Pseudoalteromonas Lipolytica Research Articles

A point mutation in a wspF-like gene in Pseudoalteromonas lipolytica enhances the anticorrosion activity.

The protection of steel based on microbial biomineralization has emerged as a novel and eco-friendly strategy for corrosion control. However, the molecular basis of the biomineralization process in mineralization bacteria remains largely unexplored. We previously reported that Pseudoalteromonas lipolytica EPS+ strain provides protection against steel corrosion by forming a hybrid biomineralization film. In this study, we identified that a point mutation in the AT00_08765 (wspF-like) gene, responsible for encoding a chemotaxis protein that regulates swimming motility and polysaccharide production, is linked to the observed anticorrosion activity in EPS+ strain. The engineered point mutation mutant strain, designated Δ08765(707A), exhibited similar phenotypes to the EPS+ strain, including colony morphology and cellulose production. Importantly, we demonstrated that moderate swimming motility in Δ08765(707A) plays a pivotal role in the development of a protective mineralization film on the steel surface. Additionally, we found that Δ08765(707A) enhances biofilm formation by rapidly forming small aggregates in the initial stage of biofilm growth. This process facilitated the assembly of more compact and larger mineralization products, effectively inhibiting steel corrosion. In addition, Δ08765(707A) formed a uniform mineralization film that completely covered the steel surface, preventing the formation of sheet-like steel corrosion products. Therefore, this study demonstrates that an engineering strain carrying a point mutation in the AT00_08765 gene can significantly enhance the anticorrosion activity. This enhancement is accomplished through the formation of small bacteria-induced aggregates, followed by the development of larger mineralization products and the creation of a uniform organic-inorganic hybrid film.IMPORTANCEIn this study, we revealed that moderate swimming motility significantly influences the anticorrosion activity of marine Pseudoalteromonas. Furthermore, our study demonstrated that overproduction of cellulose facilitates cell aggregation rapidly during the initial stages of biofilm formation, thereby promoting the development of larger mineralization products and the formation of a uniform organic-inorganic hybrid film on the steel surface. Our findings provide new insights into the biomineralization mechanisms in Pseudoalteromonas lipolytica, potentially catalyzing the advancement of an eco-friendly microbial-driven approach to marine corrosion prevention.

Enhanced production, artificial intelligence optimized three-phase partitioning extraction, and in silico characterization of extracellular neutral Bacillus cereus proteinase

The present study applied atmospheric and room temperature plasma (ARTP) mutagenesis to improve Bacillus cereus strain for neutral proteinase production. The selected improved stable strain, Phe−Tyr-ARTP-60-E demonstrated a 2.88-fold enhanced proteinase yield and 2.81-fold enhanced proteinase activity with shortened fermentation time. An artificial neural network-embedded genetic algorithm (ANN-GA) was employed to optimize the enzyme's three-phase partitioning (TPP) recovery using Python software. The optimized solution of the hybrid model recommended 38.05% wv−1 (NH4)2SO4, 1.0:2.0 crude extract/tert-butanol ratio, pH 5.84 at 23.4 °C, for the exclusive partitioning of proteinase in the intermediate phase, with 202% recovery, 13-fold purity with specific activity of 922 Umg−1. Sensitivity analysis of the model revealed that enzyme recovery was most sensitive to crude extract/tert-butanol ratio with total sensitivity (ST) of 0.253 while purification factor was most sensitive to pH with an ST of 0.444. In silico analysis using Expasy ProtParam tool revealed that mutant proteinase had 3 amino acid substitutions; Val-to-Cys; Val-to-His and Ile-to-Lys, at positions 152, 288, and 292, respectively, and 39.2 kDa molecular weight, 6.42 isoelectric point, and 33.35 instability index. The 3-Dimensional structure of the enzyme predicted by ModWeb v r273 Server revealed 39% sequence identity with Pseudoalteromonas lipolytica thermolysin. The macromolecule increased transmittance in lysozyme-coated contact lenses by 99.6% in 45 min. We conclude that ARTP mutagenesis and ANN-GA optimized TPP are effective and efficient recalcitrant strain improvement and product recovery methods, respectively. Further genetic tools and analysis of the neutral proteinase-producing gene may be required for increased improvement toward sustainable application.

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.

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.

Corrosion mechanism of copper in seawater containing the bacterial pyomelanin with redox activity

Copper has bactericidal properties, which can effectively prevent the adhesion of biofilms and thus avoid microbiologically influenced corrosion. In this study, however, pigmented Pseudoalteromonas lipolytica strain producing pyomelanin can accelerate the corrosion of copper even after the bacteria are destroyed by copper ions. Moreover, uniform and pit corrosion were most serious in an abiotic pyomelanin solution. Surface characterization and electrochemical analysis indicated that pyomelanin was the key corrosive factor, owing to its redox cycling properties. The pyomelanin may participate in the oxidation of Cu to Cu (Ⅰ). This study introduces a new perspective for inhibiting MIC of copper.

Impact of phosphate concentration on the metabolome of biofilms of the marine bacterium Pseudoalteromonas lipolytica.

Marine biofilms are the most widely distributed mode of life on Earth and drive biogeochemical cycling processes of most elements. Phosphorus (P) is essential for many biological processes such as energy transfer mechanisms, biological information storage and membrane integrity. Our aim was to analyze the effect of a gradient of ecologically relevant phosphate concentrations on the biofilm-forming capacity and the metabolome of the marine bacterium Pseudoalteromonas lipolytica TC8. In addition to the evaluation of the effect of different phosphate concentration on the biomass, structure and gross biochemical composition of biofilms of P. lipolytica TC8, untargeted metabolomics based on liquid chromatography-mass spectrometry (LC-MS) analysis was used to determine the main metabolites impacted by P-limiting conditions. Annotation of the most discriminating and statistically robust metabolites was performed through the concomitant use of molecular networking and MS/MS fragmentation pattern interpretation. At the lowest phosphate concentration, biomass, carbohydrate content and three-dimensional structures of biofilms tended to decrease. Furthermore, untargeted metabolomics allowed for the discrimination of the biofilm samples obtained at the five phosphate concentrations and the highlighting of a panel of metabolites mainly implied in such a discrimination. A large part of the metabolites of the resulting dataset were then putatively annotated. Ornithine lipids were found in increasing quantity when the phosphate concentration decreased, while the opposite trend was observed for oxidized phosphatidylethanolamines (PEs). This study demonstrated the suitability of LC-MS-based untargeted metabolomics for evaluating the effect of culture conditions on marine bacterial biofilms. More precisely, these results supported the high plasticity of the membrane of P. lipolytica TC8, while the role of the oxidized PEs remains to be clarified.

Enhanced corrosion protection action of biofilms based on endogenous and exogenous bacterial cellulose

The use of biomineralized films to protect steel surfaces is limited by the strict selection of bacteria capable of mineralization. Inspired by the different effects of the Pseudoalteromonas lipolytica mutants on steel corrosion, we found that bacterial cellulose (BC) plays an important role in corrosion protection. The endogenous cellulose was beneficial to the formation of uniform biofilms, while exogenous BC provided a stable anticorrosion activity through biomineralization. Moreover, the addition of BC extracted from the P. lipolytica ∆17125 strain into a culture medium with corrosive P. lipolytica ∆bcs or Vibrio natriegens resulted in systems with significant corrosion-inhibition abilities.

Genomic insights into Pseudoalteromonas sp. JSTW coping with petroleum-heavy metals combined pollution.

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 Cu2+ and 67% with 400 mg L-1 Cu2+ . 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.

Identification of lead (Pb(CH3COO)2)-reducing bacteria using 16S rRNA Gene

In recent years bacteria have been used as bioremediation agents to repair polluted environments. The purpose of this research is to discover the types and capabilities of bacteria in reducing lead (Pb(CH3COO)2) concentration from sediments in Karangsong Coast, Indramayu. The method used is exploratory, and the results are analyzed descriptively. In the molecular identification method, 16S rRNA Gene, a universal primer for a bacterium, is used. Bacterial sequences obtained from GenBank data are analyzed by NCBI sites. The results of this research show that 11 bacterial isolates from porewater sediments have the highest lead ( Pb(CH3COO)2) reduction capability from a concentration of 1.5 mg l−1 to 0.364 mg l−1 with an efficiency value of 75.7%. Four bacterial isolates with the highest density are identical to Albirhodobacter marinus strain N9 (ACC. No. NR 126203.1) with 98% identity value and 100% genetic relationship, Pseudoalteromonas tetraodonis strain NBRC 103034 (ACC. No. NR 114187.1) with 81% identity and 84% genetic relationship, Pseudoalteromonas lipolytica strain LMEB 39 (ACC. No. NR 116629.1) with 98% identity and 96% genetic relationship, Pseudoalteromonas lipolytica strain LMEB 39 (ACC. No. NR 116629.1) 93% identity and 87% genetic relationship.

Marine bacteria inhibit corrosion of steel via synergistic biomineralization

Metal corrosion often results in incalculable economic loss and significant safety hazards. Although numerous traditional methods have been used to mitigate the issue, such as coating and corrosion inhibitors, they are environmentally unfriendly and difficult to maintain. Therefore, in this study, an environmental approach was taken to protect steels from corrosion in a multi-species bacterial environment via synergistic biomineralization. The marine bacterium Pseudoalteromonas lipolytica mixed with Bacillus subtilis or Pseudomonas aeruginosa strains offered extraordinary corrosion protection for steel. The surface characterization and electrochemical tests showed that the biomineralized film generated by the mixed bacteria was more compact and protective than that induced by a single bacterium. Herein, we found that the synergistic mechanisms were rather different for the different bacterial groups. For Pseudoalteromonas lipolytica and Bacillus subtilis group, the related mechanisms were due to the increase of pH in the medium, secretion of carbonic anhydrase. As for Pseudoalteromonas lipolytica and Pseudomonas aeruginosa group, the synergistic mechanism was attributed to the inhibiting corrosive bacteria in biofilm by the growth advantage of Pseudoalteromonas lipolytica. Therefore, this study may introduce a new perspective for future use of biomineralization in a real marine environment.

Unveiling the CAZyme repertoire of a sponge-derived Pseudoalteromonas sp. strain for the degradation of marine polysaccharides

Despite their well‐known importance to the holobiont homeostasis, carbohydrate‐active enzymes (CAZymes) from the sponge microbiome have to date been largely understudied from an industrial perspective. Enzymes that degrade marine polysaccharides (MPs) are receiving increasing attention as alternatives in the biorefinery sector and for use in the generation of high‐value‐added hydrolysis products. Screening of the agarolytic Pseudoalteromonas sp. strain PA2MD11, isolated from the Brazilian sponge Plakina cyanorosea, on selective media indicated that the strain degrades agarose, κ‐carrageenan and sodium alginate. Subsequently in silico analysis of the strain's genome indicated that around 3 % of the total genome encoded for CAZymes. These consisted mostly of glycoside hydrolases (GH) and glycosyltransferases (GT). Four agarase genes were also present, three from the family GH50 and one from the family GH16, with sequences from other free‐living and psychrophile Pseudoalteromonas genomes as its closest relatives. These agarases are structurally related to exo‐(GH50) and endo‐β‐agarases (GH16), and appear to encode for a complete agarolytic pathway. The two alginate lyases were classified as polysaccharide lyases (PL) from the families 6 and 17, with chondroitin‐ and heparin‐like domains, respectively, and shared common homologues sequences with an estuarine Pseudoalteromonas lipolytica strain. Data on the cloning and heterologous expression of these genes will be presented. To our knowledge, this is the first time that a “genome mining” based approach has been undertaken to identify these groups of MPs‐degrading CAZymes from cultivable members of the sponge microbiome.

Electrochemical Investigations of Microbiologically Influenced Corrosion on 316L-Cu Stainless Steel by Pseudoalteromonas lipolytica

Microbiologically influenced corrosion (MIC) is becoming more often a risk factor in several industries, such as offshore, cooling water systems and construction. The Pseudoalteromonas lipolytica (P. lipolytica) is a highly corrosive aerobic marine bacterium. In this work, the 316L-Cu SS was investigated the corrosion behavior under the abiotic medium and biotic medium of P. lipolytica. Based on the electrochemical analysis, the corrosion current density (icorr) under the biotic medium is higher than that under the abiotic medium. The presence of P. lipolytica has led to the dissolution of the passive film of 316L-Cu SS, which initiates the localized pitting corrosion. However, the addition of Cu in 316L SS by melting in the vacuum induction furnace improved more corrosion-resistant than without the addition of Cu.

Pseudoalteromonas lipolytica accelerated corrosion of low alloy steel by the endogenous electron mediator pyomelanin

Endogenous electron mediators are believed to be important bridges between microorganisms and electrodes, which influence the corrosion process. In this work, the genetically engineered P. lipolytica mutant strain with hmgA gene deletion, a hmgA restoration strain and a wild-type strain were used to investigate the impact of the pyomelanin on the corrosion of steel in marine broth. Weight loss and electrochemical data demonstrated that the corrosion rate of the steel increased when the hmgA deletion strain of P. lipolytica was inoculated in marine broth to produce pyomelanin, especially with the addition of L-tyrosine as a substrate.

Biofilm formation in marine bacteria and biocidal sensitivity: interplay between a potent antibiofilm compound (AS162) and quorum-sensing autoinducers.

The capacity of two homoserine lactones to stimulate the marine bacteria Pseudoalteromonas ulvae (TC14 strain) for its capacity to form a biofilm when exposed to a potent antibiofilm compound AS162 is reported. Effective concentrations (EC50) of AS162 at 24h, 48h, and 72h were, respectively, of 4.3, 4.4, and 6.0µM. When tested in combination with HSLs, results showed that quorum-sensing signal molecules 3-oxo-C6 and 3-oxo-C8 homoserine lactones do not act directly on the biofilm formation, but are able to interfere positively with AS162 to promote biofilm growth with EC50 ranging from 30 to 50µM. The same results were obtained with two other marine bacterial strains: Pseudoalteromonas lipolytica TC8 and Paracoccus sp. 4M6. These findings suggest that HSLs can significantly affect the biocidal sensitivity of marine bacteria to antifouling agents.

Molybdenum-mediated chemotaxis of Pseudoalteromonas lipolytica enhances biofilm-induced mineralization on low alloy steel surface

Formation of biofilm and the subsequent biomineralization on the steel surface are highly influenced by the steel surface properties. Molybdenum (Mo) is a commonly used metal in steel, which can improve its strength and resistance to corrosion. Herein, the effect of Mo on the biomineralization process was investigated by designing low alloy steels containing different concentrations of Mo. Surface characterization showed that moderate concentrations of Mo (such as 0.6 wt%) significantly enhanced the biofilm formation and the mineralization process. Mechanistically, it is demonstrated that Mo ions served as a chemical attractant for Pseudoalteromonas lipolytica and activated the chemotaxis pathway.

Biofilm formation in Pseudoalteromonas lipolytica is related to IS5-like insertions in the capsular polysaccharide operon.

Bacterial capsular polysaccharides (CPSs) participate in environmental adaptation in diverse bacteria species. However, the role and regulation of CPS production in marine bacteria have remained largely unexplored. We previously reported that both wrinkled and translucent Pseudoalteromonas lipolytica variants with altered polysaccharide production were generated in pellicle biofilm-associated cells. In this study, we observed that translucent variants were generated at a rate of ∼20% in colony biofilms of P. lipolytica cultured on HSLB agar plates for 12 days. The DNA sequencing results revealed that nearly 90% of these variants had an IS5-like element inserted within the coding or promoter regions of nine genes in the cps operon. In contrast, IS5 insertion into the cps operon was not detected in planktonic cells. Furthermore, we demonstrated that the IS5 insertion event inactivated CPS production, which leads to a translucent colony morphology. The CPS-deficient variants showed an increased ability to form attached biofilms but exhibited reduced resistance to sublethal concentrations of antibiotics. Moreover, deleting the DNA repair gene recA significantly decreased the frequency of occurrence of CPS-deficient variants during biofilm formation. Thus, IS insertion into the cps operon is an important mechanism for the production of genetic variants during biofilm formation of marine bacteria.

Metabolomic and proteomic changes induced by growth inhibitory concentrations of copper in the biofilm-forming marine bacterium Pseudoalteromonas lipolytica.

Copper is an essential element for living cells but this metal is present in some marine environments at such high concentrations that it can be toxic for numerous organisms. In polluted areas, marine organisms may develop specific adaptive responses to prevent cell damage. To investigate the influence of copper on the metabolism of a single organism, a dual approach combining metabolomics and proteomics was undertaken on the biofilm-forming bacterial strain Pseudoalteromonas lipolytica TC8. In order to highlight differential adaptation according to the phenotype, the response of P. lipolytica TC8 to copper stress was studied in planktonic and biofilm culture modes under growth inhibitory copper concentrations. As expected, copper exposure led to the induction of defense and detoxification mechanisms. Specific metabolite and protein profiles were thus observed in each condition (planktonic vs. biofilm and control vs. copper-treated cultures). Copper exposure seems to induce drastic changes in the lipid composition of the bacterial cell membrane and to modulate the abundance of proteins functionally known to be involved in copper cell homeostasis in both planktonic and biofilm culture modes. Much more proteins differentially expressed after copper treatment were observed in biofilms than in planktonic cells, which could indicate a more heterogeneous response of biofilm cells to this metallic stress.

A bifunctional melamine sponge decorated with silver-reduced graphene oxide nanocomposite for oil-water separation and antibacterial applications

Modifying the commercial melamine sponge (MS) that intrinsically possesses highly porous open-cell structure with graphene oxide (GO) or GO-based nanocomposites (NCs) is a facile, effective, and low-cost route to fabricate three-dimensional (3D) porous materials with multifunctionality. Herein, we fabricated two types of 3D MSs that were separately functionalized by the reduced GO (RGO) sheets and Ag/RGO NC through combined methods of chemical reduction and immersion process. The simple immersion process brought out the transition of superhydrophilic MS to highly hydrophobic RGO-MS and Ag/RGO-MS. Due to the unique surface topography and high porosity, the RGO-MS exhibited remarkable absorption capacity of 41–91 times its own weight for a wide range of oils and organic solvents. Both the RGO-MS and Ag/RGO-MS showed excellent oil-water separation efficiencies. The Ag/RGO-MS not only exhibited outstanding absorption capacity and recyclability, but also possessed ideal antibacterial performance towards bacteria of Staphylococcus sciuri, Shewanella MR-1, Pseudoalteromonas lipolytica, and Vibrio natriegens. The Ag/RGO-MS featured with hydrophobicity and antibacterial activity is a promising candidate for potential applications in oil-spill disposal and water disinfection. This study provides an effective strategy for designing and fabricating the 3D porous materials with novel functionalities.

Marine Bacteria Provide Lasting Anticorrosion Activity for Steel via Biofilm-Induced Mineralization

Steel corrosion is a global problem in marine engineering. Numerous inhibitory treatments have been applied to mitigate the degradation of metallic materials; however, they typically have a high cost and are not environmental friendly. Here, we present a novel and "green" approach for the protection of steel by a marine bacterium Pseudoalteromonas lipolytica. This approach protects steel from corrosion in seawater via the formation of a biofilm followed by the formation of an organic-inorganic hybrid film. The hybrid film is composed of multiple layers of calcite and bacterial extracellular polymeric substances, exhibiting high and stable barrier protection efficiency and further providing an in situ self-healing activity. The process involving the key transition from biofilm to biomineralized film is essential for its lasting anticorrosion activity, which overcomes the instability of biofilm protection on corrosion. Therefore, this study introduces a new perspective and an option for anticorrosion control in marine environments.

Anti-Bacterial Adhesion Activity of Tropical Microalgae Extracts.

The evolution of regulations concerning biocidal products aimed towards an increased protection of the environment (e.g., EU Regulation No 528/2012) requires the development of new non-toxic anti-fouling (AF) systems. As the marine environment is an important source of inspiration, such AF systems inhibiting the adhesion of organisms without any toxicity could be based on molecules of natural origin. In this context, the antibiofilm potential of tropical microalgal extracts was investigated. The tropics are particularly interesting in terms of solar energy and temperatures which provide a wide marine diversity and a high production of microalgae. Twenty microalgal strains isolated from the Indian Ocean were studied. Their extracts were characterized in terms of global chemical composition by high resolution magic angle spinning (HR-MAS) and nuclear magnetic resonance (NMR) spectroscopy, toxicity against marine bacteria (viability and growth) and anti-adhesion effect. The different observations made by confocal laser scanning microscopy (CLSM) showed a significant activity of three extracts from Dinoflagellate strains against the settlement of selected marine bacteria without any toxicity at a concentration of 50 μg/mL. The Symbiodinium sp. (P-78) extract inhibited the adhesion of Bacillus sp. 4J6 (Atlantic Ocean), Shewanella sp. MVV1 (Indian Ocean) and Pseudoalteromonas lipolytica TC8 (Mediterranean Ocean) at 60, 76 and 52%, respectively. These results underlined the potential of using microalgal extracts to repel fouling organisms.