Pseudoalteromonas Lipolytica Research Articles

Peptide stabilized gold and silver nanoparticles derived from the mangrove isolate Pseudoalteromonas lipolytica mediate dye decolorization

A mangrove associated halotolerant bacterium identified as Pseudoalteromonas lipolytica mediated the synthesis of gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs). Interaction of P. lipolytica cells with chloroauric acid (HAuCl4) resulted in the synthesis of cell associated AuNPs that could be released in an extracellular manner after sonication. For obtaining extracellular AgNPs, pre-grown cells were subjected to osmotic shock treatment by re-suspending cells in distilled water. The preparation was centrifuged and the filtered supernatant was challenged with 4 mM AgNO3. The biogenic NPs were characterized on the basis of UV–vis spectroscopy, Field Emission Scanning Electron Microscope observations, Transmission Electron Microscope studies, X-Ray Diffraction profiles and Energy-Dispersive X-ray spectroscopy. The size of AuNPs and AgNPs ranged between 10–30 nm and 5–15 nm, respectively. The active components in the preparation after osmotic shock treatment were subjected to thin layer chromatography, Fourier Transform Infrared spectroscopy and liquid chromatography mass spectrometry (LCMS) analysis. Five oligopeptides, Ile-Arg, His-Asp-Asp, Glu-Ile-Lys, Lys-Asp-Asn, and Arg-Arg-Gln were identified in the active band. AuNPs brought about 91% decolorization of methylene blue and congo red dyes. With AgNPs, 92% of congo red was decolorized. The rate constants for methylene blue and congo red decolorization using AuNPs were 0.2545 and 0.1587 min−1, respectively. For congo red dye decolorization by AgNPs the rate constant was 0.1882 min−1. These peptide stabilized NPs could be used as effective catalysts for sodium borohydride mediated decolorization of methylene blue and congo red dyes.

Metabolome and proteome changes between biofilm and planktonic phenotypes of the marine bacterium Pseudoalteromonas lipolytica TC8

A number of bacteria adopt various lifestyles such as planktonic free-living or sessile biofilm stages. This enables their survival and development in a wide range of contrasting environments. With the aim of highlighting specific metabolic shifts between these phenotypes and to improve the overall understanding of marine bacterial adhesion, a dual metabolomics/proteomics approach was applied to planktonic and biofilm cultures of the marine bacterium Pseudoalteromonas lipolytica TC8. The liquid chromatography mass spectrometry (LC-MS) based metabolomics study indicated that membrane lipid composition was highly affected by the culture mode: phosphatidylethanolamine (PEs) derivatives were over-produced in sessile cultures while ornithine lipids (OLs) were more specifically synthesized in planktonic samples. In parallel, differences between proteomes revealed that peptidases, oxidases, transcription factors, membrane proteins and the enzymes involved in histidine biosynthesis were over-expressed in biofilms while proteins involved in heme production, nutrient assimilation, cell division and arginine/ornithine biosynthesis were specifically up-regulated in free-living cells.

ISOLATION AND SCREENING OF BIOSURFACTANT-PRODUCING MARINE BACTERIA FROM KUANTAN PORT, PAHANG, MALAYSIA

Biosurfactants play an important role in bioremediation of organic pollutants such as petroleum hydrocarbon. The unique properties of biosurfactants make them possible to be used in the remediation of hydrocarbon contaminated sites. Therefore, the existence of indigenous microorganisms that have the ability to consume petroleum hydrocarbon as carbon source and simultaneously produce biosurfactants in order to facilitate the hydrocarbon metabolism can be manipulated for bioremediation purposes. In this study, isolation and screening of potential biosurfactant-producing bacteria from two sampling points in Kuantan Port seawater were successfully done. Amongst the isolates, 4 out of 7 isolates from Point A were Gram negative bacteria and 2 out 5 isolates from Point B were Gram negative bacteria. The positive oxidase test resulted for all isolates from Point A and only B5 from Point B produced negative result. Catalase test conducted produced positive results on isolates from Point A (A3, A5, A6& A7) and Point B (B1, B2, B4 & B5).The highest percentage emulsification index measured belonged to isolate B4 and B5 which are 67%, thus make these isolates to be the most promising biosurfactant producers. Further identification by 16S rRNA gene found that isolates were closely related to Rhodococcus erythropolis (A1), Psedomonas stutzeri (A2), Pseudoalteromonas lipolytica (A3, A6 and B4), Vibrio brasiliensis (A4 and B2), Vibrio tubiashii (B1), Marinobacter salsuginis (A5), Labrenzia aggregate (A7), Marinococcus halophilus (B3) and Thalassospira xianmenensis(B5). Hence, through biosurfactant activities exhibited by isolates, B4 and B5 were the most potential isolates to produce biosurfactant. Therefore, these isolates can potentially be exploited to aid in bioremediation of petroleum hydrocarbon contaminated sites and would also be useful to enhance oil recovery in petroleum industry.

Pyomelanin from Pseudoalteromonas lipolytica reduces biofouling.

SummaryMembers of the marine bacterial genus Pseudoalteromonas are efficient producers of antifouling agents that exert inhibitory effects on the settlement of invertebrate larvae. The production of pigmented secondary metabolites by Pseudoalteromonas has been suggested to play a role in surface colonization. However, the physiological characteristics of the pigments produced by Pseudoalteromonas remain largely unknown. In this study, we identified and characterized a genetic variant that hyperproduces a dark‐brown pigment and was generated during Pseudoalteromonas lipolytica biofilm formation. Through whole‐genome resequencing combined with targeted gene deletion and complementation, we found that a point mutation within the hmgA gene, which encodes homogentisate 1,2‐dioxygenase, is solely responsible for the overproduction of the dark‐brown pigment pyomelanin. In P. lipolytica, inactivation of the hmgA gene led to the formation of extracellular pyomelanin and greatly reduced larval settlement and metamorphosis of the mussel Mytilus coruscus. Additionally, the extracted pyomelanin from the hmgA deletion mutant and the in vitro‐synthesized pyomelanin also reduced larval settlement and metamorphosis of M. coruscus, suggesting that extracellular pyomelanin released from marine Pseudoalteromonas biofilm can inhibit the settlement of fouling organisms.

Adhesion of Bacillus subtilis and Pseudoalteromonas lipolytica to steel in a seawater environment and their effects on corrosion

In a marine environment, Bacillus subtilis and Pseudoalteromonas lipolytica are commonly found in the biofilms adherent to low-alloy engineering steel, and they have distinct effects on corrosion. In the present work, this phenomenon was investigated through the study of various materials characterization methods, electrochemical techniques, and contact angle measurements. It was found that the surface film formed on the steel in the presence of B. subtilis was compact, uniform, free of cracks, and hydrophobic. However, the film formed in the presence of P. lipolytica was loose, rough, heterogeneous, and hydrophilic. The main components of the films formed in the presence of B. subtilis and P. lipolytica were polysaccharides/TasA amyloid fibers and proteins/carboxylic acid, respectively. The composition, structure, and properties of the surface films formed on the steel were associated with different effects on corrosion. The presence of B. subtilis enhances the steel’s resistance to corrosion, whereas corrosion was increased by the presence of P. lipolytica. In short, the compact and hydrophobic biofilm of B. subtilis appears to inhibit the corrosion of steel, while the loose, hydrophilic film of P. lipolytica tends to induce pitting corrosion.

Isolation and Complete Genome Sequence of a Novel Pseudoalteromonas Phage PH357 from the Yangtze River Estuary.

Phage PH357, a novel lytic Pseudoalteromonas lipolytica phage belonging to the Myoviridae family was isolated from the Yangtze River estuary. The microbiological characterization demonstrated that phage PH357 is stable from -20 to 60°C and the optimal pH 7. The one-step growth curve showed a latent period of 20min, a rise period of 20min, and the average burst size was about 85 virions per cell. Complete genome of phage PH357 was determined. Genome of phage PH357 consisted of a linear, double-stranded 136,203bp DNA molecule with 34.58% G+C content, and 242 putative open reading frames (ORFs) without tRNA. All the predicted ORFs were classified into eight functional groups, including DNA replication, regulation and nucleotide metabolism, transcription, translation, phage packaging, phage structure, lysis, host or phage interactions, and hypothetical protein. A phylogenetic analysis showed that phage PH357 had similarity to the previously published Pseudoalteromonas phage PH101 and Vibrio phages. Furthermore, the study of phage PH357 genome will provide useful information for further research on the interaction between phages and their hosts.

Discrimination of Four Marine Biofilm-Forming Bacteria by LC-MS Metabolomics and Influence of Culture Parameters.

Most marine bacteria can form biofilms, and they are the main components of biofilms observed on marine surfaces. Biofilms constitute a widespread life strategy, as growing in such structures offers many important biological benefits. The molecular compounds expressed in biofilms and, more generally, the metabolomes of marine bacteria remain poorly studied. In this context, a nontargeted LC-MS metabolomics approach of marine biofilm-forming bacterial strains was developed. Four marine bacteria, Persicivirga (Nonlabens) mediterranea TC4 and TC7, Pseudoalteromonas lipolytica TC8, and Shewanella sp. TC11, were used as model organisms. The main objective was to search for some strain-specific bacterial metabolites and to determine how culture parameters (culture medium, growth phase, and mode of culture) may affect the cellular metabolism of each strain and thus the global interstrain metabolic discrimination. LC-MS profiling and statistical partial least-squares discriminant analyses showed that the four strains could be differentiated at the species level whatever the medium, the growth phase, or the mode of culture (planktonic vs biofilm). A MS/MS molecular network was subsequently built and allowed the identification of putative bacterial biomarkers. TC8 was discriminated by a series of ornithine lipids, while the P. mediterranea strains produced hydroxylated ornithine and glycine lipids. Among the P. mediterranea strains, TC7 extracts were distinguished by the occurrence of diamine derivatives, such as putrescine amides.

Cloning, expression and characterization of a lipase gene from marine bacterium Pseudoalteromonas lipolytica SCSIO 04301

A lipase gene, lip1233, isolated from Pseudoalteromonas lipolytica SCSIO 04301, was cloned and expressed in E. coli. The enzyme comprised 810 amino acid residues with a deduced molecular weight of 80 kDa. Lip1233 was grouped into the lipase family X because it contained a highly conserved motif GHSLG. The recombinant enzyme was purified with Ni-NTA affinity chromatography. The optimal temperature and pH value of Lip1233 were 45°C and 8.0, respectively. It retained more than 70% of original activity after being incubated in pH ranging from 6.0 to 9.5 for 30 min. It was stable when the temperature was below 45°C, but was unstable when the temperature was above 55°C. Most metal ions tested had no significant effect on the activity of Lip1233. Lip1233 remained more than original activity in some organic solvents at the concentration of 30% (v/v). It retained more than 30% activity after incubated in pure organic solvents for 12 h, while in hexane the activity was nearly 100%. Additionally, Lip1233 exhibited typical halotolerant characteristic as it was active under 4M NaCl. Lip1233 powder could catalyze efficiently the synthesis of fructose esters in hexane at 40°C. These characteristics demonstrated that Lip1233 is applicable to elaborate food processing and organic synthesis.

Pseudoalteromonas profundi sp. nov., isolated from a deep-sea seamount.

A Gram-stain-negative, rod-shaped, strictly aerobic, motile bacterial strain, designated TP162T, was isolated from a seamount near the Yap Trench in the tropical western Pacific. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain TP162T was related to the genus Pseudoalteromonas and had highest 16S rRNA gene sequence similarities with the type strains Pseudoalteromonas shioyasakiensis SE3T (98.2 %), Pseudoalteromonas lipolytica LMEB 39T (97.7 %), Pseudoalteromonas arabiensis k53T (97.4 %) and Pseudoalteromonas aliena KMM 3562T (97.2 %). The predominant cellular fatty acids were summed feature 3 (composed of iso-C15 : 0 2-OH and/or C16 : 1ω7c), C17 : 1ω8c and C16 : 0. The quinone system for strain TP162T comprised predominantly ubiquinone-8, and the polar lipid profile contained phosphatidylethanolamine, phosphatidylglycerol, one unidentified phospholipid and four unidentified lipids. The genomic DNA G+C content of strain TP162T was 46.7 mol%. Strain TP162T shared 28 % DNA-DNA relatedness with P.shioyasakiensis JCM 18891T, 21 % with P. lipolytica JCM 15903T, 35 % with P.arabiensis JCM 17292T and 18 % with P.aliena LMG 22059T. Combined data from phenotypic, chemotaxonomic, phylogenetic and DNA-DNA relatedness studies demonstrated that strain TP162T is a representative of a novel species of the genus Pseudoalteromonas, for which we propose the name Pseudoalteromonas profundi sp. nov. (type strain TP162T=KACC 18554T=CGMCC 1.15394T).

Screening of bromotyramine analogues as antifouling compounds against marine bacteria

Rapid and efficient synthesis of 23 analogues inspired by bromotyramine derivatives, marine natural products, by means of CuSO4-catalysed [3+2] alkyne–azide cycloaddition is described. The final target was then assayed for anti-biofilm activity against three Gram-negative marine bacteria, Pseudoalteromonas ulvae (TC14), Pseudoalteromonas lipolytica (TC8) and Paracoccus sp. (4M6). Most of the synthesised bromotyramine/triazole derivatives are more active than the parent natural products Moloka’iamine (A) and 3,5-dibromo-4-methoxy-β-phenethylamine (B) against biofilm formation by the three bacterial strains. Some of these compounds were shown to act as non-toxic inhibitors of biofilm development with EC50 < 200 μM without any effect on bacterial growth even at high concentrations (200 μM).

Characterization of self-generated variants in Pseudoalteromonas lipolytica biofilm with increased antifouling activities

Pseudoalteromonas is widespread in various marine environments, and most strains can affect invertebrate larval settlement and metamorphosis by forming biofilms. However, the impact and the molecular basis of population diversification occurring in Pseudoalteromonas biofilms are poorly understood. Here, we show that morphological diversification is prevalent in Pseudoalteromonas species during biofilm formation. Two types of genetic variants, wrinkled (frequency of 12 ± 5 %) and translucent (frequency of 5 ± 3 %), were found in Pseudoalteromonas lipolytica biofilms. The inducing activities of biofilms formed by the two variants on larval settlement and metamorphosis of the mussel Mytilus coruscus were significantly decreased, suggesting strong antifouling activities. Using whole-genome re-sequencing combined with genetic manipulation, two genes were identified to be responsible for the morphology alternations. A nonsense mutation in AT00_08765 led to a wrinkled morphology due to the overproduction of cellulose, whereas a point mutation in AT00_17125 led to a translucent morphology via a reduction in capsular polysaccharide production. Taken together, the results suggest that the microbial behavior on larval settlement and metamorphosis in marine environment could be affected by the self-generated variants generated during the formation of marine biofilms, thereby rendering potential application in biocontrol of marine biofouling.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6865-x) contains supplementary material, which is available to authorized users.

Pseudoalteromonas lipolytica sp. nov., isolated from the Yangtze River estuary

A strictly aerobic, Gram-negative, non-pigmented bacterial strain, designated LMEB 39(T), was isolated from a seawater sample collected from the Yangtze River estuary near the East China Sea and was examined physiologically, chemotaxonomically and phylogenetically. The novel isolate was motile by a single polar flagellum and positive for nitrate reduction and decomposition of casein, gelatin, Tween 20 and Tween 80, but negative for indole production. Chemotaxonomic analysis revealed ubiquinone-8 as the predominant respiratory quinone and phosphatidylethanolamine and phosphatidylglycerol as major polar lipids. The major fatty acids were C(16 : 1) ω 7c/iso-C(15 : 0) 2-OH, C(16 : 0), C(18 : 1) ω 7c, C(12 : 0) 3-OH, C(17 : 1)ω 8c and C(17 : 0). The genomic DNA G+C content was 42.3 mol%. Comparative 16S rRNA gene sequence analysis revealed that the isolate belongs to the genus Pseudoalteromonas. Strain LMEB 39(T) exhibited the closest phylogenetic affinity to Pseudoalteromonas byunsanensis JCM 12483(T) (97.4 % sequence similarity). The DNA-DNA reassociation values between strain LMEB 39(T) and P. byunsanensis JCM 12483(T) and Pseudoalteromonas undina DSM 6065(T) (97.2 % sequence similarity) were 31.7 and 30.3 %, respectively. On the basis of phenotypic and genotypic data, strain LMEB 39(T) represents a novel species of the genus Pseudoalteromonas, for which the name Pseudoalteromonas lipolytica sp. nov. is proposed; the type strain is LMEB 39(T) (=CGMCC 1.8499(T)=JCM 15903(T)).