Search

Pseudoalteromonas is a genus of marine bacteria and a promising source of natural products with antibacterial, antifungal, and antifouling bioactivities. To accelerate the exploration of new compounds from this genus, we applied the gene-first approach to study 632 public Pseudoalteromonas genomes. We identified 3968 biosynthetic gene clusters (BGCs) involved in the biosynthesis of secondary metabolites and classified them into 995 gene cluster families (GCFs). Surprisingly, only 9 GCFs (0.9 %) included an experimentally identified reference biosynthetic gene cluster from the Minimum Information about a Biosynthetic Gene cluster database (MIBiG), suggesting a striking novelty of secondary metabolites in Pseudoalteromonas. Bioinformatic analysis of the biosynthetic diversity encoded in the identified BGCs uncovered six dominant species of this genus, P. citrea, P. flavipulchra, P. luteoviolacea, P. maricaloris, P. piscicida, and P. rubra, that encoded more than 17 BGCs on average. Moreover, each species exhibited a species-specific distribution of BGC. However, a deep analysis revealed two BGCs conserved across five of the six dominant species. These BGCS encoded an unknown lanthipeptide and the siderophore myxochelin B implying an essential role of antibiotics for Pseudoalteromonas. We chemically profiled 11 strains from the 6 dominant species and identified four new antibiotics, korormicins L-O (1-4), from P. citrea WJX-3. Our results highlight the unexplored biosynthetic potential for bioactive compounds in Pseudoalteromonas and provide an important guideline for targeting exploration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Chemical cues mediate interactions between marine phytoplankton and bacteria, underpinning ecosystem-scale processes including nutrient cycling and carbon fixation. Phage infection alters host metabolism, stimulating the release of chemical cues from intact plankton, but how these dynamics impact ecology and biogeochemistry is poorly understood. Here we determine the impact of phage infection on dissolved metabolite pools from marine cyanobacteria and the subsequent chemotactic response of heterotrophic bacteria using time-resolved metabolomics and microfluidics. Metabolites released from intact, phage-infected Synechococcus elicited strong chemoattraction from Vibrio alginolyticus and Pseudoalteromonas haloplanktis, especially during early infection stages. Sustained bacterial chemotaxis occurred towards live-infected Synechococcus, contrasted by no discernible chemotaxis towards uninfected cyanobacteria. High-throughput microfluidics identified 5'-deoxyadenosine and 5'-methylthioadenosine as key attractants. Our findings establish that, before lysis, phage-infected picophytoplankton release compounds that attract motile heterotrophic bacteria, suggesting a mechanism for resource transfer that might impact carbon and nutrient fluxes across trophic levels., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Domoic acid (DA) is a compound generated as a secondary metabolite during harmful algal blooms, has historically received attention as the potent neurotoxicity in marine environment. However, the aerobic degradation mechanism of DA and the DA-degrader remain largely unknown. Here, we revealed the mechanism of aerobic degradation of DA by a ubiquitous marine Pseudoalteromonas sp., and more importantly, we confirmed that the degradation of DA is mediated by biogenic reactive oxygen species (ROS), rather than direct enzyme-mediated as traditionally conceived. Results indicated that DA degradation was caused by biogenic O 2 - and OH, where DA underwent reactions of decarboxylation, hydroxylation, and oxidation to yield the detoxification terminal product. Besides, whole genome sequencing and RT-qPCR analysis revealed that the genes conferring to encoding leucine dehydrogenase (ldh) and Na+-translocated NADH-quinone oxidoreductase (nqrA, nqrF) are responsible for biogenic ROS production. Finally, we found through comparative proteomic analysis that biogenic ROS mediated the DA degradation may be prevalent in the environment. Overall, this work not only reveals aerobic biotransformation mechanism of DA, but also identifies a novel mechanism of DA degradation, which provides new perspective into the environmental fate of DA and the artificial bioremediation of DA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Brown algae are one of the most abundant biomasses on Earth. To recycle them as blue carbon sources, an effective decomposition system is necessary. This study focused on microorganisms present in seawater that decompose brown algae which contain laminarin and alginate. Where Undaria and Sargassum spp. were present, genera Psychromonas, Psychrobacter, and Pseudoalteromonas were predominant in seawater, while genera Arcobacter and Fusobacterium increased in abundance during the process of decomposition. The inoculation of Undaria samples into laminarin-minimal media led to a predominance of Pseudoalteromonas species. A Pseudoalteromonas isolate, identified as Pseudoalteromonas distincta, possesses genes encoding a putative laminarinase, polysaccharide lyase family 6 (PL6) alginate lyases, and a PL7 alginate lyase. The culture media of P. distincta contained no monosaccharides, suggesting the rapid conversion of polysaccharides to metabolites. These findings indicated that Pseudoalteromonas species play a major role in the decomposition of brown algae and affect the microbiota associated with them., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Plastics dumped in the environment are fragmented into microplastics by various factors (UV, weathering, mechanical abrasion, animal chewing, etc.). However, little is known about plastic fragmentation and degradation mediated by deep-sea microflora. To obtain deep-sea bacteria that can degrade plastics, we enriched in situ for 1 year in the Western Pacific using PS as a carbon source. Subsequently, two deep-sea prevalent bacteria of the genus Pseudoalteromonas (Pseudoalteromonas lipolytica and Pseudoalteromonas tetraodonis) were isolated after 6 months enrichment in the laboratory under low temperature (15 °C). Both showed the ability to degrade polystyrene (PS) and polypropylene (PP), and biodegradation accelerated the generation of micro- and nanoplastics. Plastic biodegradation was evidenced by the formation of carboxyl and carboxylic acid groups, heat resistance decrease and plastic weight loss. After 80 days incubation at 15 °C, the microplastic concentration of PS and PP could be up to 1.94 × 10 7 /L and 5.83 × 10 7 /L, respectively, and the proportion of nanoplastics (< 1 μm) could be up to 65.8 % and 73.6 %. The film weight loss were 5.4 % and 4.5 % of the PS films, and 2.3 % and 1.8 % of the PP films by P. lipolytica and P. tetraodonis, respectively; thus after discounting the weight loss of microplastics, the only 3.9 % and 2.8 % of the PS films, and 1.3 % and 0.7 % of the PP films, respectively, were truly degraded by the two bacteria respectively after 80 days of incubation. This study highlights the role of Pseudoalteromonas in fragmentation and degradation of plastics in cold dark pelagic deep sea., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 (C 4 D) were tested with CE, and a combination of direct UV and C 4 D 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., (© 2024 Wiley‐VCH GmbH.)

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., Competing Interests: The authors declare no conflict of interest.

Plasmids orchestrate bacterial adaptation across diverse environments and facilitate lateral gene transfer within bacterial communities. Their presence can perturb host metabolism, creating a competitive advantage for plasmid-free cells. Plasmid stability hinges on efficient replication and partition mechanisms. While plasmids commonly encode histone-like nucleoid-structuring (H-NS) family proteins, the precise influence of plasmid-encoded H-NS proteins on stability remains elusive. In this study, we examined the conjugative plasmid pMBL6842, harboring the hns gene, and observed its positive regulation of parAB transcription, critical for plasmid segregation. Deletion of hns led to rapid plasmid loss, which was remedied by hns complementation. Further investigations unveiled adverse effects of hns overexpression on the bacterial host. Transcriptome analysis revealed hns's role in regulating numerous bacterial genes, impacting both host growth and swimming motility in the presence of the hns gene. Therefore, our study unveils the multifaceted roles of H-NS in both plasmid stability and host physiology, underscoring its biological significance and paving the way for future inquiries into the involvement of H-NS in horizontal gene transfer events., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The prevalence of antimicrobial resistance in Gram-negative bacteria poses a greater challenge due to their intrinsic resistance to many antibiotics. Recently, darobactins have emerged as a novel class of antibiotics originating from previously unexplored Gram-negative bacterial species such as Photorhabdus , Vibrio , Pseudoalteromonas and Yersinia . Darobactins belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) class of antibiotics, exhibiting selective activity against Gram-negative bacteria. They target the β-barrel assembly machinery (BAM), which is crucial for the maturation and insertion of outer membrane proteins in Gram-negative bacteria. The dar operon in the producer's genome encodes for the synthesis of darobactins, which are characterized by a fused ring system connected via an alkyl-aryl ether linkage (C-O-C) and a C-C cross-link. The enzyme DarE, using the radical S -adenosyl-l-methionine (rSAM), facilitates the formation of these bonds. Biosynthetic manipulation of the darobactin gene cluster, along with its expression in a surrogate host, has enabled access to diverse darobactin analogues with variable antibiotic activities. Recently, two independent research groups successfully achieved the total synthesis of darobactin, employing Larock heteroannulation to construct the bicyclic structure. This paper presents a comprehensive review of darobactins, encompassing their discovery through to the most recent advancements.

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-β-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., Competing Interests: The authors declare no conflict of interest.

Microorganisms in the waterline zone can secrete pigments to avoid damage caused by ultraviolet radiation, some of which have corrosive effects. In this work, we found that the secretion of pyomelanin by P3 strain of Pseudoalteromonas lipolytica significantly increases under strong lighting conditions, accelerating the corrosion of the material. Molecular mechanisms indicate that strong light, as a stressful environmental factor, enhances the expression of melanin secretion-related genes to prevent bacteria from being damaged by ultraviolet radiation. Therefore, this work proposes a new corrosion mechanism in the waterline zone, pigment-producing microorganisms are also involved in the waterline corrosion process., Competing Interests: Declaration of competing interest All authors declare no financial or personal relationships that may be perceived as influencing our work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Due to the low temperature, the Antarctic marine environment is challenging for protein functioning. Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologs, resulting in enhanced reaction rates at low temperatures. The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) genome is one of the few examples of coexistence of multiple hemoglobin genes encoding, among others, two constitutively transcribed 2/2 hemoglobins (2/2Hbs), also named truncated Hbs (TrHbs), belonging to the Group II (or O), annotated as PSHAa0030 and PSHAa2217. In this work, we describe the ligand binding kinetics and their interrelationship with the dynamical properties of globin Ph-2/2HbO-2217 by combining experimental and computational approaches and implementing a new computational method to retrieve information from molecular dynamic trajectories. We show that our approach allows us to identify docking sites within the protein matrix that are potentially able to transiently accommodate ligands and migration pathways connecting them. Consistently with ligand rebinding studies, our modeling suggests that the distal heme pocket is connected to the solvent through a low energy barrier, while inner cavities play only a minor role in modulating rebinding kinetics., (© 2024 The Protein Society.)

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., (© 2024 John Wiley & Sons Ltd.)

Escherichia coli , one of the most efficient expression hosts for recombinant proteins, is widely used in chemical, medical, food, and other industries. De novo engineering of gene regulation circuits and cell density-controlled E. coli cell lysis are promising directions for the release of intracellular bioproducts. Here, we developed an E. coli autolytic system, named the quorum sensing-mediated bacterial autolytic (QS-BA) system, by incorporating an acyl-homoserine lactone (AHL)-based YasI/YasR-type quorum sensing circuit from Pseudoalteromonas into E. coli cells. The results showed that the E. coli QS-BA system can release the intracellular bioproducts into the cell culture medium in terms of E. coli cell density, which offers an environmentally-friendly, economical, efficient, and flexible E. coli lysis platform for production of recombinant proteins. The QS-BA system has the potential to serve as an integrated system for the large-scale production of target products in E. coli for medical and industrial applications.

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., (© 2024. The Author(s).)

Naphthenic acids (NAs) have been found to exert serious threats on offshore sediment ecosystems and human health in recent years, which entails us the urgent need for NAs remediation. Bioremediation is considered an ideal method for sediment remediation due to ecological sustainability and economic feasibility. However, current bioremediation efficiency of offshore sediments suffers from relatively slow and there has never any attempts to bioremediate offshore sediment NAs contamination hitherto. In this study, the green synthetic iron oxides (gFeOx) based on Laminaria extracts was employed to enhance the biodegradation of NAs (Cyclohexylacetic acid, CHAA) in offshore sediments by Pseudoalteromonas sp. JSTW (an indigenous microorganism). The results showed that CHAA (20 mg·kg -1 ) in offshore sediments was removed almost 100% within 7 days at 30 mg·kg -1 gFeOx and 0.6 mg·kg -1 Strain JSTW. High-throughput sequencing results revealed that the structure and function of sediment microbial community were essentially restored to uncontaminated levels after bioremediation, highlighting the joint remediation approach is an efficient and eco-friendly method. Overall, this work has firstly provided insights into the application for NAs in situ bioremediation in offshore sediments., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

When cells are exposed to freezing temperatures, high concentrations of cryoprotective agents (CPA) prevent ice crystal formation, thus enhancing cell survival. However, high concentrations of CPAs can also cause cell toxicity. Exopolysaccharides (EPSs) from polar marine environments exhibit lower toxicity and display effects similar to traditional CPA. In this study, we sought to address these issues by i) selecting strains that produce EPS with novel cryoprotective activity, and ii) optimizing culture conditions for EPS production. Sixty-six bacteria producing mucous substances were isolated from the Ross Sea (Antarctic Ocean) using solid marine agar plates. Among them, Pseudoalteromonas sp. RosPo-2 was ultimately selected based on the rheological properties of the produced EPS (p-CY02). Cryoprotective activity experiments demonstrated that p-CY02 exhibited significantly cryoprotective activity at a concentration of 0.8% (w/v) on mammalian cells (HaCaT). This activity was further improved when combined with various concentrations of dimethyl sulfoxide (DMSO) compared to using DMSO alone. Moreover, the survival rate of HaCaT cells treated with 5% (v/v) DMSO and 0.8% (w/v) p-CY02 was measured at 87.9 ± 2.8% after freezing treatment. This suggests that p-CY02 may be developed as a more effective, less toxic, and novel non-permeating CPA. To enhance the production of EPS with cryoprotective activity, Response Surface Methodology (RSM) was implemented, resulting in a 1.64-fold increase in production of EPS with cryoprotective activity.

The sea squirt Ciona robusta (formerly Ciona intestinalis type A) has been the subject of many interdisciplinary studies. Known as a vanadium-rich ascidian, C. robusta is an ideal model for exploring microbes associated with the ascidian and the roles of these microbes in vanadium accumulation and reduction. In this study, we discovered two bacterial strains that accumulate large amounts of vanadium, CD2-88 and CD2-102, which belong to the genera Pseudoalteromonas and Vibrio, respectively. The growth medium composition impacted vanadium uptake. Furthermore, pH was also an important factor in the accumulation and localization of vanadium. Most of the vanadium(V) accumulated by these bacteria was converted to less toxic vanadium(IV). Our results provide insights into vanadium accumulation and reduction by bacteria isolated from the ascidian C. robusta to further study the relations between ascidians and microbes and their possible applications for bioremediation or biomineralization., (© 2024. The Author(s).)

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., Competing Interests: The authors declare no conflict of interest.

The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is considered an interesting alternative host for the recombinant protein production, that can be explored when the conventional bacterial expression systems fail. Indeed, the manufacture of all the difficult-to-express proteins produced so far in this bacterial platform gave back soluble and active products. Despite these promising results, the low yield of recombinant protein production achieved is hampering the wider and industrial exploitation of this psychrophilic cell factory. All the expression plasmids developed so far in PhTAC125 are based on the origin of replication of the endogenous pMtBL plasmid and are maintained at a very low copy number. In this work, we set up an experimental strategy to select mutated OriR sequences endowed with the ability to establish recombinant plasmids at higher multiplicity per cell. The solution to this major production bottleneck was achieved by the construction of a library of psychrophilic vectors, each containing a randomly mutated version of pMtBL OriR, and its screening by fluorescence-activated cell sorting (FACS). The selected clones allowed the identification of mutated OriR sequences effective in enhancing the plasmid copy number of approximately two orders of magnitude, and the production of the recombinant green fluorescent protein was increased up to twenty times approximately. Moreover, the molecular characterization of the different mutant OriR sequences allowed us to suggest some preliminary clues on the pMtBL replication mechanism that deserve to be further investigated in the future. KEY POINTS: • Setup of an electroporation procedure for Pseudoalteromonas haloplanktis TAC125. • Two order of magnitude improvement of OriR-derived psychrophilic expression systems. • Almost twenty times enhancement in Green fluorescent protein production., (© 2023. The Author(s).)

Small RNAs (sRNAs) play a very important role in gene regulation at the posttranscriptional level. However, sRNAs from nonmodel microorganisms, extremophiles in particular, have been rarely explored. We discovered a putative sRNA, termed Pf1 sRNA, in Pseudoalteromonas fuliginea BSW20308 isolated from the polar regions in our previous work. In this study, we identified the sRNA and investigated its regulatory role in gene expression under different temperatures. Pf1 sRNA was confirmed to be a new member of the CsrB family but has little sequence similarity with Escherichia coli CsrB. However, Pf1 sRNA was able to bind to CsrA from E. coli and P. fuliginea BSW20308 to regulate glycogen synthesis. The Pf1 sRNA knockout strain (Δ Pf1 ) affected motility, fitness, and global gene expression in transcriptomes and proteomes at 4°C and 32°C. Genes related to carbon metabolism, amino acid metabolism, salinity tolerance, antibiotic resistance, oxidative stress, motility, chemotaxis, biofilm, and secretion systems were differentially expressed in the wild-type strain and the Δ Pf1 mutant. Our study suggested that Pf1 sRNA might play an important role in response to environmental changes by regulating global gene expression. Specific targets of the Pf1 sRNA-CsrA system were tentatively proposed, such as genes involved in the type VI secretion system, TonB-dependent receptors, and response regulators, but most of them have an unknown function. Since this is the first study of CsrB family sRNA in Pseudoalteromonas and microbes from the polar regions, it provides a novel insight at the posttranscriptional level into the responses and adaptation to temperature changes in bacteria from extreme environments. This study also sheds light on the evolution of sRNA in extreme environments and expands the bacterial sRNA database. IMPORTANCE Previous research on microbial temperature adaptation has focused primarily on functional genes, with little attention paid to posttranscriptional regulation. Small RNAs, the major posttranscriptional modulators of gene expression, are greatly underexplored, especially in nonpathogenic and nonmodel microorganisms. In this study, we verified the first Csr sRNA, named Pf1 sRNA, from Pseudoalteromonas , a model genus for studying cold adaptation. We revealed that Pf1 sRNA played an important role in global regulation and was indispensable in improving fitness. This study provided us a comprehensive view of sRNAs from Pseudoalteromonas and expanded our understanding of bacterial sRNAs from extreme environments.

Organisms need sufficient intracellular iron to maintain biological processes. However, cells can be damaged by excessive iron-induced oxidation stress. Therefore, iron homeostasis must be strictly regulated. In general, bacteria have evolved complex mechanisms to maintain iron homeostasis. In this study, we showed that Pseudoalteromonas sp. R3 has four sets of iron uptake systems. Among these, the siderophore pyoverdine-dependent iron uptake system and the ferrous iron transporter Feo system are more important for iron uptake and prodiginine biosynthesis. Stringent starvation protein SspA positively controls iron uptake and iron-dependent prodiginine biosynthesis by regulating the expression of all iron uptake systems. In turn, the expression of SspA can be induced and repressed by extracellular iron deficiency and excess, respectively. Interestingly, extracytoplasmic function sigma factor PvdS also regulates iron uptake and prodiginine production and responds to extracellular iron levels, exhibiting a similar phenomenon as SspA. Notably, not only do SspA and PvdS function independently, but they can also compensate for each other, and their expression can be affected by the other. All of these findings demonstrate that SspA and PvdS coordinate iron homeostasis and prodiginine biosynthesis in strain R3. More importantly, our results also showed that SspA and PvdS homologs in Pseudomonas aeruginosa PAO1 have similar functions in iron uptake to their counterparts in Pseudoalteromonas , suggesting that coordination between SspA and PvdS on iron homeostasis could be conserved in typical Gram-negative bacteria. Since master regulation of iron homeostasis is extremely important for cell survival, this cross talk between SspA and PvdS may be environmentally significant. IMPORTANCE Both deficiency and excess of intracellular iron can be harmful, and thus, the iron homeostasis needs to be tightly regulated in organisms. At present, the ferric uptake regulator (Fur) is the best-characterized regulator involved in bacterial iron homeostasis, while other regulators of iron homeostasis remain to be further explored. Here, we demonstrated that the stringent starvation protein SspA and the extracytoplasmic function sigma factor PvdS coordinate iron uptake and iron-dependent prodiginine biosynthesis in Pseudoalteromonas sp. R3. These two regulators work independently, but their functions can compensate for the other and their expression can be affected by the other. Moreover, their expression can be activated and repressed by extracellular iron deficiency and excess, respectively. Notably, SspA and PvdS homologs in Pseudomonas aeruginosa PAO1 exhibit similar functions in iron uptake to their counterparts in Pseudoalteromonas , suggesting that this novel fine-tuned mode of iron homeostasis could be conserved in typical Gram-negative bacteria.

A synergistic approach using cultivation methods, chemical, and bioinformatic analyses was applied to explore the potential of Pseudoalteromonas sp. S8-8 in the production of extracellular polymeric substances (EPSs) and the possible physiological traits related to heavy metal and/or antibiotic resistance. The effects of different parameters (carbon source, carbon source concentration, temperature, pH and NaCl supplement) were tested to ensure the optimization of growth conditions for EPS production by the strain S8-8. The highest yield of EPS was obtained during growth in culture medium supplemented with glucose (final concentration 2%) and NaCl (final concentration 3%), at 15 °C and pH 7. The EPS was mainly composed of carbohydrates (35%), followed by proteins and uronic acids (2.5 and 2.77%, respectively) and showed a monosaccharidic composition of glucose: mannose: galactosamine: galactose in the relative molar proportions of 1:0.7:0.5:0.4, as showed by the HPAE-PAD analysis. The detection of specific molecular groups (sulfates and uronic acid content) supported the interesting properties of EPSs, i.e. the emulsifying and cryoprotective action, heavy metal chelation, with interesting implication in bioremediation and biomedical fields. The analysis of the genome allowed to identify a cluster of genes involved in cellulose biosynthesis, and two additional gene clusters putatively involved in EPS biosynthesis. KEY POINTS: • A cold-adapted Pseudoalteromonas strain was investigated for EPS production. • The EPS showed emulsifying, cryoprotective, and heavy metal chelation functions. • Three gene clusters putatively involved in EPS biosynthesis were evidenced by genomic insights., (© 2022. The Author(s).)

Background: A significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related to their catalytic and regulatory domains. Human CDKL5 belongs to this category. It is a serine/threonine protein kinase whose mutations are involved in CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental pathology still lacking a therapeutic intervention. The lack of successful CDKL5 manufacture hampered the exploitation of the otherwise highly promising enzyme replacement therapy. As almost two-thirds of the enzyme sequence is predicted to be intrinsically disordered, the recombinant product is either subjected to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Therefore, the use of an unconventional expression system can constitute a valid alternative to solve these issues., Results: Using a multiparametric approach we managed to optimize the transcription of the CDKL5 gene and the synthesis of the recombinant protein in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 applying a bicistronic expression strategy, whose generalization for recombinant expression in the cold has been here confirmed with the use of a fluorescent reporter. The recombinant protein largely accumulated as a full-length product in the soluble cell lysate. We also demonstrated for the first time that full-length CDKL5 produced in Antarctic bacteria is catalytically active by using two independent assays, making feasible its recovery in native conditions from bacterial lysates as an active product, a result unmet in other bacteria so far. Finally, the setup of an in cellulo kinase assay allowed us to measure the impact of several CDD missense mutations on the kinase activity, providing new information towards a better understanding of CDD pathophysiology., Conclusions: Collectively, our data indicate that P. haloplanktis TAC125 can be a valuable platform for both the preparation of soluble active human CDKL5 and the study of structural-functional relationships in wild type and mutant CDKL5 forms. Furthermore, this paper further confirms the more general potentialities of exploitation of Antarctic bacteria to produce "intractable" proteins, especially those containing large intrinsically disordered regions., (© 2022. The Author(s).)

The genomic and carbohydrate metabolic features of Pseudoalteromonas agarivorans Hao 2018 ( P. agarivorans Hao 2018) were investigated through pan-genomic and transcriptomic analyses, and key enzyme genes that may encode the process involved in its extracellular polysaccharide synthesis were screened. The pan-genome of the P. agarivorans strains consists of a core-genome containing 2331 genes, an accessory-genome containing 956 genes, and a unique-genome containing 1519 genes. Clusters of Orthologous Groups analyses showed that P. agarivorans harbors strain-specifically diverse metabolisms, probably representing high evolutionary genome changes. The Kyoto Encyclopedia of Genes and Genomes and reconstructed carbohydrate metabolic pathways displayed that P. agarivorans strains can utilize a variety of carbohydrates, such as d-glucose, d-fructose, and d-lactose. Analyses of differentially expressed genes showed that compared with the stationary phase (24 h), strain P. agarivorans Hao 2018 had upregulated expression of genes related to the synthesis of extracellular polysaccharides in the logarithmic growth phase (2 h), and that the expression of these genes affected extracellular polysaccharide transport, nucleotide sugar synthesis, and glycosyltransferase synthesis. This is the first investigation of the genomic and metabolic features of P. agarivorans through pan-genomic and transcriptomic analyses, and these intriguing discoveries provide the possibility to produce novel marine drug lead compounds with high biological activity.

Five strains of Pseudoalteromonas, isolated from oyster haemolymph, have exhibited antibacterial activity against several Gram-negative bacteria. Bioactive compounds have been identified in their cell-free supernatant and characterised as alterins, which are cyclolipopeptides comprising a heptapeptidic ring connected to a fatty acid chain. Using ultra-performance liquid chromatography-high-resolution mass spectrometry, this paper describes 37 structural analogues differing from each other by one or more amino acid residue, the length of the fatty acid chain, its hydroxylation and the presence of unsaturation., Competing Interests: Declaration of Competing Interests None declared., (Copyright © 2022. Published by Elsevier Ltd.)

As the most abundant d-amino acid (DAA) in the ocean, d-alanine (d-Ala) is a key component of peptidoglycan in the bacterial cell wall. However, the underlying mechanisms of bacterial metabolization of d-Ala through the microbial food web remain largely unknown. In this study, the metabolism of d-Ala by marine bacterium Pseudoalteromonas sp. strain CF6-2 was investigated. Based on genomic, transcriptional, and biochemical analyses combined with gene knockout, d-Ala aminotransferase was found to be indispensable for the catabolism of d-Ala in strain CF6-2. Investigation on other marine bacteria also showed that d-Ala aminotransferase gene is a reliable indicator for their ability to utilize d-Ala. Bioinformatic investigation revealed that d-Ala aminotransferase sequences are prevalent in genomes of marine bacteria and metagenomes, especially in seawater samples, and Gammaproteobacteria represents the predominant group containing d-Ala aminotransferase. Thus, Gammaproteobacteria is likely the dominant group to utilize d-Ala via d-Ala aminotransferase to drive the recycling and mineralization of d-Ala in the ocean. IMPORTANCE As the most abundant d-amino acid in the ocean, d-Ala is a component of the marine DON (dissolved organic nitrogen) pool. However, the underlying mechanism of bacterial metabolization of d-Ala to drive the recycling and mineralization of d-Ala in the ocean is still largely unknown. The results in this study showed that d-Ala aminotransferase is specific and indispensable for d-Ala catabolism in marine bacteria and that marine bacteria containing d-Ala aminotransferase genes are predominantly Gammaproteobacteria widely distributed in global oceans. This study reveals marine d-Ala-utilizing bacteria and the mechanism of their metabolization of d-Ala. The results shed light on the mechanisms of recycling and mineralization of d-Ala driven by bacteria in the ocean, which are helpful in understanding oceanic microbial-mediated nitrogen cycle.

As an important enzyme involved in the marine carbon cycle, alginate lyase has received extensive attention because of its excellent degradation ability on brown algae, which is widely utilized for alginate oligosaccharide preparation or bioethanol production. In comparison with endo-type alginate lyases (PL-5, PL-7, and PL-18 families), limited studies have focused on PL-17 family alginate lyases, especially for those with special characteristics. In this study, a novel PL-17 family alginate lyase, Aly23, was identified and cloned from the marine bacterium Pseudoalteromonas carrageenovora ASY5. Aly23 exhibited maximum activity at 35 °C and retained 48.93% of its highest activity at 4 °C, representing an excellent cold-adaptation property. Comparative molecular dynamics analysis was implemented to explore the structural basis for the cold-adaptation property of Aly23. Aly23 had a high substrate preference for poly β-D-mannuronate and exhibited both endolytic and exolytic activities; its hydrolysis reaction mainly produced monosaccharides, disaccharides, and trisaccharides. Furthermore, the enzymatic hydrolyzed oligosaccharides displayed good antioxidant activities to reduce ferric and scavenge radicals, such as hydroxyl, ABTS + , and DPPH. Our work demonstrated that Aly23 is a promising cold-adapted biocatalyst for the preparation of natural antioxidants from brown algae.

Exopolysaccharides (EPSs) are carbohydrate polymers produced and secreted by microorganisms. In a changing marine environment, EPS secretion can reduce damage from external environmental disturbances to microorganisms. Meanwhile, EPSs have promising application prospects in the fields of food, cosmetics, and pharmaceuticals. Changes in external environmental pH have been shown to affect the synthesis of EPSs in microorganisms. In this study, we analyzed the effects of different initial fermentation pHs on the production, monosaccharide composition, and antioxidant activity of the EPSs of Pseudoalteromonas agarivorans Hao 2018. In addition, the transcriptome sequence of P. agarivorans Hao 2018 under different initial fermentation pH levels was determined. GO and KEGG analyses showed that the differentially expressed genes were concentrated in the two-component regulatory system and bacterial chemotaxis pathways. We further identified the expression of key genes involved in EPS synthesis during pH changes. In particular, the expression of genes encoding the glucose/galactose MFS transporter, phosphomannomutase, and mannose-1-phosphate guanylyltransferase was upregulated when the environmental pH increased, thus promoting EPS synthesis. This study not only contributes to elucidating the environmental adaptation mechanisms of P. agarivorans , but also provides important theoretical guidance for the directed development of new products using biologically active polysaccharides.

The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 is an unconventional protein production host displaying a notable proficiency in the soluble production of difficult proteins, especially of human origin. Furthermore, the accumulation of recombinant products in insoluble aggregates has never been observed in this bacterium, indicating that its cellular physicochemical conditions and/or folding processes are rather different from those observed in mesophilic bacteria. The ability of this cell factory was challenged by producing a human protein, the cyclin-dependent kinase-like 5 (hCDKL5) in the bacterium cytoplasm at 0 °C. Human CDKL5 is a serine/threonine protein kinase characterized by the absence of a defined structure for the last two/third of its sequence, one of the largest intrinsically disordered regions so far observed in a human protein. This large unstructured domain makes difficult its production in most of the conventional hosts since the recombinant product accumulates as insoluble aggregates and/or is heavily proteolyzed. As the full-length hCDKL5 production is of great interest both for basic science and as protein drug for an enzyme replacement therapy, its production in the Antarctic bacterium was tested by combining the use of a regulated psychrophilic gene expression system with the use of a defined growth medium optimized for the host growth at subzero temperature. This is the first report of soluble and full-length recombinant production of hCDKL5 protein in a bacterium., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Pseudoalteromonas (BB2-AT2) is a ubiquitous marine heterotroph, often associated with labile organic carbon sources in the ocean (e.g. phytoplankton blooms and sinking particles). Heterotrophs hydrolyze exported photosynthetic materials, components of the biological carbon pump, with the use of diverse metalloenzymes containing zinc (Zn), manganese (Mn), cobalt (Co), and nickel (Ni). Studies on the metal requirements and cytosolic utilization of metals for marine heterotrophs are scarce, despite their relevance to global carbon cycling. Here, we characterized the Zn, Mn, Co, and Ni metallome of BB2-AT2. We found that the Zn metallome is complex and cytosolic Zn is associated with numerous proteins for transcription (47.2% of the metallome, obtained from singular value decomposition of the metalloproteomic data), translation (33.5%), proteolysis (12.8%), and alkaline phosphatase activity (6.4%). Numerous proteolytic enzymes also appear to be putatively associated with Mn, and to a lesser extent, Co. Putative identification of the Ni-associated proteins, phosphoglucomutase and a protein in the cupin superfamily, provides new insights for Ni utilization in marine heterotrophs. BB2-AT2 relies on numerous transition metals for proteolytic and phosphatase activities, inferring an adaptative potential to metal limitation. Our field observations of increased alkaline phosphatase activity upon addition of Zn in field incubations suggest that such metal limitation operates in sinking particulate material collected from sediment traps. Taken together, this study improves our understanding of the Zn, Mn, Co, and Ni metallome of marine heterotrophic bacteria and provides novel and mechanistic frameworks for understanding the influence of nutrient limitation on biogeochemical cycling., (© The Author(s) 2021. Published by Oxford University Press.)

Pseudoalteromonas rubra S4059 produces the red pigment prodigiosin, which has pharmaceutical and industrial potential. Here, we targeted a putative prodigiosin-synthesizing transferase PigC, and a pigC in-frame deletion mutant did not produce prodigiosin. However, extractions of the pigC mutant cultures retained antibacterial activity, and bioassay-guided fractionation found antibacterial activity in two fractions of blue color. A precursor of prodigiosin, 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC), was the dominant compound in both the fractions and likely caused the antibacterial activity. Also, a stable blue pigment, di-pyrrolyl-dipyrromethene prodigiosin, was identified from the two fractions. We also discovered antibacterial activity in the sterile filtered (nonextracted) culture supernatant of both wild type and mutant, and both contained a heat-sensitive compound between 30 and 100 kDa. Deletion of prodigiosin production did not affect growth rate or biofilm formation of P. rubra and did not change its fitness, as the mutant and wild type coexisted in equal levels in mixed cultures. In conclusion, a prodigiosin biosynthetic gene cluster (BGC) was identified and verified genetically and chemically in P. rubra S4059 and a stable blue pigment was isolated from the pigC mutant of S4059, suggesting that this strain may produce several prodigiosin-derived compounds of pharmaceutical and/or industrial potential. IMPORTANCE Pigmented Pseudoalteromonas strains are renowned for their production of secondary metabolites, and genome mining has revealed a high number of biosynthetic gene clusters (BGCs) for which the chemistry is unknown. Identification of those BGCs is a prerequisite for linking products to gene clusters and for further exploitation through heterologous expression. In this study, we identified the BGCs for the red, bioactive pigment prodigiosin using genomic, genetic, and metabolomic approaches. We also report here for the first time the production of a stable blue pigment, di-pyrrolyl-dipyrromethene prodigiosin (Dip-PDG), being produced by the pigC mutant of Pseudoalteromonas rubra S4059.

Worldwide marine compound contamination by petroleum products and heavy metals is a burgeoning environmental concern. Pseudoalteromonas, prevalently distributed in marine environment, has been proven to degrade petroleum and plays an essential role in the fate of oil pollution under the combined pollution. Nevertheless, the research on the reference genes is still incomplete. Therefore, this study aims to thoroughly investigate the reference genes represented by Pseudoalteromonas sp. JSTW via whole-genome sequencing. Next-generation sequencing technology unfolded a genome of 4,026,258 bp, database including Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were utilized to annotate the genes and metabolic pathways conferring to petroleum hydrocarbon degradation. The results show that common alkane and aromatic hydrocarbon degradation genes (alkB, ligB, yqhD, and ladA), chemotaxis gene (MCP, cheA, cheB, pcaY, and pcaR), heavy-metal resistance, and biofilm genes (σ54, merC, pcoA, copB, etc.) were observed in whole-genome sequence (WGS) of JSTW, which indicated that strain JSTW could potentially cope with combined pollution. The degradation efficiency of naphthalene in 60 h by JSTW was 99% without Cu 2+ and 67% with 400 mg L -1 Cu 2+ . Comparative genome analysis revealed that genomes of Pseudoalteromonas lipolytica strain LEMB 39 and Pseudoalteromonas donghaensis strain HJ51 shared similarity with strain JSTW, suggesting they are also the potential degradater of petroleum hydrocarbons under combined pollution. Therefore, this study provides a WGS annotation and reveals the mechanism of response to combined pollution of Pseudoalteromonas sp. JSTW., (© 2021 Wiley-VCH GmbH.)

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 Mg 2+ concentrations (2 mM). We further analyzed an exceptionally stable folding trap of the G,C-rich Tth P RNA., (© 2021 Marszalkowski et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Pseudoalteromonas spp. are Gram-negative bacteria which are ubiquitous in marine environments. Our previous work found that there is a classic LuxI/LuxR-type quorum sensing (QS) system which was named YasI/YasR in Pseudoalteromonas sp. R3, but the factors that control QS in strain R3 are unclear yet. Here, we found that the deficiency of hfq encoding RNA chaperon Hfq down-regulated the transcription levels of yasI encoding acyl-homoserine lactones (AHLs) synthase and yasR encoding AHLs receptor in strain R3. The assay based on fusion reporter of yasI-lacZ showed that Hfq regulates the expression of yasR at both transcriptional and translational levels. In addition, Hfq affects the expression of yasI via yasR. Further analysis indicated that the 5'UTR region of yasR is necessary for Hfq to control QS. In addition, the deletion of hfq increases the unstability of the target yasR mRNA. Based on transcriptome sequencing and bioinformatic analysis together with molecular experiments, Hfq-dependent sRNA00002 was identified to be involved in positively regulating QS in Pseudoalternas sp. R3. It was found that sRNA00002 deficiency causes the decrease in expression of yasI and yasR, and thus abolishes the production of AHLs in strain R3. It was concluded that Hfq-dependent sRNA00002 regulates yasR expression by base-pairing with target yasR mRNA at 5'UTR region and altering the stability of yasR mRNA. Our work paves the way for understanding the regulation mechanism of Hfq-dependent sRNAs on QS in Pseudoalteromonas., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The molecular mechanism underlying modulation of metamorphosis of the bivalve Mytilus coruscus by bacteria remains unclear. Here, the functional role of the thioesterase gene tesA of the bacterium Pseudoalteromonas marina in larval metamorphosis was examined. The aim was to determine whether inactivation of the tesA gene altered the biofilm-inducing capacity, bacterial cell motility, biopolymers, or the intracellular c-di-GMP levels. Complete inactivation of tesA increased the c-di-GMP content in P. marina , accompanied by a reduced fatty acid content, weaker motility, upregulation of bacterial aggregation, and biofilm formation. The metamorphosis rate of mussel larvae on Δ tesA biofilms was reduced by ∼ 80% compared with those settling on wild-type P. marina . Exogenous addition of a mixture of extracted fatty acids from P. marina into the Δ tesA biofilms promoted the biofilm-inducing capacity. This study suggests that the bacterial thioesterase gene tesA altered the fatty acid composition of Δ tesA P. marina biofilms (BF) through regulation of its c-di-GMP, subsequently impacting mussel metamorphosis.

A cultivated form of bacteria (strain 2202) was isolated from the hemal fluid of the bivalve mollusk Modiolus kurilensis. Based on the set of data collected by genetic and physiological/biochemical analyses, the strain was identified as the species Pseudoalteromonas piscicida. Strain 2202 exhibits antimicrobial activity against Staphylococcus aureus, Candida albicans, and Bacillus subtilis but not against Escherichia coli and Pseudomonas aeruginosa. These activities characterize the behavior of strain 2202 as predator-like and classify it as a facultative predator. Being part of the normal microflora in the hemolymph of M. kurilensis, when external conditions change, strain 2202 shows features of opportunistic microflora. The strain 2202 exhibits selective toxicity towards larvae of various invertebrates: it impairs the early development of Mytilus edulis, but not of Strongylocentrotus nudus. Thus, the selective manner in which P. piscicida strains interact with various species of microorganisms and eukaryotes should be taken into consideration when using their biotechnological potential as a probiotic in aquaculture, source of antimicrobial substances, and factors that prevent fouling., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Alginate, which is mainly produced by brown algae and decomposed by heterotrophic bacteria, is an important marine organic carbon source. The genus Pseudoalteromonas contains diverse forms of heterotrophic bacteria that are widely distributed in marine environments and are an important group in alginate degradation. In this review, the diversity of alginate-degrading Pseudoalteromonas is introduced, and the characteristics of Pseudoalteromonas alginate lyases, including their sequences, enzymatic properties, structures, and catalytic mechanisms, and the synergistic effect of Pseudoalteromonas alginate lyases on alginate degradation are introduced. The acquisition of the alginate degradation capacity and the alginate utilization pathways of Pseudoalteromonas are also introduced. This paper provides a comprehensive overview of alginate degradation by Pseudoalteromonas, which will contribute to the understanding of the degradation and recycling of marine algal polysaccharides driven by marine bacteria.

Reliable and accurate in situ sensors capable of detecting and quantifying troublesome marine biofilms on metallic surfaces are increasingly necessary. A 0.2 mm diameter gold electrochemical sensor was fully characterized using cyclic voltammetry in abiotic and biotic artificial seawater media within a continuous culture flow cell to detect the growth and development of an aerobic Pseudoalteromonas sp. biofilm. Deconvolution of the abiotic and biotic responses enable the constituent extracellular electron transfer and biofilm responses to be resolved. Differentiation of enhanced oxygen reduction kinetics within the aerobic bacterial biofilm is linked to enzyme and redox mediator activities.

Microbial degradation of organic matter along the vertical profile of the water column is a major process driving the carbon cycle in the ocean. Pseudoalteromonas has been identified as a dominant genus in pelagic marine environments worldwide, playing important roles in the remineralization of organic carbon. However, the current understanding of Pseudoalteromonas was mainly based on shallow water populations or cultivated species. This study analyzed for the first time the structure, activity potential and ecotypes differentiation of Pseudoalteromonas in the water column of the New Britain Trench (NBT) down to 6000 m. Analysis on diversities of the 16S rRNA gene and their transcripts showed that Pseudoalteromonas was greatly enriched in deep-sea waters and showed high activity potentials. The deep-sea Pseudoalteromonas were significantly different from their shallow-water counterparts, suggesting an obvious ecotype division along with the vertical profile. Phylogenetic analysis on the 16S rRNA gene and hsp60 gene of 219 Pseudoalteromonas strains isolated from different depths further showed that the vertical ecotype division could even occur at the strain level, which might be a result of long-term adaptation to environmental conditions at different depths. The discovered depth-specific strains provide valuable models for further studies on adaptation, evolution and functions of the deep-sea Pseudoalteromonas., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

κ-Carrageenan oligosaccharides with many excellent biological properties could be produced by κ-carrageenases selectively. In this study, based on the encoding gene of full length κ-carrageenase obtained from Pseudoalteromonas sp. ZDY3 and the reported mature secreted κ-carrageenase composed of 275 amino acid residues (N26-T300), CgkPZ&#95;GH16 was expressed in E. coli, but no soluble active protein could be detected. Fortunately, the signal peptide of wild-type κ-carrageenase was recognized, and cleaved in the soluble and folding form in E. coli, the K m and k cat values of CgkPZ&#95;SP&#95;GH16 was 1.007 mg/mL and 362.8 s -1 . By molecular dynamics simulations, it was showed that YjdB domain might affect the activity of κ-carrageenase. Due to the absence of mature processing modification system in E. coli, YjdB was remained in recombinant full length κ-carrageenase, and the lost catalytic efficiency of CgkPZ was compensated by expression level and thermal stability. Interestingly, CgkPZ&#95;GH16&#95;YjdB was expressed soluble without the signal peptide, which indicated that YjdB could contribute to the expression and folding of κ-carrageenase. These results provide new insight into the effects of different modules of κ-carrageenase on the expression and properties of enzyme., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cadmium (Cd) is a common toxic heavy metal in the environment, and bacteria have evolved different strategies to deal with Cd toxicity. Here, a bacterium designated Pseudoalteromonas sp. MT33b possessing strong Cd resistance was isolated from the Mariana Trench sediments. Supplement of cysteine significantly increased bacterial Cd resistance and removal rate. Biofilm formation was demonstrated to play a positive role toward bacterial Cd resistance. Transcriptome analysis showed the supplement of cysteine effectively prevented Cd 2+ from entering bacterial cells, promoted saccharide metabolism and thereby facilitating energy production, which consists well with bacterial growth trend analysed under the same conditions. Notably, the expressions of many biofilm formation related genes including flagellar assembly, signal transduction, bacterial secretion and TonB-dependent transfer system were significantly upregulated when facing Cd stress, indicating their important roles in determining bacterial biofilm formation and enhancing Cd resistance. Overall, this study indicates the formation of insoluble CdS precipitates and massive biofilm is the major strategy adopted by Pseudoalteromonas sp. MT33b to eliminate Cd stress. Our results provide a good model to investigate how heavy metals impact biofilm formation in the deep-sea ecosystems, which may facilitate a deeper understanding of microbial environmental adaptability and better utilization of these microbes for bioremediation purposes in the future., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Antioxidants prevent ageing and are usually quantified and screened using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. However, this assay cannot be used for salt-containing samples, such as the cell-free supernatants of marine microorganisms that are aggregated under these conditions. Herein, the DPPH solvent (methanol or ethanol) and its water content were optimized to enable the analysis of salt-containing samples, aggregation was observed for alcohol contents of >70%. The water content of methanol influenced the activities of standard antioxidants but did not significantly affect that of the samples. Based on solution stability considerations, 70% aqueous methanol was chosen as the optimal DPPH solvent. The developed method was successfully applied to the cell-free supernatants of marine bacteria ( Pseudoalteromonas rubra and Pseudoalteromonas xiamenensis ), revealing their high antioxidant activities. Furthermore, it was concluded that this method would be useful for the screening of marine microorganism-derived antioxidants, which also has numerous potential applications, such as salt-fermented foods.

Prodiginines are a family of red-pigmented secondary metabolites with multiple biological activities. The biosynthesis of prodiginines is affected by various physiological and environmental factors. Thus, prodiginine biosynthesis regulation is highly complex and multifaceted. Although the regulatory mechanism for prodiginine biosynthesis has been extensively studied in Serratia and Streptomyces species, little is known about that in the marine betaproteobacterium Pseudoalteromonas In this study, we report that stringent starvation protein A (SspA), an RNA polymerase-associated regulatory protein, is required for the biosynthesis of prodiginine in Pseudoalteromonas sp. strain R3. The strain lacking sspA (Δ sspA ) fails to produce prodiginine, which resulted from the downregulation of the prodiginine biosynthetic gene ( pig ) cluster. The effect of SspA on prodiginine biosynthesis is independent of histone-like nucleoid structuring protein (H-NS) and RpoS (σ S ). Further analysis demonstrates that the Δ sspA strain has a significant decrease in the transcription of the siderophore biosynthesis gene ( pvd ) cluster, leading to the inhibition of siderophore production and iron uptake. The Δ sspA strain regains the ability to synthesize prodiginine by cocultivation with siderophore producers or the addition of iron. Therefore, we conclude that SspA-regulated prodiginine biosynthesis is due to decreased siderophore levels and iron deficiency. We further show that the iron homeostasis master regulator Fur is also essential for pig transcription and prodiginine biosynthesis. Overall, our results suggest that SspA indirectly regulates the biosynthesis of prodiginine, which is mediated by the siderophore-dependent iron uptake pathway. IMPORTANCE The red-pigmented prodiginines are attracting increasing interest due to their broad biological activities. As with many secondary metabolites, the biosynthesis of prodiginines is regulated by both environmental and physiological factors. At present, studies on the regulation of prodiginine biosynthesis are mainly restricted to Serratia and Streptomyces species. This work focused on the regulatory mechanism of prodiginine biosynthesis in Pseudoalteromonas sp. R3. We found that stringent starvation protein A (SspA) positively regulates prodiginine biosynthesis via affecting the siderophore-dependent iron uptake pathway. The connections among SspA, iron homeostasis, and prodiginine biosynthesis were investigated. These findings uncover a novel regulatory mechanism for prodigiosin biosynthesis., (Copyright © 2021 American Society for Microbiology.)

Extremophilic microbes have adapted to thrive in ecological niches characterized by harsh chemical/physical conditions such as, for example, very low/high temperature. Living organisms inhabiting these environments have developed peculiar mechanisms to cope with extreme conditions, in such a way that they mark the chemical-physical boundaries of life on Earth. Studying such mechanisms is stimulating from a basic research viewpoint and because of biotechnological applications. Pseudoalteromonas species are a group of marine gamma-proteobacteria frequently isolated from a range of extreme environments, including cold habitats and deep-sea sediments. Since deep-sea floors constitute almost 60% of the Earth's surface and cold temperatures represent the most common of the extreme conditions, the genus Pseudoalteromonas can be considered one of the most important model systems for studying microbial adaptation. Particularly, among all Pseudoalteromonas representatives, P. haloplanktis TAC125 has recently gained a central role. This bacterium was isolated from seawater sampled along the Antarctic ice-shell and is considered one of the model organisms of cold-adapted bacteria. It is capable of thriving in a wide temperature range and it has been suggested as an alternative host for the soluble overproduction of heterologous proteins, given its ability to rapidly multiply at low temperatures. In this review, we will present an overview of the recent advances in the characterization of Pseudoalteromonas strains and, more importantly, in the understanding of their evolutionary and chemical-physical strategies to face such a broad array of extreme conditions. A particular attention will be given to systems-biology approaches in the study of the above-mentioned topics, as genome-scale datasets (e.g. genomics, proteomics, phenomics) are beginning to expand for this group of organisms. In this context, a specific section dedicated to P. haloplanktis TAC125 will be presented to address the recent efforts in the elucidation of the metabolic rewiring of the organisms in its natural environment (Antarctica)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pseudoalteromonas species produce a diverse range of biologically active compounds, including those biosynthesized by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here, we report the biochemical and genomic analysis of Pseudoalteromonas sp. strain HM-SA03, isolated from the blue-ringed octopus, Hapalochlaena sp. Genome mining for secondary metabolite pathways revealed seven putative NRPS/PKS biosynthesis gene clusters, including those for the biosynthesis of alterochromides, pseudoalterobactins, alteramides, and four novel compounds. Among these was a novel siderophore biosynthesis gene cluster with unprecedented architecture (NRPS-PKS-NRPS-PKS-NRPS-PKS-NRPS). Alterochromide production in HM-SA03 was also confirmed by liquid chromatography-mass spectrometry. An investigation of the biosynthetic potential of 42 publicly available Pseudoalteromonas genomes indicated that some of these gene clusters are distributed throughout the genus. Through the phylogenetic analysis, a particular subset of strains formed a clade with extraordinary biosynthetic potential, with an average density of 10 biosynthesis gene clusters per genome. In contrast, the majority of Pseudoalteromonas strains outside this clade contained an average of three clusters encoding complex biosynthesis. These results highlight the underexplored potential of Pseudoalteromonas as a source of new natural products. IMPORTANCE This study demonstrates that the Pseudoalteromonas strain HM-SA03, isolated from the venomous blue-ringed octopus, Hapalochalaena sp., is a biosynthetically talented organism, capable of producing alterochromides and potentially six other specialized metabolites. We identified a pseudoalterobactin biosynthesis gene cluster and proposed a pathway for the production of the associated siderophore. A novel siderophore biosynthesis gene cluster with unprecedented architecture was also identified in the HM-SA03 genome. Finally, we demonstrated that HM-SA03 belongs to a phylogenetic clade of strains with extraordinary biosynthetic potential. While our results do not support a role of HM-SA03 in Hapalochalaena sp. venom (tetrodotoxin) production, they emphasize the untapped potential of Pseudoalteromonas as a source of novel natural products., (Copyright © 2021 American Society for Microbiology.)

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas , we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.

Pigmented bacterial symbionts play major roles in the health of coral holobionts. However, there is scarce knowledge on the diversity of these microbes for several coral species. To gain further insights into holobiont health, pigmented bacterial isolates of Fabibacter pacificus (Bacteroidetes; n = 4), Paracoccus marcusii (Alphaproteobacteria; n = 1), and Pseudoalteromonas shioyasakiensis (Gammaproteobacteria; n = 1) were obtained from the corals Mussismilia braziliensis and Montastraea cavernosa in Abrolhos Bank, Brazil. Cultures of these bacterial symbionts produced strong antioxidant activity (catalase, peroxidase, and oxidase). To explore these bacterial isolates further, we identified their major pigments by HPLC and mass spectrometry. The six phylogenetically diverse symbionts had similar pigment patterns and produced myxol and keto-carotene. In addition, similar carotenoid gene clusters were confirmed in the whole genome sequences of these symbionts, which reinforce their antioxidant potential. This study highlights the possible roles of bacterial symbionts in Montastraea and Mussismilia holobionts.

Discovery after discovery, host-associated microbiota reveal a growing list of positive effects on host homeostasis by contributing to host nutrition, improving hosts' immune systems and protecting hosts against pathogens. In that context, a collection of oyster associated bacteria producing antibacterial compounds have been established to evaluate their role in non-host-derived immunity. Here, we described alterins; potent anti-Gram negative compounds produced by Pseudoalteromonas h Cg -6 and h Cg -42 isolated from different healthy oyster hemolymph. The strains h Cg -6 and h Cg -42 produce a set of at least seven antibacterial compounds, ranging from 926 to 982 Da structurally characterized as cyclolipopeptides (CLPs). Alterins share the same cationic heptapeptidic cycle connected via an amido bond to different hydrophobic hydrocarbon tails. Their MICs disclosed a potent antibacterial activity directed against Gram-negative bacteria including oyster and human pathogens that may confer a beneficial defense mechanism to the host but also represents an untapped source of new antibiotics. The alterins' mechanisms of action have been deciphered: after binding to lipopolysaccharides (LPS), alterins provoke a membrane depolarization and permeabilization leading to bacterial lysis. As h Cg -6 and h Cg -42 produced a set of natural derivatives, the structure/activity relationship linked to the carbon tail is clarified. We showed that the hydrocarbon tail determines the LPS-binding properties of alterins and consequently their antibacterial activities. Its length and saturation seem to play a major role in this interaction.

Novel antimicrobials are urgently needed due to the rapid spread of antibiotic resistant bacteria. In a genome-wide analysis of Pseudoalteromonas strains, one strain (S4498) was noticed due to its potent antibiotic activity. It did not produce the yellow antimicrobial pigment bromoalterochromide, which was produced by several related type strains with which it shared less than 95% average nucleotide identity. Also, it produced a sweet-smelling volatile not observed from other strains. Mining the genome of strain S4498 using the secondary metabolite prediction tool antiSMASH led to eight biosynthetic gene clusters with no homology to known compounds, and synteny analyses revealed that the yellow pigment bromoalterochromide was likely lost during evolution. Metabolome profiling of strain S4498 using HPLC-HRMS analyses revealed marked differences to the type strains. In particular, a series of quinolones known as pseudanes were identified and verified by NMR. The characteristic odor of the strain was linked to the pseudanes. The highly halogenated compound tetrabromopyrrole was detected as the major antibacterial component by bioassay-guided fractionation. Taken together, the polyphasic analysis demonstrates that strain S4498 belongs to a novel species within the genus Pseudoalteromonas, and we propose the name Pseudoalteromonas galatheae sp. nov. (type strain S4498 T  = NCIMB 15250 T  = LMG 31599 T ).