Cloning and Characterization of Extracellular Metal Protease Gene of the Algicidal Marine Bacterium Pseudoalteromonas sp. Strain A28

The gene (empI) encoding an extracellular metal protease was isolated from a Pseudoalteromonas sp. strain A28 DNA library. The recombinant EmpI protein was expressed in E. coli and purified. Paper-disk assays showed that the purified protease had potent algicidal activity. A skim milk-polyacrylamide gel electrophoresis protease assay showed that the 38-kDa band of protease activity, which co-migrated with purified EmpI and was sensitive to 1,10-phenathroline, was detected in the extracellular supernatant of A28.     

Antibiofilm Activity of the Marine Bacterium Pseudoalteromonas sp. Strain 3J6

Biofilm formation results in medical threats or economic losses and is therefore a major concern in a variety of domains. In two-species biofilms of marine bacteria grown under dynamic conditions, Pseudoalteromonas sp. strain 3J6 formed mixed biofilms with Bacillus sp. strain 4J6 but was largely predominant over Paracoccus sp. strain 4M6 and Vibrio sp. strain D01. The supernatant of Pseudoalteromonas sp. 3J6 liquid culture (SN_3J6) was devoid of antibacterial activity against free-living Paracoccus sp. 4M6 and Vibrio sp. D01 cells, but it impaired their ability to grow as single-species biofilms and led to higher percentages of nonviable cells in 48-h biofilms. Antibiofilm molecules of SN_3J6 were able to coat the glass surfaces used to grow biofilms and reduced bacterial attachment about 2-fold, which might partly explain the biofilm formation defect but not the loss of cell viability. SN_3J6 had a wide spectrum of activity since it affected all Gram-negative marine strains tested except other Pseudoalteromonas strains. Biofilm biovolumes of the sensitive strains were reduced 3- to 530-fold, and the percentages of nonviable cells were increased 3- to 225-fold. Interestingly, SN_3J6 also impaired biofilm formation by three strains belonging to the human-pathogenic species Pseudomonas aeruginosa, Salmonella enterica, and Escherichia coli. Such an antibiofilm activity is original and opens up a variety of applications for Pseudoalteromonas sp. 3J6 and/or its active exoproducts in biofilm prevention strategies.Biofilms are defined as microbial communities of cells that are irreversibly attached to a substratum, to an interface, or to each other and are embedded into a matrix of extracellular polymeric substances that they have produced (8). It is now considered that most (if not all) bacteria are capable of forming biofilms and that this is their predominant bacterial life-style. Biofilm formation is a complex biological phenomenon and has been generally described as a temporal process involving a succession of distinct stages: a reversible and then irreversible attachment of planktonic bacteria onto a surface, the formation of microcolonies either by the clonal growth of attached cells or by the active translocation of cells across the surface, the coalescence of growing microcolonies to form a macrocolony, and cell dispersal. It should, however, be noted that this developmental model still requires further experimental validation, especially concerning the possibility of a hierarchical order of genetic pathways (26). Furthermore, Karatan and Watnick (17) pointed out that there are as many different types of biofilms as there are bacteria and that a single bacterium may even make several different types of biofilms under different environmental conditions. Biofilm formation is associated with the virulence of pathogenic bacteria, and cells included within a biofilm are generally more resistant (up to 1,000-fold) to antibiotics and disinfectants than free-living bacteria (8, 26). Biofilms are therefore a major concern in medicine and in medical environments but also in all domains where their growth constitutes a source of contamination for humans or animals (food industry, cooling towers, and water pipes, etc.) or leads to economical losses (biofouling of boats and immersed structures and material biocorrosion, etc.). The development of antibiofilm strategies is therefore of major interest and currently constitutes an important field of investigation in which environmentally friendly antibiofilm molecules or organisms are highly valuable (5, 7, 9).Marine bacteria belonging to the genus Pseudoalteromonas of the class Gammaproteobacteria are often found in association with marine eukaryotes, and their ability to produce a variety of biological activities has attracted particular attention (2, 11, 13, 15, 28). We previously isolated marine bacteria attached to solid surfaces (glass in most cases) immersed for 3 or 6 h in the Morbihan Gulf or in the Bay of Brest, France (10, 20, 21, 27). Out of the three Pseudoalteromonas strains isolated, we were able to tag strain 3J6 with a green fluorescent protein (GFP)-encoding plasmid. This allowed us to investigate whether Pseudoalteromonas sp. strain 3J6 affected the biofilm growth of other marine bacterial isolates. Here, we report that strain 3J6 predominated in two-species biofilms over Paracoccus sp. strain 4M6 and Vibrio sp. strain D01. Although devoid of antibacterial activity against planktonic cells, Pseudoalteromonas sp. 3J6 exoproducts impaired biofilm formation by Paracoccus sp. 4M6 and Vibrio sp. D01. We characterized the effects of these exoproducts on the latter strains and on other bacteria.     

Involvement of an extracellular protease in algicidal activity of the marine bacterium Pseudoalteromonas sp. strain A28

The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M(w)-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N'-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30 degrees C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium.     

A Denitrifying Activity in an Aerobic Photosynthetic Bacterium, Erythrobacter sp. Strain OCh 114

An aerobic photosynthetic bacterium, Erythrobacter sp. strainOCh 114, was capable of growth under anaerobic conditions inthe dark with nitrate as a terminal electron acceptor. The optimalnitrate concentration was about 6 mM for anaerobic growth, althougha wide range of concentrations from 1 to 400 mM were effective.A large amount of N₂O gas was released during this anaerobicgrowth, indicating a denitrifying activity in this bacterium.Light had no stimulating or inhibiting effect on the rates ofanaerobic growth and gas release. The enzymes responsible forthe denitrifying activity, dissimilatory nitrate and nitritereductases, were present in aerobically grown cells. (Received February 19, 1988; Accepted May 16, 1988)     

Genome Sequence of the Agar-Degrading Marine Bacterium Alteromonadaceae sp. Strain G7

Here, we present the high-quality draft genome sequence of the agar-degrading marine gammaproteobacterium Alteromonadaceae sp. strain G7, which was isolated from coastal seawater to be utilized as a bioresource for production of agar-derived biofuels. The 3.91-Mb genome contains a number of genes encoding algal polysaccharide-degrading enzymes such as agarases and sulfatases.     

Algicidal Activity of Marine Alteromonas sp. KNS-16 and Isolation of Active Compounds

The KNS-16 algicidal strain was isolated from a harmful alga bloom (HAB) area and identified as Alteromonas sp. based on 16S rDNA sequencing. The KNS-16 strain was found to control HABs by producing algicidal compounds in an indirect interaction. Four active compounds were isolated from KNS-16 culture, and their structures were analyzed by interpreting nuclear magnetic resonance and mass spectroscopy data. The structures were identified as 2-undecen-1'-yl-4-quinolone (1), 2-undecyl-4-quinolone (2), 3-hexyl-6-pentyl-4-hydroxyl-2H-pyran-2-one (3), and 6-heptyl-3-hexyl-4-hydroxyl-2H-pyran-2-one (4). Compound 1 was most active against HABs such as Heterosigma akashiwo, Cochlodinium polykrikoides, and Alexandrium tamarense with LC(50) values of 0.5-1.1 μg/mL. The four compounds exhibited high LC(50) values against aquaculture algae such as Tetaselmis suecica, Isochrysis galbana, and Pavlova lutheri at 39-66 μg/mL. Based on toxicity tests on the brine shrimp Artemia salina and the rotifer Brachionus rotundiformis, the four compounds showed ranges of 409-608 and 189-224 μg/mL of LC(50) for the two organisms, respectively. The LC(50) values for juvenile fish of Sebastes schlegelii were 284-304 μg/mL.     

Extracellular secretion of Pseudoalteromonas sp. cold-adapted esterase in Escherichia coli in the presence of Pseudoalteromonas sp. components of ABC transport system

Recently we described identification and characterization of GDSL esterase EstA from psychrotrophic bacterium Pseudoalteromonas sp. 643A. Attempts to obtain heterologous overexpression of this enzyme in Escherichia coli system were not satisfactory. The EstA protein was expressed as inclusion bodies, most of that were inactive after purification step, and the recovery of esterolytic activity was very low after refolding. Based on the sequence analysis we found that the esterase EstA gene is clustered with three genes encoding components of ABC transport system. These genes, designated abc1, abc2, and abc3 encode an ATP-binding protein (ABC1) and two permease proteins (ABC2 and ABC3). In present study, to obtain larger amounts of the active cold-adapted EstA esterase from Pseudoalteromonas sp. 643A, we designed a two-plasmid E. coli expression system where the gene encoding EstA enzyme was cloned into pET30b(+) expression vector and three genes encoding components of ABC transport system were cloned into pACYC-pBAD vector. It was shown that the created expression system was useful for extracellular production of active EstA enzyme which was purified from the culture medium. In the presence of all the three transporter proteins the secretion of EstA was at the highest level. When one or two of these components were missing, EstA secretion was also possible, but not so effective. It indicates that ABC2 and ABC3 proteins of Pseudoalteromonas sp. 643A could be replaced with their homologous proteins of E. coli.     

Purification and Characterization of an Alginate Lyase from Marine Bacterium Vibrio sp. Mutant Strain 510-64

Marine Vibrio sp. 510 was chosen as a parent strain for screening high producers of alginate lyase using the complex mutagenesis of Ethyl Methanesulphonate and UV radiation treatments. The mutant strain Vibrio sp. 510-64 was selected and its alginate lyase activity was increased by 3.87-fold (reaching 46.12 EU/mg) over that of the parent strain. An extracellular alginate lyase was purified from Vibrio sp. 510-64 cultural supernatant by successive fractionation on DEAE Sepharose FF and two steps of Superdex 75. The purified enzyme yielded a single band on SDS-PAGE with the molecular weight of 34.6 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel alginate lyase. The substrate specificity results demonstrated that the alginate lyase had the specificity for poly G block.     

Biofilm Development and Cell Death in the Marine Bacterium Pseudoalteromonas tunicata

The newly described green-pigmented bacterium Pseudoalteromonas tunicata (D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. As part of our studies on the ecology of P. tunicata and its interaction with marine surfaces, we examined the ability of P. tunicata to form biofilms under continuous culture conditions within the laboratory. P. tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels. Remarkably, we observed a repeatable pattern of cell death during biofilm development of P. tunicata, similar to that recently reported for biofilms of Pseudomonas aeruginosa (J. S. Webb et al., J. Bacteriol. 185:4585-4595, 2003). Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures. A subpopulation of viable cells was always observed within the regions of killing in the biofilm. Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum. A novel 190-kDa autotoxic protein produced by P. tunicata, designated AlpP, was found to be involved in this biofilm killing and detachment. A ΔalpP mutant derivative of P. tunicata was generated, and this mutant did not show cell death during biofilm development. We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of P. tunicata and subsequent dispersal of surviving cells within the marine environment.     

Competitive Interactions in Mixed-Species Biofilms Containing the Marine Bacterium Pseudoalteromonas tunicata

Pseudoalteromonas tunicata is a biofilm-forming marine bacterium that is often found in association with the surface of eukaryotic organisms. It produces a range of extracellular inhibitory compounds, including an antibacterial protein (AlpP) thought to be beneficial for P. tunicata during competition for space and nutrients on surfaces. As part of our studies on the interactions between P. tunicata and the epiphytic bacterial community on the marine plant Ulva lactuca, we investigated the hypothesis that P. tunicata is a superior competitor compared with other bacteria isolated from the plant. A number of U. lactuca bacterial isolates were (i) identified by 16S rRNA gene sequencing, (ii) characterized for the production of or sensitivity to extracellular antibacterial proteins, and (iii) labeled with a fluorescent color tag (either the red fluorescent protein DsRed or green fluorescent protein). We then grew single- and mixed-species bacterial biofilms containing P. tunicata in glass flow cell reactors. In pure culture, all the marine isolates formed biofilms containing microcolony structures within 72 h. However, in mixed-species biofilms, P. tunicata removed the competing strain unless its competitor was relatively insensitive to AlpP (Pseudoalteromonas gracilis) or produced strong inhibitory activity against P. tunicata (Roseobacter gallaeciensis). Moreover, biofilm studies conducted with an AlpP⁻ mutant of P. tunicata indicated that the mutant was less competitive when it was introduced into preestablished biofilms, suggesting that AlpP has a role during competitive biofilm formation. When single-species biofilms were allowed to form microcolonies before the introduction of a competitor, these microcolonies coexisted with P. tunicata for extended periods of time before they were removed. Two marine bacteria (R. gallaeciensis and P. tunicata) were superior competitors in this study. Our data suggest that this dominance can be attributed to the ability of these organisms to rapidly form microcolonies and their ability to produce extracellular antibacterial compounds.     

1株金黄杆菌分离、鉴定及其胞外蛋白酶活性的初步研究

从无指盘臭蛙(Odorrana grahami)皮肤表面分离到1株具有胞外蛋白水解酶活性的菌株,命名为CW-E2T。初步研究发现CW-E2T系革兰阴性,菌体短杆状,无鞭毛,菌落呈圆形,边缘光滑并相互粘连,表面突起,产生黄色素,有金黄色光泽细菌。通过形态学、生理生化和16SrRNA基因序列分析,确定CW-E2T为金黄杆菌属内的一个未定种。在此基础上对该菌株蛋白水解酶进行了初步研究,确定菌株CW-E2T胞外蛋白酶为金属蛋白酶。以上对CW-E2T细菌分类学研究和胞外蛋白酶的初步研究,为该菌株的大量培养以及胞外蛋白酶的进一步研究提供理论依据。     

Intracellular Alginolytic Enzymes of the Marine Bacterium Pseudoalteromonas citrea KMM 3297

The marine bacterium Pseudoalteromonas citrea KMM 3297 is an associate of the holothurian Apostichopus japonicus. When grown in a medium containing glucose, the strain produces two intracellular alginolytic enzymes, AlI and AlII. Fucoidan from the brown alga Fucus evanescens induces synthesis of one more alginolytic enzyme, AlIII. These enzymes were separated using anion-exchange chromatography. The alginate lyase AlI completely retains its activity at 35 degrees C, AlII and AlIII being stable at 45 degrees C. The alginate lyases exhibit maximal activities in the range of pH 7-8. The molecular weights of AlI, AlII, and AlIII determined by gel filtration are 25, 79, and 61 kD, respectively. All the investigated enzymes are endo-type alginate lyases. They catalyze degradation of polyguluronate (poly-G) and polymannuronate (poly-M) yielding oligosaccharides of the polymerization degree of 5 > or = n > or = 3 with the unsaturated bond between the C4 and C5 atoms of the non-reducing terminus. A mixture of these three enzymes exhibits synergism while acting on the polymeric substrate. The Km values of the alginate lyase AlI for poly-G and poly-M are 24 and 34 micro g/ml, respectively. Alginate lyase AlIII exhibits less affinity to poly-M (Km = 130.0 microg/ml) than to poly-G (Km = 40.0 microg/ml). NaCl (0.2 M), MgCl2 and MgSO4 (0.01 M) activate all three enzymes more than twofold. The presence of several alginolytic enzymes of different specificity provides efficient destruction of alginic acids of brown algae by the strain P. citrea KMM 3297.     

Binding of Extracellular Carboxymethylcellulase Activity from the Marine Shipworm Bacterium to Insoluble Cellulosic Substrates

The binding of extracellular endoglucanase, a carboxymethylcellulase (CMCase), produced by the marine shipworm bacterium to insoluble cellulose substrates was investigated. Up to 70% of CMCase activity bound to cellulosic substrates, and less than 10% bound to noncellulosic substrates. CMCase binding to cellulose was enhanced in basal salt medium or sodium phosphate buffer containing 0.5 M NaCl. Increased cellulose particle size correlated with decreased CMCase binding. Also, cellulose treated with either 5 N NaOH or commercial cellulase reduced the CMCase binding to these surfaces. Pretreatment of CMCase preparations with 0.01% sodium dodecyl sulfate, 5% β-mercaptoethanol, and 5 mM EDTA or ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) had little effect on binding to cellulose. While pretreatment of CMCase with trypsin, chymotrypsin, and pronase had little effect on CMCase enzymatic activity, the ability to bind to cellulose was greatly diminished by these treatments.     

Induction of Protease Release of the Resistant Diatom Chaetoceros didymus in Response to Lytic Enzymes from an Algicidal Bacterium

Marine lytic bacteria can have a substantial effect on phytoplankton and are even capable to terminate blooms of microalgae. The bacterium Kordia algicida was reported to lyse cells of the diatom Skeletonema costatum and several other diatoms by a quorum sensing controlled excretion of proteases. However the diatom Chaetoceros didymus is fully resistant against the bacterial enzymes. We show that the growth curve of this diatom is essentially unaffected by addition of bacterial filtrates that are active against other diatoms. By monitoring proteases from the medium using zymography and fluorescence based activity assays we demonstrate that C. didymus responds to the presence of the lytic bacteria with the induced production of algal proteases. These proteases exhibit a substantially increased activity compared to the bacterial counterparts. The induction is also triggered by signals in the supernatant of a K. algicida culture. Size fractionation shows that only the >30 kD fraction of the bacterial exudates acts as an inducing cue. Implications for a potential induced defense of the diatom C. didymus are discussed.     

A Gliding Bacterium Strain Inhibits Adhesion and Motility of Another Gliding Bacterium Strain in a Marine Biofilm

Two species of gliding bacteria were isolated from a marine biofilm. They were described and identified as members of the genus Cytophaga. One of them (RB1057) produced an extracellular inhibitor of colony expansion of the other (RB1058). The inhibitor was characterized as a glycoprotein with an apparent molecular mass of 60 kDa. It inhibited RB1058 adhesion to and gliding on substrata. Motility and adhesion of several other aquatic gliding bacteria were not measurably affected by this agent.     

A Metalloprotease (MprIII) Involved in the Chitinolytic System of a Marine Bacterium, Alteromonas sp. Strain O-7

Alteromonas sp. strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction. In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain. The gene encoding MprIII was cloned in Escherichia coli. The open reading frame of mprIII encoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da. Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function. Western and real-time quantitative PCR analyses demonstrated that mprIII was induced in the presence of insoluble polysaccharides, such as chitin and cellulose. Native MprIII was purified to homogeneity from the culture supernatant of Alteromonas sp. strain O-7 and characterized. The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa. The optimum pH and temperature of MprIII were 7.5 and 50°C, respectively, when gelatin was used as a substrate. Pretreatment of native chitin with MprIII significantly promoted chitinase activity. Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.