Development of a Quorum Sensing-Mediated Bacterial Autolytic System in Escherichia coli for Automatic Release of Intracellular Products.

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.

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

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

Kinetic and dynamical properties of truncated hemoglobins of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

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.

Increased secretion of bacterial pyomelanin caused by light accelerates corrosion of low alloy steel.

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.

Optimization of Culture Medium for the Production of an Exopolysaccharide (p-CY02) with Cryoprotective Activity by Pseudoalteromonas sp. RosPo-2 from the Antarctic Sea.

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.

Vanadium Accumulation and Reduction by Vanadium-Accumulating Bacteria Isolated from the Intestinal Contents of Ciona robusta.

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.

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.

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.

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.

Characterization of the exopolymer-producing Pseudoalteromonas sp. S8-8 from Antarctic sediment.

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.

Pan-Genomic and Transcriptomic Analyses of Marine Pseudoalteromonas agarivorans Hao 2018 Revealed Its Genomic and Metabolic Features.

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.

Effect of Different Initial Fermentation pH on Exopolysaccharides Produced by Pseudoalteromonas agarivorans Hao 2018 and Identification of Key Genes Involved in Exopolysaccharide Synthesis via Transcriptome Analysis.

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.

Biochemical Characterization and Cold-Adaption Mechanism of a PL-17 Family Alginate Lyase Aly23 from Marine Bacterium Pseudoalteromonas sp. ASY5 and Its Application for Oligosaccharides Production.

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 ß-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.

Alterins, a new family of marine antibacterial cyclolipopeptides.

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.

Soluble Recombinant Protein Production in Pseudoalteromonas haloplanktis TAC125: The Case Study of the Full-Length Human CDKL5 Protein.

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.

d-Alanine Metabolism via d-Ala Aminotransferase by a Marine Gammaproteobacterium, Pseudoalteromonas sp. Strain CF6-2.

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.

Identification and Verification of the Prodigiosin Biosynthetic Gene Cluster (BGC) in Pseudoalteromonas rubra S4059.

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.

Reduction of mussel metamorphosis by inactivation of the bacterial thioesterase gene via alteration of the fatty acid composition.

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 ΔtesA 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 ΔtesA biofilms promoted the biofilm-inducing capacity. This study suggests that the bacterial thioesterase gene tesA altered the fatty acid composition of ΔtesA P. marina biofilms (BF) through regulation of its c-di-GMP, subsequently impacting mussel metamorphosis.