modulating adhesion and biofilm formation depending on the microorganism. This study gives insights for further developments of novel functionalized artificial reefs made of biocomposites.

Pseudoalteromonas simplex sp. nov. Isolated from the Skin of Bandtail Puffer Fish (Sphoeroides spengleri).

A novel bacterial isolate A520T (A520T = CBAS 737T = CAIM 1944T) was obtained from the skin of bandtail puffer fish Sphoeroides spengleri (Tetraodontidae Family), collected in Arraial do Cabo (Rio de Janeiro, Brazil). A520T is Gram-stain-negative, flagellated and aerobic bacteria. Optimum growth occurs at 25-30 °C in the presence of 3% NaCl. The genome sequence of the novel isolate consisted of 4.5 Mb (4082 coding genes and G+C content of 41.1%). The closest phylogenetic neighbor was Pseudoalteromonas shioyasakiensis JCM 18891T (97.9% 16S rRNA sequence similarity, 94.8% Average Amino Acid Identity, 93% Average Nucleotide Identity and 51.8% similarity in Genome-to-Genome-Distance). Several in silico phenotypic features are useful to differentiate A520T from its closest phylogenetic neighbors, including trehalose, D-mannose, cellobiose, pyrrolidonyl-beta-naphthylamide, starch hydrolysis, D-xylose, lactose, tartrate utilization, sucrose, citrate, glycerol, mucate and acetate utilization, malonate, glucose oxidizer, gas from glucose, nitrite to gas, L-rhamnose, ornithine decarboxylase, lysine decarboxylase and yellow pigment. The genome of the novel species contains 3 gene clusters (~ 66.81 Kbp in total) coding for different types of bioactive compounds that could indicate ecological roles pertaining to the bandtail puffer fish host. Based on genome-based taxonomic approach, strain A520T (A520T = CBAS 737T = CAIM 1944T) is proposed as a new species, Pseudoalteromonas simplex sp. nov.

The Description of Pseudoalteromonas apostichopi sp. nov., Vibrio apostichopi sp. nov., and Marinobacter apostichopi sp. nov. from the Fertilized Eggs and Larvae of Apostichopus japonicus.

Three novel bacterial strains, FE4T, FE10T, and LA51T, which are phylogenetically affiliated to the genera Pseudoalteromonas, Vibrio, or Marinobacter, respectively, isolated from fertilized eggs and juveniles of sea cucumber Apostichopus japonicus were characterized by a genome-based taxonomical approach including multilocus sequence analysis (MLSA) combined with classical phenotypic and chemotaxonomic characterizations. A molecular network reconstructed on the basis of nucleotide sequences of four phylogenetic maker protein genes revealed that the strains FE4T, FE10T, and LA51T were closely related to Pseudoalteromonas shioyasakiensis, Vibrio lentus, and Marinobacter similis, respectively. Average nucleotide identity (ANI) comparisons against phylogenetically related species to FE4T, FE10T, and LA51T demonstrated that each newly described strain could not be identified as any previously described species within each genus showing < 95% ANI: 91.3% of FE4T against P. shioyasakiensis JCM 18891 T, 92.6% of FE10T against "V. bathopelagicus" Sal10, and 92.6% of LA51T against M. similis A3d10T, in maximum, respectively. Here, we show molecular phylogenetic, genomic, phenotypic, and chemotaxonomic features of the newly described species FE4T, FE10T, and LA51T. We also propose Pseudoalteromonas apostichopi sp. nov. with FE4T (JCM 36173 T = LMG 33143 T) as the type strain, Vibrio apostichopi sp. nov. with FE10T (JCM 36174 T = LMG 33144 T) as the type strain, and Marinobacter apostichopi sp. nov. with LA51T (JCM 36175 T = LMG 33145 T) as the type strain.

Functional Genomics of a Collection of Gammaproteobacteria Isolated from Antarctica.

Antarctica, one of the most extreme environments on Earth, hosts diverse microbial communities. These microbes have evolved and adapted to survive in these hostile conditions, but knowledge on the molecular mechanisms underlying this process remains limited. The Italian Collection of Antarctic Bacteria (Collezione Italiana Batteri Antartici (CIBAN)), managed by the University of Messina, represents a valuable repository of cold-adapted bacterial strains isolated from various Antarctic environments. In this study, we sequenced and analyzed the genomes of 58 marine Gammaproteobacteria strains from the CIBAN collection, which were isolated during Italian expeditions from 1990 to 2005. By employing genome-scale metrics, we taxonomically characterized these strains and assigned them to four distinct genera: Pseudomonas, Pseudoalteromonas, Shewanella, and Psychrobacter. Genome annotation revealed a previously untapped functional potential, including secondary metabolite biosynthetic gene clusters and antibiotic resistance genes. Phylogenomic analyses provided evolutionary insights, while assessment of cold-shock protein presence shed light on adaptation mechanisms. Our study emphasizes the significance of CIBAN as a resource for understanding Antarctic microbial life and its biotechnological potential. The genomic data unveil new horizons for insight into bacterial existence in Antarctica.

Conjugative plasmid-encoded toxin-antitoxin system PrpT/PrpA directly controls plasmid copy number.

Toxin-antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin-antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

Characteristics of inhibitory active substances produced by Pseudoalteromonas SW-1.

To confirm the antagonistic activity characterization of the strain Pseudoalteromonas SW-1 (P. SW-1), its cell-free supernatant (CFS) was studied against a clam pathogenic strain of Vibrio Alginolyticu MP-1 (V.MP-1). The CFS of P. SW-1 exhibited evident antagonistic activities against the pathogens, and the absorbance value (600 nm) of V. MP-1 remained at a lower level at 24 h when compared with the control. The results showed that the inhibitory activities of strain P. SW-1 CFS showed differences after treatment with heat, acid and alkali, and proteinase K. The CFS of P. SW-1 inhibitory activities decreased after treatment with heat, but the inhibitory activities of strain P. SW-1 CFS were still effective after treatment with proteinase K for 24 h. The acid and alkali treatments could increase the inhibitory activities of strain P. SW-1 CFS. Therefore, the ammonium sulfate precipitation test also indicated that P. SW-1 could produce some active protein compounds to antagonize pathogenic V. MP-1.

Improvement of thermostability by increasing rigidity in the finger regions and flexibility in the catalytic pocket area of Pseudoalteromonas porphyrae κ-carrageenase.

Poor thermostability reduces the industrial application value of κ-carrageenase. In this study, the PoPMuSiC algorithm combined with site-directed mutagenesis was applied to improve the thermostability of the alkaline κ-carrageenase from Pseudoalteromonas porphyrae. The mutant E154A with improved thermal stability was successfully obtained using this strategy after screening seven rationally designed mutants. Compared with the wild-type κ-carrageenase (WT), E154A improved the activity by 29.4% and the residual activity by 51.6% after treatment at 50 °C for 30 min. The melting temperature (Tm) values determined by circular dichroism were 66.4 °C and 64.6 °C for E154A and WT, respectively. Molecular dynamics simulation analysis of κ-carrageenase showed that the flexibility decreased within the finger regions (including F1, F2, F3, F5 and F6) and the flexibility improved in the catalytic pocket area of the mutant E154A. The catalytic tunnel dynamic simulation analysis revealed that E154A led to enlarged catalytic tunnel volume and increased rigidity of the enzyme-substrate complex. The increasing rigidity within the finger regions and more flexible catalytic pocket of P. porphyrae κ-carrageenase might be a significant factor for improvement of the thermostability of the mutant κ-carrageenase E154A. The proposed rational design strategy could be applied to improve the enzyme kinetic stability of other industrial enzymes. Moreover, the hydrolysates of κ-carrageenan digested by the mutant E154A demonstrated increased scavenging activities against hydroxyl (OH) radicals and 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radicals compared with the undigested κ-carrageenan.

Comparative study on tertiary contacts and folding of RNase P RNAs from a psychrophilic, a mesophilic/radiation-resistant, and a thermophilic bacterium.

In most bacterial type A RNase P RNAs (P RNAs), two major loop-helix tertiary contacts (L8-P4 and L18-P8) help to orient the two independently folding S- and C-domains for concerted recognition of precursor tRNA substrates. Here, we analyze the effects of mutations in these tertiary contacts in P RNAs from three different species: (i) the psychrophilic bacterium Pseudoalteromonas translucida (Ptr), (ii) the mesophilic radiation-resistant bacterium Deinococcus radiodurans (Dra), and (iii) the thermophilic bacterium Thermus thermophilus (Tth). We show by UV melting experiments that simultaneous disruption of these two interdomain contacts has a stabilizing effect on all three P RNAs. This can be inferred from reduced RNA unfolding at lower temperatures and a more concerted unfolding at higher temperatures. Thus, when the two domains tightly interact via the tertiary contacts, one domain facilitates structural transitions in the other. P RNA mutants with disrupted interdomain contacts showed severe kinetic defects that were most pronounced upon simultaneous disruption of the L8-P4 and L18-P8 contacts. At 37°C, the mildest effects were observed for the thermostable Tth RNA. A third interdomain contact, L9-P1, makes only a minor contribution to P RNA tertiary folding. Furthermore, D. radiodurans RNase P RNA forms an additional pseudoknot structure between the P9 and P12 of its S-domain. This interaction was found to be particularly crucial for RNase P holoenzyme activity at near-physiological Mg2+ concentrations (2 mM). We further analyzed an exceptionally stable folding trap of the G,C-rich Tth P RNA.