Marine Pseudoalteromonas Sp Research Articles

Agarase Production by Marine Pseudoalteromonas sp. MHS: Optimization, and Purification

Agar is an essential polysaccharide that has been utilized in numerous fields. Many kinds of literature have been published regarding agarolytic microorganisms’ isolation and agarases biochemical studies. In this search, a local marine agarolytic bacterium associated with marine alga Ulva lactuca surface was isolated and identified as Pseudoalteromonas sp. MHS. The agarase production was parallel to the growth of Pseudoalteromonas sp. MHS as cells displayed a lag phase (2 h), subsequently an exponential growth that prolonged till 10 h where maximum growth (OD550nm = 3.9) was achieved. The enzyme activity increased rapidly as cells increased exponentially where the maximum activity of 0.22 U/mL was achieved after 8h and remained constant till 12 h during the stationary phase of growth. Agarase production was optimized using Plackett-Burman statistical design by measuring enzyme activity as a response and the design was validated using a verification experiment; the activity of the enzyme increased from 0.22 U/mL to 0.29 U/mL. Pseudoalteromonas sp. MHS agarase was partially purified and its molecular weight (MW) was determined by SDSPAGE (15-25 kDa). Agarase showed approximately 94% of its activity at 40 °C. The enzyme stability decreased as the temperature increased; the enzyme could retain about 98, 90, 80, 75, and 60% of its activity at 20, 30, 40, 50, and 60 °C, respectively. Biomass of the red alga Pterocladia capillacea proved to be a suitable substrate for agarase production using Pseudoalteromonas sp. MHS; the enzyme activity recorded after 24 h of incubation was 0.35 U/mL compared to 0.29 U/mL from the optimized medium.

A novel degradation mechanism of naphthenic acids by marine Pseudoalteromonas sp.

Naphthenic acids (NAs) are a persistent toxic organic pollutant that occur in different environment worldwide and cause serious threat to the ecosystem and public health. However, knowledge on the behavior and fate of NAs in marine environments still remains unknown. In this study, the degradation mechanism of NAs (cyclohexylacetic acid, CHAA) was investigated using an common indigenous marine Pseudoalteromonas sp. The results showed that CHAA could be degraded completely under aerobic condition, but could not be utilized directly under anaerobic condition. Interestingly, transcriptome and key enzyme activity results showed the CHAA degradation pathway induced under aerobic condition could still work in anaerobic condition. The degradation was activated by acetyl-CoA transferase and sequentially formed the corresponding cyclohexene, alcohol, and ketone with the assistance of related enzymes, and finally cleaved by hydroxymethylglutarate-CoA lyase. Besides, there was a positive correlation between chemotaxis and aerobic degradation genes (r = 0.976, P < 0.05), the chemotaxis would enhance bacterium movement and NAs biodegradation. It is proposed that bacterium could translocate to NAs and accomplish biodegradation from aerobic to anaerobic environments, which was a new anaerobic degradation pathway of NAs. This study provides new insights into the fate of NAs and other organic contaminants in marine environment.

Extracellular degradation of tetrabromobisphenol A via biogenic reactive oxygen species by a marine Pseudoalteromonas sp.

Tetrabromobisphenol A (TBBPA) has attracted considerable attention due to its ubiquitous presence in different environmental compartments worldwide. However, information on its aerobic biodegradability in coastal environments remains unknown. Here, the aerobic biodegradation of TBBPA using a Pseudoalteromonas species commonly found in the marine environment was investigated. We found that extracellular biogenic siderophore, superoxide anion radical (O2•−), hydrogen peroxide (H2O2), and hydroxyl radical (•OH) were involved in TBBPA degradation. Upregulation of genes (nqrA and lodA) encoding Na+-translocating NADH-quinone oxidoreductase and l-lysine-ε-oxidase supported the extracellular O2•− and H2O2 production. The underlying mechanism of TBBPA biodegradation presumably involves both O2•− reduction and •OH-based advanced oxidation process (AOP). Furthermore, TBBPA intermediates of tribromobisphenol A, 4-isopropylene-2,6-dibromophenol, 4-(2-hydroxyisopropyl)-2,6-dibromophenol, 2,4,6-tribromophenol (TBP), 4-hydroxybenzoic acid, and 2-bromobenzoic acid were detected in the culture medium. Debromination and β-scission pathways of TBBPA biodegradation were proposed. Additionally, membrane integrity assays revealed that the increase of intracellular catalase (CAT) activity and the extracellular polymeric substances (EPS) might account for the alleviation of oxidative damage. These findings could deepen understanding of the biodegradation mechanism of TBBPA and other related organic pollutants in coastal and artificial bioremediation systems.

Biosorption isotherm study of Cd2 +, Pb2 + and Zn2 + biosorption onto marine bacterium Pseudoalteromonas sp. SCSE709-6 in multiple systems

To overcome the low efficiency and prerequisites conditions of simultaneous metals removal and the unsuitability of single metal adsorption isotherm models in multiple metals, the multiple biosorption of Cd2+, Pb2+ and Zn2+ onto a new marine Pseudoalteromonas sp. SCSE709-6 from aqueous solution was studied and compared with single metal system. Results showed that Pseudoalteromonas sp. SCSE709-6 was a competitive biosorbent for metals from aqueous solutions in single system (maximum adsorption capacity: 1.38mmolg−1 for Cd2+, 1.05mmolg−1 for Pb2+ and 0.91mmolg−1 for Zn2+, respectively). The affinity between metals and biosorbent calculated by the separation factor in binary metals systems followed the order: Pb2+>Cd2+>Zn2+. In multiple metals systems, Cd2+ biosorption was the most sensitive, while Pb2+ biosorption was the least sensitive. In addition, Pb2+ biosorption had less pH dependence than the biosorption of Cd2+ and Zn2+. Among six isotherm models applied in this study, the extended Freundlich isotherm model best described the binary metal biosorption behaviors. Isotherm analysis revealed that binary biosorption was a complex process which occurred both mono- and multi-layer biosorption. FT-IR result spectrum revealed that carboxyl, amide, acyl and hydroxyl played a significant role in metals biosorption. This study showed superior performance of metal removal by Pseudoalteromonas sp. SCSE709-6 in multiple metals systems.

Characterization of a New Cold-Adapted and Salt-Activated Polysaccharide Lyase Family 7 Alginate Lyase from Pseudoalteromonas sp. SM0524.

Marine bacterial alginate lyases play a role in marine alginate degradation and carbon cycling. Although a large number of alginate lyases have been characterized, reports on alginate lyases with special characteristics are still rather less. Here, a gene alyPM encoding an alginate lyase of polysaccharide lyase family 7 (PL7) was cloned from marine Pseudoalteromonas sp. SM0524 and expressed in Escherichia coli. AlyPM shows 41% sequence identity to characterized alginate lyases, indicating that AlyPM is a new PL7 enzyme. The optimal pH for AlyPM activity was 8.5. AlyPM showed the highest activity at 30°C and remained 19% of the highest activity at 5°C. AlyPM was unstable at temperatures above 30°C and had a low Tm of 37°C. These data indicate that AlyPM is a cold-adapted enzyme. Moreover, AlyPM is a salt-activated enzyme. AlyPM activity in 0.5–1.2 M NaCl was sixfolds higher than that in 0 M NaCl, probably caused by a significant increase in substrate affinity, because the Km of AlyPM in 0.5 M NaCl decreased more than 20-folds than that in 0 M NaCl. AlyPM preferably degraded polymannuronate and mainly released dimers and trimers. These data indicate that AlyPM is a new PL7 endo-alginate lyase with special characteristics.

An Extra Peptide within the Catalytic Module of a β-Agarase Affects the Agarose Degradation Pattern

Agarase hydrolyzes agarose into a series of oligosaccharides with repeating disaccharide units. The glycoside hydrolase (GH) module of agarase is known to be responsible for its catalytic activity. However, variations in the composition of the GH module and its effects on enzymatic functions have been minimally elucidated. The agaG4 gene, cloned from the genome of the agarolytic Flammeovirga strain MY04, encodes a 503-amino acid protein, AgaG4. Compared with elucidated agarases, AgaG4 contains an extra peptide (Asn(246)-Gly(302)) within its GH module. Heterologously expressed AgaG4 (recombinant AgaG4; rAgaG4) was determined to be an endo-type β-agarase. The protein degraded agarose into neoagarotetraose and neoagarohexaose at a final molar ratio of 1.5:1. Neoagarooctaose was the smallest substrate for rAgaG4, whereas neoagarotetraose was the minimal degradation product. Removing the extra fragment from the GH module led to the inability of the mutant (rAgaG4-T57) to degrade neoagarooctaose, and the final degradation products of agarose by the truncated protein were neoagarotetraose, neoagarohexaose, and neoagarooctaose at a final molar ratio of 2.7:2.8:1. The optimal temperature for agarose degradation also decreased to 40 °C for this mutant. Bioinformatic analysis suggested that tyrosine 276 within the extra fragment was a candidate active site residue for the enzymatic activity. Site-swapping experiments of Tyr(276) to 19 various other amino acids demonstrated that the characteristics of this residue were crucial for the AgaG4 degradation of agarose and the cleavage pattern of substrate.

Cloning, expression and characterization of a new agarase-encoding gene from marine Pseudoalteromonas sp.

The beta-agarase gene agaA, cloned from a marine bacterium, Pseudoalteromonas sp. CY24, consists of 1,359 nucleotides encoding 453 amino acids in a sequence corresponding to a catalytic domain of glycosyl hydrolase family 16 (GH16) and a carbohydrate-binding module type 13 (CBM13). The recombinant enzyme is an endo-type agarase that hydrolyzes beta-1,4-linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the predominant products. In two cleavage patterns, AgaA digested the smallest substrate, neoagarooctaose, into neoagarobiose, neoagarotetraose and neoagarohexaose. Site directed mutation was performed to investigate the differences between AgaA and AgaD of Vibrio sp. PO-303, identifying residues V(109)VTS(112) as playing a key role in the enzyme reaction.

Accelerator analysis of tributyltin adsorbed onto the surface of a tributyltin resistant marine Pseudoalteromonas sp. cell.

Tributyltin (TBT) released into seawater from ship hulls is a stable marine pollutant and obviously remains in marine environments. We isolated a TBT resistant marine Pseudoalteromonas sp. TBT1 from sediment of a ship’s ballast water. The isolate (109.3 ± 0.2 colony-forming units mL−1) adsorbed TBT in proportion to the concentrations of TBTCl externally added up to 3 mM, where the number of TBT adsorbed by a single cell was estimated to be 108.2. The value was reduced to about one-fifth when the lysozyme-treated cells were used. The surface of ethanol treated cells became rough, but the capacity of TBT adsorption was the same as that for native cells. These results indicate that the function of the cell surface, rather than that structure, plays an important role to the adsorption of TBT. The adsorption state of TBT seems to be multi-layer when the number of more than 106.8 TBT molecules is adsorbed by a single cell.

Correlation between Chemical Structures and Inhibitory Activities of Anti-bacterial Substances from Marine Pseudoalteromonas sp. A1-J11

Correlation between Chemical Structures and Inhibitory Activities of Anti-bacterial Substances from Marine Pseudoalteromonas sp. A1-J11

Effects of commercial enzymes on the adhesion of a marine biofilm-forming bacterium

The antifouling potential of commercial hydrolases, four proteases, seven glycosidases and one lipase was evaluated on the adhesion of marine Pseudoalteromonas sp. D41. The experimental method, adapted to screen antifouling agents, was based on bacterial adhesion in natural sterile sea water in a microtiter plate and on total biomass quantification by the fluorescent dye DAPI (4[prime]6-diamidino-2-phenylindole). Savinase (subtilisin) was the most effective hydrolase in both the prevention of bacterial adhesion and the removal of adhered bacteria. However, some enzymatic preparations tested such as Amano protease were not only ineffective but also increased the number of adhered bacterial cells. Enumeration using epifluorescence microscopy of CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and DAPI stained adhered D41 cells confirmed these observations. Overall, these results demonstrated that hydrolases could either prevent adhesion and remove adhered bacterial cells effectively, or conversely increase bacterial adhesion, depending on enzymatic concentrations and the type of enzymes tested.

Molecular Cloning and Characterization of a Novel β-Agarase, AgaB, from Marine Pseudoalteromonas sp. CY24

Agarases are generally classified into glycoside hydrolase families 16, 50, and 86 and are found to degrade agarose to frequently generate neoagarobiose, neoagarotetraose, or neoagarohexaose as the main products. In this study we have cloned a novel endo-type beta-agarase gene, agaB, from marine Pseudoalteromonas sp. CY24. The novel agarase encoded by agaB gene has no significant sequence similarity with any known proteins including all glycoside hydrolases. It degrades agarose to generate neoagarooctaose and neoagarodecaose as the main end products. Based on the analyses of enzymatic kinetics and degradation patterns of different oligosaccharides, the agarase AgaB appears to have a large substrate binding cleft that accommodates 12 sugar units, with 8 sugar units toward the reducing end spanning subsites +1 to +8 and 4 sugar units toward the non-reducing end spanning subsites -4 to -1, and enzymatic cleavage taking place between subsites -1 and +1. In addition, 1H NMR analysis shows that this enzyme hydrolyzes the glycosidic bond with inversion of anomeric configuration, in contrast to other known agarases that are retaining. Altogether, AgaB is structurally and functionally different from other known agarases and appears to represent a new family of glycoside hydrolase.

Calcium influences cellular and extracellular product formation during biofilm-associated growth of a marine Pseudoalteromonas sp.

Bacteria undergo a variety of physiological changes following a switch from planktonic growth to surface-associated biofilm growth. Here, it is shown that biofilm development of a marine isolate, Pseudoalteromonas sp. 1398, results in global changes in its cytosolic and extracellular proteomes. Calcium influences these proteome responses, and affects the amount of surface-associated biomass and extracellular matrix material produced by Pseudoalteromonas sp. 1398. Four extracellular proteins, characterized by N-terminal sequencing, showed increased abundances, while one protein, flagellin, showed reduced abundance at higher [Ca(2+)]. Immunoblotting and transmission-electron-microscopy analysis confirmed that higher [Ca(2+)] and surface-associated growth results in the repression of flagella production. Two-dimensional gel electrophoresis (2DGE) studies combined with cluster analysis of global proteome responses demonstrated that Ca(2+) had a greater regulatory influence on Pseudoalteromonas sp. growing in biofilms than on planktonic cultures. Approximately 22 % of the total cytosolic proteins resolved by 2DGE had differing abundances in response to a switch from planktonic growth to surface-associated growth when the cells were cultivated in 1 mM Ca(2+). At higher [Ca(2+)] this number increased to 38 %. Fifteen cellular proteins that were differentially expressed in response to biofilm growth and/or Ca(2+) were analysed by N-terminal sequencing and/or MS/MS. These proteins were identified as factors involved in cellular metabolic functions, putative proteases and transport proteins, although there were several proteins that had not been previously characterized. These results indicate that Ca(2+) causes global changes in matrix material, as well as in cellular and extracellular protein profiles of Pseudoalteromonas sp. 1398. These changes are more pronounced when the bacterium grows in biofilms than when it grows in planktonic culture.

Marine Pseudoalteromonas sp. Composes Most of the Bacterial Population Developed in Oysters (Tiostrea chilensis) Spoiled During Storage

ABSTRACT: To identify bacteria associated with spoilage in oysters, changes in both the load and composition of the bacterial community occurring in storage were followed. Oyster spoilage was accompanied by a 100‐fold increase in the number of CFU. Molecular analysis revealed that a large proportion of the bacteria present in spoiled oysters possessed an intergenic 16S‐23S rDNA spacer of approximately 400 bp. A spacer with the same size was also found in spoiled oysters of different origin and dates of harvest. The bacterial strains containing this spacer may constitute a common cause of the oyster spoilage. These strains were salt‐dependent marine Pseudoalteromonas species and were probably present in oysters before harvest.