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.

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.

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.

Characterization and genomic analysis of a novel Pseudoalteromonas phage PS_L5.

Pseudoalteromonas is a widely distributed bacterial genus that is associated with marine algae. However, there is still limited knowledge about their bacteriophage. In this study, we reported the isolation of a novel lytic bacteriophage that infects Pseudoalteromonas marina. Transmission electron microscopy revealed that PS_L5 had an icosahedral head of 52.6 ± 2 nm and a non-contractile tail with length of 96.5 ± 2 nm. The genome sequence of this phage was 34, 257 bp and had a GC content of 40.75%. Furthermore, this genome contained 61 predicted open reading frames (ORFs), which involved in various functions such as phage structure, packaging, DNA metabolism, host lysis and other additional functions. Additionally, the phylogenetic analysis based on major capsid protein showed that the phage PS_L5 was closely related to five other Pseudoalteromonas phages, namely PHS3, PHS21, AL, SL25 and Pq0 which also possessed the non-contractile long tail. This study provided the fundamental insights into the evolutionary dynamics of Pseudoalteromonas phages and the interaction between phage and host.

Isolation and characterization of polyhydroxyalkanoate-degrading bacteria in seawater at two different depths from Suruga Bay.

IMPORTANCE: Polyhydroxyalkanoate (PHA) is a highly biodegradable microbial polyester, even in marine environments. In this study, we incorporated an enrichment culture-like approach in the process of isolating marine PHA-degrading bacteria. The resulting 91 isolates were suggested to fall into five genera (Alloalcanivorax, Alteromonas, Arenicella, Microbacterium, and Pseudoalteromonas) based on 16S rRNA analysis, including two novel genera (Arenicella and Microbacterium) as marine PHA-degrading bacteria. Microbacterium schleiferi (DSM 20489) and Alteromonas macleodii (NBRC 102226), the type strains closest to the several isolates, have an extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase homolog that does not fit a marine-type domain composition. However, A. macleodii exhibited no PHA degradation ability, unlike M. schleiferi. This result demonstrates that the isolated Alteromonas spp. are different species from A. macleodii. P(3HB) depolymerase homologs in the genus Alteromonas should be scrutinized in the future, particularly about which ones work as the depolymerase.

Genomic Analysis of Two Cold-Active Pseudoalteromonas Phages Isolated from the Continental Shelf in the Arctic Ocean.

Cold-active bacteriophages are bacterial viruses that infect and replicate at low temperatures (≤4 °C). Understanding remains limited of how cold-active phage-host systems sustain high viral abundance despite the persistently low temperatures in pelagic sediments in polar seas. In this study, two Pseudoalteromonas phages, ACA1 and ACA2, were isolated from sediment core samples of the continental shelf in the western Arctic Ocean. These phages exhibited successful propagation at a low temperature of 1 °C and displayed typical myovirus morphology with isometric icosahedral heads and contractile tails. The complete genome sequences of phages ACA1 and ACA2 were 36,825 bp and 36,826 bp in size, respectively, sharing almost the same gene content. These are temperate phages encoding lysogeny-related proteins such as anti-repressor, immunity repressor and integrase. The absence of cross-infection between the host strains, which were genomically distinct Pseudoalteromonas species, can likely be attributed to heavy divergence in the anti-receptor apparently mediated by an associated diversity-generating retroelement. HHpred searching identified genes for all of the structural components of a P2-like phage (family Peduoviridae), although the whole of the Peduoviridae family appeared to be divided between two anciently diverged tail modules. In contrast, Blast matching and whole genome tree analysis are dominated by a nonstructural gene module sharing high similarity with Pseudoalteromonas phage C5a (founder of genus Catalunyavirus). This study expands the knowledge of diversity of P2-like phages known to inhabit Peudoalteromonas and demonstrates their presence in the Arctic niche.

Molecular basis of the phenotypic variants arising from a Pseudoalteromonas lipolytica mutator.

Bacterial deficiencies in the DNA repair system can produce mutator strains that promote adaptive microevolution. However, the role of mutator strains in marine Pseudoalteromonas, capable of generating various gain-of-function genetic variants within biofilms, remains largely unknown. In this study, inactivation of mutS in Pseudoalteromonas lipolytica conferred an approximately 100-fold increased resistance to various antibiotics, including ciprofloxacin, rifampicin and aminoglycoside. Furthermore, the mutator of P. lipolytica generated variants that displayed enhanced biofilm formation but reduced swimming motility, indicating a high phenotypic diversity within the ΔmutS population. Additionally, we observed a significant production rate of approximately 50 % for the translucent variants, which play important roles in biofilm formation, when the ΔmutS strain was cultured on agar plates or under shaking conditions. Using whole-genome deep-sequencing combined with genetic manipulation, we demonstrated that point mutations in AT00_17115 within the capsular biosynthesis cluster were responsible for the generation of translucent variants in the ΔmutS subpopulation, while mutations in flagellar genes fliI and flgP led to a decrease in swimming motility. Collectively, this study reveals a specific mutator-driven evolution in P. lipolytica, characterized by substantial genetic and phenotypic diversification, thereby offering a reservoir of genetic attributes associated with microbial fitness.

Marine Bacterioplankton Community Dynamics and Potentially Pathogenic Bacteria in Seawater around Jeju Island, South Korea, via Metabarcoding.

Understanding marine bacterioplankton composition and distribution is necessary for improving predictions of ecosystem responses to environmental change. Here, we used 16S rRNA metabarcoding to investigate marine bacterioplankton diversity and identify potential pathogenic bacteria in seawater samples collected in March, May, September, and December 2013 from two sites near Jeju Island, South Korea. We identified 1343 operational taxonomic units (OTUs) and observed that community diversity varied between months. Alpha- and Gamma-proteobacteria were the most abundant classes, and in all months, the predominant genera were Candidatus Pelagibacter, Leisingera, and Citromicrobium. The highest number of OTUs was observed in September, and Vibrio (7.80%), Pseudoalteromonas (6.53%), and Citromicrobium (6.16%) showed higher relative abundances or were detected only in this month. Water temperature and salinity significantly affected bacterial distribution, and these conditions, characteristic of September, were adverse for Aestuariibacter but favored Citromicrobium. Potentially pathogenic bacteria, among which Vibrio (28 OTUs) and Pseudoalteromonas (six OTUs) were the most abundant in September, were detected in 49 OTUs, and their abundances were significantly correlated with water temperature, increasing rapidly in September, the warmest month. These findings suggest that monthly temperature and salinity variations affect marine bacterioplankton diversity and potential pathogen abundance.

Isolation and genome sequencing of a novel lytic Pseudoalteromonas phage SL20.

Phage SL20, a novel lytic Pseudoalteromonas phage, was isolated from the coastal waters of the Yellow Sea, China. The microbiological characterization demonstrated that phage SL20 was relatively stable from 35 to 55 °C and the optimal pH was approximately 6.0. A latent period of approximately 24 min was indicated by a one-step growth curve. The burst size was approximately 12 ± 3 PFU/cell. The genome had a length of 120,295 bp with a G + C content of 35.84%, and predicted 95 ORFs. The phylogenetic tree based on DNA helicase showed that Pseudoalteromonas phage SL20 was related to the Pseudoalteromonas phage H101 and was a member of the family Shandongvirus. The isolation and genomic analysis of SL20 has improved our understanding of host-phage interactions and the ecology of the marine bacteria Pseudoalteromonas.

Deciphering the Biosynthesis of Novel Class I Lanthipeptides from Marine Pseudoalteromonas Reveals a Dehydratase PsfB with Dethiolation Activity.

Lanthipeptides are a representative class of RiPPs that possess characteristic lanthionine and/or methyllanthionine thioether cross-links. The biosynthetic potentials of marine-derived lanthipeptides remain largely unexplored. In this study, we characterized three novel lanthipeptides pseudorosin A-C by heterologous expression of a class I lanthipeptide biosynthetic gene cluster from marine Pseudoalteromonas flavipulchra S16. Interestingly, pseudorosin C contains a large loop spanning 18 amino acid residues, which is rare in lanthipeptides. Unexpectedly, the dehydratase PsfB could catalyze the dethiolation of specific Cys residues in all three core peptides, thereby generating dehydroalanines in the absence of LanC cyclase. To the best of our knowledge, we identified the first member of the LanB dehydratase family to perform glutamylation and subsequent elimination on Cys thiol groups, which likely represents a new bypass for class I lanthipeptide biosynthesis. Furthermore, we employed mutagenesis to determine the important motif of the core peptide for dethiolation activity. Moreover, sequence analysis revealed that PsfB exhibited a distinct phylogenetic distance from the characterized LanBs from Gram-positive bacteria. Our findings, therefore, pave the way for further genome mining of lanthipeptides, novel post-translational modification enzymes from marine Gram-negative bacteria, and bioengineering applications.

Complete genome sequence of Pseudoalteromonas sp. PS1M3, a psychrotrophic bacterium isolated from deep-sea sediment off the Boso Peninsula, Japan Trench.

Herein, we report the complete genome sequence of Pseudoalteromonas sp. PS1M3 (= NCBI 87791), which is a psychrotrophic bacterium that inhabits in seabed off the Boso Peninsula, Japan Trench. Analysis of the genomic sequence revealed that PS1M3 possesses 2 circular chromosomal DNAs and 2 circular plasmid DNAs. The genome of PS1M3 had a total size of 4,351,630 bp, an average GC content of 39.9%, and contained a total of 3811 predicted protein coding sequences, 28 rRNAs, and 100 tRNAs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was utilized to annotate the genes and KofamKOALA within KEGG assigned a gene cluster involved in glycogen biosynthesis and metabolic pathways with regard to heavy metal resistance (copper; cop and mercury; mer), indicating that PS1M3 can potentially use a stored glycogen as an energy source under oligotrophic environment and cope with multi-heavy metal contamination. To assess available genome relatedness indices, whole-genome average nucleotide identity analysis was examined using the complete genome sequences of Pseudoalteromonas spp., showing that 67.29-97.40% sequence similarity with PS1M3. This study may be useful in understanding the roles of a psychrotrophic Pseudoalteromonas in cold deep-sea sediment adaptation mechanisms.

Simultaneous enhancement of thermostability and catalytic activity of κ-carrageenase from Pseudoalteromonas tetraodonis by rational design.

κ-Carrageenase provides an attractive enzymatic approach to preparation of κ-carrageenan oligosaccharides. Pseudoalteromonas tetraodonis κ-carrageenase is active at the alkaline conditions but displays low thermostability. To further improve its enzymatic performance, two mutants of Q42V and I51H exhibiting both improved thermostability and enzyme activity were screened by the PoPMuSiC algorithm. Compared with the wild-type κ-carrageenase (WT), Q42V and I51H increased the enzyme activity by 20.9% and 25.4%, respectively. After treatment at 50 â„ƒ for 40 min, Q42V and I51H enhanced the residual activity by 31.1% and 25.9%, respectively. The Tm values of Q42V, I51H, and WT determined by differential scanning calorimetry were 58.2 â„ƒ, 54.8 â„ƒ, and 51.2 â„ƒ, respectively. Compared with untreated and HCl-treated κ-carrageenans, Q42V-treated κ-carrageenan exhibited higher pancreatic lipase inhibitory activity. Molecular docking analysis indicated that the additional pi-sigma force and hydrophobic interaction in the enzyme-substrate complex could account for the increased catalytic activity of Q42V and I51H, respectively. Molecular dynamics analysis indicated that the improved thermostability of mutants Q42V and I51H could be attributed to the less structural deviation and the flexible changes of enzyme conformation at high temperature. This study provides new insight into κ-carrageenase performance improvement and identifies good candidates for their industrial applications.

Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125.

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.

Computational Insight into Intraspecies Distinctions in Pseudoalteromonas distincta: Carotenoid-like Synthesis Traits and Genomic Heterogeneity.

Advances in the computational annotation of genomes and the predictive potential of current metabolic models, based on more than thousands of experimental phenotypes, allow them to be applied to identify the diversity of metabolic pathways at the level of ecophysiology differentiation within taxa and to predict phenotypes, secondary metabolites, host-associated interactions, survivability, and biochemical productivity under proposed environmental conditions. The significantly distinctive phenotypes of members of the marine bacterial species Pseudoalteromonas distincta and an inability to use common molecular markers make their identification within the genus Pseudoalteromonas and prediction of their biotechnology potential impossible without genome-scale analysis and metabolic reconstruction. A new strain, KMM 6257, of a carotenoid-like phenotype, isolated from a deep-habituating starfish, emended the description of P. distincta, particularly in the temperature growth range from 4 to 37 °C. The taxonomic status of all available closely related species was elucidated by phylogenomics. P. distincta possesses putative methylerythritol phosphate pathway II and 4,4'-diapolycopenedioate biosynthesis, related to C30 carotenoids, and their functional analogues, aryl polyene biosynthetic gene clusters (BGC). However, the yellow-orange pigmentation phenotypes in some strains coincide with the presence of a hybrid BGC encoding for aryl polyene esterified with resorcinol. The alginate degradation and glycosylated immunosuppressant production, similar to brasilicardin, streptorubin, and nucleocidines, are the common predicted features. Starch, agar, carrageenan, xylose, lignin-derived compound degradation, polysaccharide, folate, and cobalamin biosynthesis are all strain-specific.

Identification and Regulatory Roles of a New Csr Small RNA from Arctic Pseudoalteromonas fuliginea BSW20308 in Temperature Responses.

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 (ΔPf1) 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 ΔPf1 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.

Stringent Starvation Protein SspA and Iron Starvation Sigma Factor PvdS Coordinately Regulate Iron Uptake and Prodiginine Biosynthesis in Pseudoalteromonas sp. R3.

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.

Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

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.

Mobile genetic elements used by competing coral microbial populations increase genomic plasticity.

Intraspecies diversification and niche adaptation by members of the Vibrio genus, one of the most diverse bacterial genera, is thought to be driven by horizontal gene transfer. However, the intrinsic driving force of Vibrio species diversification is much less explored. Here, by studying two dominant and competing cohabitants of the gastric cavity of corals, we found that a phenotype influencing island (named VPII) in Vibrio alginolyticus was eliminated upon coculturing with Pseudoalteromonas. The loss of VPII reduced the biofilm formation and phage resistance, but activated motility, which may allow V. alginolyticus to expand to other niches. Mechanistically, we discovered that the excision of this island is mediated by the cooperation of two unrelated mobile genetic elements harbored in Pseudoalteromonas spp., an integrative and conjugative element (ICE) and a mobilizable genomic island (MGI). More importantly, these mobile genetic elements are widespread in cohabitating Gram-negative bacteria. Altogether, we discovered a new strategy by which the mobilome is employed by competitors to increase the genomic plasticity of rivals.

Isolation and Complete Sequence of One Novel Marine Bacteriophage PHS21 Infecting Pseudoalteromonas marina.

PHS21 against Pseudoalteromonas is isolated from Qingdao offshore seawater. The phage was characterized and identified by morphological examination, stability, whole genome sequencing, and bioinformatics analysis. Morphological analysis of PHS21 by transmission electron microscopy shows that belonged to the Siphoviridae family. PHS21 showed strong tolerance with a wide range of temperatures and pH. One-step growth assay indicates that the latent period is about 48 min and the burst size is approximately 218 PFU/cell (plaque forming unit/cell). Its complete genomic sequence is 35,802-bp long with 50 putative open reading frames. Phage PHS21 and PHS3 displayed a very close evolutionary relationship; however, having different DNA packaging proteins indicates that they may have already evolved distinct ways to package DNA in host cells. This study provides the detailed description and genomic characterization of a novel Pseudoalteromonas phage.

Novel Salt-Tolerant Leucine Dehydrogenase from Marine Pseudoalteromonas rubra DSM 6842.

This study reported the cloning, expression, and characterization of a new salt-tolerant leucine dehydrogenase (PrLeuDH) from Pseudoalteromonas rubra DSM 6842. A codon-optimized 1038 bp gene encoding PrLeuDH was successfully expressed on pET-22b( +) in E. coli BL21(DE3). The purified recombinant PrLeuDH showed a single band of about 38.7 kDa on SDS-PAGE. It exhibited the maximum activity at 40 °C and pH 10.5, while kept high activities in the range of 25-45 °C and pH 9.5-12. The Km value and turnover number kcat for leucine of PrLeuDH were 2.23 ± 0.12 mM and 35.39 ± 0.05 s-1, respectively, resulting in a catalytic efficiency kcat/Km of 15.87 s-1/mM. Importantly, PrLeuDH remained 92.1 ± 2.67% active in the presence of 4.0 M NaCl. The study provides the first in-depth understanding of LeuDH from marine Pseudoalteromonas rubra, meanwhile the unique properties of high activity at low temperature and high salt tolerance make it a promising biocatalyst for the synthesis of non-protein amino acids and α-ketoacids under special conditions in pharmaceutical industry.