Antibacterial Activity of Pseudoalteromonas in the Coral Holobiont

Corals harbor diverse and abundant prokaryotic populations. Bacterial communities residing in the coral mucus layer may be either pathogenic or symbiotic. Some species may produce antibiotics as a method of controlling populations of competing microbial species. The present study characterizes cultivable Pseudoalteromonas sp. isolated from the mucus layer of different coral species from the northern Gulf of Eilat, Red Sea, Israel. Six mucus-associated Pseudoalteromonas spp. obtained from different coral species were screened for antibacterial activity against 23 tester strains. Five of the six Pseudoalteromonas strains demonstrated extracellular antibacterial activity against Gram-positive—but not Gram-negative—tester strains. Active substances secreted into the cell-free supernatant are heat-tolerant and inhibit growth of Bacillus cereus, Staphylococcus aureus, and of ten endogenous Gram-positive marine bacteria isolated from corals. The Pseudoalteromonas spp. isolated from Red sea corals aligned in a phylogenetic tree with previously isolated Pseudoalteromonas spp. of marine origin that demonstrated antimicrobial activity. These results suggest that coral mucus-associated Pseudoalteromonas may play a protective role in the coral holobiont's defense against potential Gram-positive coral pathogens.     

Bioactivity,Chemical Profiling,and 16S rRNA-Based Phylogeny of <Emphasis Type="BoldItalic">Pseudoalteromonas</Emphasis> Strains Collected on a Global Research Cruise

One hundred one antibacterial Pseudoalteromonas strains that inhibited growth of a Vibrio anguillarum test strain were collected on a global research cruise (Galathea 3), and 51 of the strains repeatedly demonstrated antibacterial activity. Here, we profile secondary metabolites of these strains to determine if particular compounds serve as strain or species markers and to determine if the secondary metabolite profile of one strain represents the bioactivity of the entire species. 16S rRNA gene similarity divided the strains into two primary groups: One group (51 strains) consisted of bacteria which retained antibacterial activity, 48 of which were pigmented, and another group (50 strains) of bacteria which lost antibacterial activity upon sub-culturing, two of which were pigmented. The group that retained antibacterial activity consisted of six clusters in which strains were identified as Pseudoalteromonas luteoviolacea, Pseudoalteromonas aurantia, Pseudoalteromonas phenolica, Pseudoalteromonas ruthenica, Pseudoalteromonas rubra, and Pseudoalteromonas piscicida. HPLC-UV/VIS analyses identified key peaks, such as violacein in P. luteoviolacea. Some compounds, such as a novel bromoalterochromide, were detected in several species. HPLC-UV/VIS detected systematic intra-species differences for some groups, and testing several strains of a species was required to determine these differences. The majority of non-antibacterial, non-pigmented strains were identified as Pseudoalteromonas agarivorans, and HPLC-UV/VIS did not further differentiate this group. Pseudoalteromonas retaining antibacterial were more likely to originate from biotic or abiotic surfaces in contrast to planktonic strains. Hence, the pigmented, antibacterial Pseudoalteromonas have a niche specificity, and sampling from marine biofilm environments is a strategy for isolating novel marine bacteria that produce antibacterial compounds.     

Identification of Bacterial Strains Isolated from the Mediterranean Sea Exhibiting Different Abilities of Biofilm Formation

The Mediterranean Sea has rarely been investigated for the characterization of marine bacteria as compared to other marine environments such as the Atlantic or Pacific Ocean. Bacteria recovered from inert surfaces are poorly studied in these environments, when it has been shown that the community structure of attached bacteria can be dissimilar from that of planktonic bacteria present in the water column. The objectives of this study were to identify and characterize marine bacteria isolated from biofilms developed on inert surfaces immersed in the Mediterranean Sea and to evaluate their capacity to form a biofilm in vitro. Here, 13 marine bacterial strains have been isolated from different supports immersed in seawater in the Bay of Toulon (France). Phylogenetic analysis and different biological and physico-chemical properties have been investigated. Among the 13 strains recovered, 8 different genera and 12 different species were identified including 2 isolates of a novel bacterial species that we named Persicivirga mediterranea and whose genus had never been isolated from the Mediterranean Sea. Shewanella sp. and Pseudoalteromonas sp. were the most preponderant genera recovered in our conditions. The phenotypical characterization revealed that one isolate belonging to the Polaribacter genus differed from all the other ones by its hydrophobic properties and poor ability to form biofilms in vitro. Identifying and characterizing species isolated from seawater including from Mediterranean ecosystems could be helpful for example, to understand some aspects of bacterial biodiversity and to further study the mechanisms of biofilm (and biofouling) development in conditions approaching those of the marine environment.     

Purification and Partial Identification of Novel Antimicrobial Protein from Marine Bacterium <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> Species Strain X153

A marine bacterium, X153, was isolated from a pebble collected at St. Anne du Portzic (France). By 16S ribosomal DNA gene sequence analysis, X153 strain was identified as a Pseudoalteromonas sp. close to P. piscicida. The crude culture of X153 was highly active against human pathogenic strains involved in dermatologic diseases, and marine bacteria including various ichthyopathogenic Vibrio strains. The active substance occurred both in bacterial cells and in culture supernatant. An antimicrobial protein was purified to homogeneity by a 4-step procedure using size-exclusion and ion-exchange chromatography. The highly purified P-153 protein is anionic, and sodium dodecylsulfate polyacrylamide gel electrophoresis gives an apparent molecular mass of 87 kDa. The X153 bacterium protected bivalve larvae against mortality, following experimental challenges with ichthyopathogenic Vibrio. Pseudoalteromonas sp. X153 may be useful in aquaculture as a probiotic bacterium.     

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1–M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.     

Diversity and antimicrobial activities of microbes from two Irish marine sponges, Suberites carnosus and Leucosolenia sp

Aims: To evaluate the diversity and antimicrobial activity of bacteria from the marine sponges Suberites carnosus and Leucosolenia sp. Methods and Results: Two hundred and thirty‐seven bacteria were isolated from the sponges S. carnosus (Demospongiae) and Leucosolenia sp. (Calcarea). Isolates from the phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria were obtained. Isolates of the genus Pseudovibrio were dominant among the bacteria from S. carnosus, whereas Pseudoalteromonas and Vibrio were the dominant genera isolated from Leucosolenia sp. Approximately 50% of the isolates from S. carnosus displayed antibacterial activity, and c. 15% of the isolates from Leucosolenia sp. demonstrated activity against the test fungal strains. The antibacterial activity observed was mostly from Pseudovibrio and Spongiobacter isolates, while the majority of the antifungal activity was observed from the Pseudoalteromonas, Bacillus and Vibrio isolates. Conclusions: Both sponges possess a diverse range of bioactive and potentially novel bacteria. Differences observed from the sponge‐derived groups of isolates in terms of bioactivity suggest that S. carnosus isolates may be a better source of antibacterial compounds, while Leucosolenia sp. isolates appear to be a better source of antifungal compounds. Significance and Impact of the Study: This is the first study in which cultured bacterial isolates from the marine sponges S. carnosus and a Leucosolenia sp. have been evaluated for their antibacterial activity. The high percentage of antibacterial isolates from S. carnosus and of antifungal isolates from Leucosolenia sp. suggests that these two sponges may be good sources for potentially novel marine natural products.     

大连海域沉积物中可培养海洋链霉菌活性及其多样性

以大肠杆菌、金黄色葡萄球菌和尖孢镰刀枯萎病菌作为测试靶目标,采用9种分离培养基从大连海域13个不同采样点的海洋沉积物样品中分离到165株海洋链霉菌。从165株海洋放线菌中筛选到对金黄色葡萄球菌具有抑制活性的菌株85株,占总菌株数的51.5%;对大肠杆菌具有抑制活性的菌株27株,占总菌株数的16.4%;对尖孢镰刀枯萎病菌具有抑制活性的菌株仅有6株,占总菌株数的3.6%。因此,海洋链霉菌的活性更多地表现为对细菌的抗性,尤其对革兰氏阳性细菌具有更高的抑制活性。对其中具有抑制活性或形态独特的菌株进行了16S r DNA序列分析,并构建系统发育树,显示活性海洋链霉菌具有丰富的种类多样性和广谱抗菌活性。同种海洋链霉菌与土壤链霉菌活性比较结果也表明,海洋链霉菌多表现抗革兰氏阳性细菌活性。     

Species-level identification of <Emphasis Type="Italic">Bacillus</Emphasis> strains isolates from marine sediments by conventional biochemical, 16S rRNA gene sequencing and inter-tRNA gene sequence lengths analysis

The aim of this study was to compare the ability of commonly used conventional biochemical tests, sequencing analysis of 16S rRNA genes and tDNA-intergenic spacer length polymorphism (tDNA-PCR) to identify species of the genus Bacillus recovered from marine sediments. While biochemical tests were not sufficiently sensitive to distinguish between the 23 marine strains analyzed, partial 16S rRNA gene sequences allowed a correct identification, clustering them into four species belonging to Bacillus licheniformis (n = 6), Bacillus cereus (n = 9), Bacillus subtilis (n = 7) and Bacillus pumilus (n = 1). The identification results obtained with 16S rRNA sequencing were validated by tDNA-PCR analysis of 23 marine isolates that were identified by the similarities of their fingerprints to those of reference strains. tDNA-PCR fingerprinting was as discriminatory as 16S rRNA sequencing analysis. Although it was not able to distinguish among the species of the B. cereus and B. subtilis groups, it should be considered a rapid and easy approach for the reliable identification of unknown Bacillus isolates or at least for the primary differentiation of Bacillus groups.     

Antibacterial Activity of Marine Culturable Bacteria Collected from a Global Sampling of Ocean Surface Waters and Surface Swabs of Marine Organisms

The purpose of the present study was to isolate marine culturable bacteria with antibacterial activity and hence a potential biotechnological use. Seawater samples (244) and 309 swab samples from biotic or abiotic surfaces were collected on a global Danish marine research expedition (Galathea 3). Total cell counts at the seawater surface were 5 × 10⁵ to 10⁶ cells/ml, of which 0.1–0.2% were culturable on dilute marine agar (20°C). Three percent of the colonies cultured from seawater inhibited Vibrio anguillarum, whereas a significantly higher proportion (13%) of colonies from inert or biotic surfaces was inhibitory. It was not possible to relate a specific kind of eukaryotic surface or a specific geographic location to a general high occurrence of antagonistic bacteria. Five hundred and nineteen strains representing all samples and geographic locations were identified on the basis of partial 16S rRNA gene sequence homology and belonged to three major groups: Vibrionaceae (309 strains), Pseudoalteromonas spp. (128 strains), and the Roseobacter clade (29 strains). Of the latter, 25 strains were identified as Ruegeria mobilis or pelagia. When re-testing against V. anguillarum, only 409 (79%) retained some level of inhibitory activity. Many strains, especially Pseudoalteromonas spp. and Ruegeria spp., also inhibited Staphylococcus aureus. The most pronounced antibacterial strains were pigmented Pseudoalteromonas strains and Ruegeria spp. The inhibitory, pigmented Pseudoalteromonas were predominantly isolated in warmer waters from swabs of live or inert surfaces. Ruegeria strains were isolated from all ocean areas except for Arctic and Antarctic waters and inhibitory activity caused by production of tropodithietic acid.     

The effect of bacterial and diatom biofilms on the settlement of the bryozoan Bugula neritina

Biofilms of marine bacteria and diatoms and their combinations were examined in laboratory choice assays to determine their effects on the attachment and successful metamorphosis of the larvae of the bryozoan Bugula neritina (Linnéus). The larval settlement in response to unfilmed surfaces, a natural biofilm (NBF) and adsorbed cells of three strains of bacteria, five strains of pennate diatoms and combinations of the two at different densities. Bacterial and diatom strains showed different effects on the larval settlement of B. neritina. Bacterial monospecific strains of an unidentified α-Proteobacterium and Vibrio sp. mediated the same percentage of settlement as a filtered seawater control. Biofilms of Pseudoalteromonas sp. caused significantly lower larval settlement. Larval settlement of B. neritina was negatively correlated with increasing densities of Pseudoalteromonas sp. The highest percentages of settlement were mediated by the biofilms of the diatom species Achnanthes sp., Amphora cofeaeformis, Amphora tenerrima, Nitzschia constricta and a 5-day-old natural biofilm, while the lowest settlement was found on a N. frustulum film. A three-way analysis of variance demonstrated that the density of bacteria and the presence of particular species of diatoms and bacteria in combined biofilms, significantly affected the settlement of B. neritina larvae. High settlement of larvae (50-90%) at all treatments indicated that B. neritina larvae are much more indiscriminate settlers than previously expected. Hence, using this species as a monitoring organism to trace ecologically relevant subtle changes of settlement cues in the natural environment should be carefully re-examined.     

Isolation and characterization of algicidal bacteria from <Emphasis Type="Italic">Cochlodinium polykrikoides</Emphasis> culture

In this study, we analyzed a bacterial community closely associated with Cochlodinium polykrikoides that caused harmful algal blooming in the sea. Filtration using a plankton mesh and percoll gradient centrifugation were performed to eliminate free-living bacteria. Attached bacteria were analyzed by culture-dependent and culture-independent methods. Five culturable bacterial strains were isolated and identified from the C. polykrikoides mixed bacterial community. The isolates belonged to α-Proteobacteria (Nautella sp., Sagittula sp., and Thalassobius sp.) and γ-Proteobacteria (Alteromonas sp. and Pseudoalteromonas sp.). All of the 5 isolates showed algicidal activity against C. polykrikoides and produced extracellular compounds responsible for algicidal properties after entering the stationary phase. The algicidal compounds produced by the 5 isolates were heat-stable and had molecular masses of less than 10,000 Da. Furthermore, the algicidal compounds were relatively specific for C. polykrikoides in terms of their algicidal activities. Culture-independent analysis of the bacterial community in association with C. polykrikoides was performed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). On the basis of the PCR-DGGE profile, Sagittula sp. was identified as a dominant species in the bacterial community of C. polykrikoides.     

Metalloid Reducing Bacteria Isolated from Deep Ocean Hydrothermal Vents of the Juan de Fuca Ridge, Pseudoalteromonas telluritireducens sp. nov. and Pseudoalteromonas spiralis sp. nov

Five strains of Gram-negative, rod, curved rod and spiral-shaped bacteria were isolated from the vicinity of deep ocean hydrothermal vents along the Main Endeavour Segment of the Juan de Fuca Ridge in the Pacific Ocean. All strains showed remarkable resistance to high levels of toxic metalloid oxyanions, and were capable of reducing the oxyanions tellurite and selenite to their less toxic elemental forms. Phylogenetic analysis of four strains identified these isolates as close relatives of the genus Pseudoalteromonas within the class Gammaproteobacteria. Pseudoalteromonas agarivorans was the closest relative of strains Te-1-1 and Se-1-2-red^T, with, respectively, 99.5 and 99.8% 16S rDNA sequence similarity. Strain Te-2-2^T was most closely related to Pseudoalteromonas paragorgicola, with 99.8% 16S rDNA sequence similarity. The DNA G+C base composition was 39.6 to 41.8 mol%, in agreement with other members of the genus Pseudoalteromonas. However, the isolates showed important morphological and physiological differences from previously described species of this genus, with one group forming rod-shaped bacteria typical of Pseudoalteromonas and the other forming vibrioid- to spiral-shaped cells. Based on these differences, and on phylogenetic data, we propose the creation of the new species Pseudoalteromonas telluritireducens sp. nov., with strain Se-1-2-red^T (DSMZ=16098^T=VKM B-2382^T) as the type strain, and Pseudoalteromonas spiralis sp. nov., with strain Te-2-2^T (DSMZ=16099^T=VKM B-2383^T) as the type strain. The EMBL accession numbers for the 16S rDNA sequences are: Te-1-1, AJ314843; Te-2-2^T, AJ314842; Se-1-2-or, AJ314844; Se-1-2-red^T, AJ314845.     

Diversity of Thiosulfate-Oxidizing Bacteria from Marine Sediments and Hydrothermal Vents

Species diversity, phylogenetic affiliations, and environmental occurrence patterns of thiosulfate-oxidizing marine bacteria were investigated by using new isolates from serially diluted continental slope and deep-sea abyssal plain sediments collected off the coast of New England and strains cultured previously from Galapagos hydrothermal vent samples. The most frequently obtained new isolates, mostly from 10³- and 10⁴-fold dilutions of the continental slope sediment, oxidized thiosulfate to sulfate and fell into a distinct phylogenetic cluster of marine alpha-Proteobacteria. Phylogenetically and physiologically, these sediment strains resembled the sulfate-producing thiosulfate oxidizers from the Galapagos hydrothermal vents while showing habitat-related differences in growth temperature, rate and extent of thiosulfate utilization, and carbon substrate patterns. The abyssal deep-sea sediments yielded predominantly base-producing thiosulfate-oxidizing isolates related to Antarctic marine Psychroflexus species and other cold-water marine strains of the Cytophaga-Flavobacterium-Bacteroides phylum, in addition to gamma-proteobacterial isolates of the genera Pseudoalteromonas and Halomonas-Deleya. Bacterial thiosulfate oxidation is found in a wide phylogenetic spectrum of Flavobacteria and Proteobacteria.     

Life-Style and Genome Structure of Marine Pseudoalteromonas Siphovirus B8b Isolated from the Northwestern Mediterranean Sea

Marine viruses (phages) alter bacterial diversity and evolution with impacts on marine biogeochemical cycles, and yet few well-developed model systems limit opportunities for hypothesis testing. Here we isolate phage B8b from the Mediterranean Sea using Pseudoalteromonas sp. QC-44 as a host and characterize it using myriad techniques. Morphologically, phage B8b was classified as a member of the Siphoviridae family. One-step growth analyses showed that this siphovirus had a latent period of 70 min and released 172 new viral particles per cell. Host range analysis against 89 bacterial host strains revealed that phage B8b infected 3 Pseudoalteromonas strains (52 tested, >99.9% 16S rRNA gene nucleotide identity) and 1 non-Pseudoaltermonas strain belonging to Alteromonas sp. (37 strains from 6 genera tested), which helps bound the phylogenetic distance possible in a phage-mediated horizontal gene transfer event. The Pseudoalteromonas phage B8b genome size was 42.7 kb, with clear structural and replication modules where the former were delineated leveraging identification of 16 structural genes by virion structural proteomics, only 4 of which had any similarity to known structural proteins. In nature, this phage was common in coastal marine environments in both photic and aphotic layers (found in 26.5% of available viral metagenomes), but not abundant in any sample (average per sample abundance was 0.65% of the reads). Together these data improve our understanding of siphoviruses in nature, and provide foundational information for a new ‘rare virosphere’ phage–host model system.     

Chemical Characterization of Lipid A from Some Marine Proteobacteria

Lipids A from type and wild strains of marine Proteobacteria belonging to Alteromonadaceae (Alteromonas (1 species), Idiomarina (1 species), and Pseudoalteromonas (8 species) genera) and Vibrionaceae (Shewanella (1 species) and Vibrio (1 species) genera) families and Marinomonas genus (1 species) were isolated by hydrolysis of their respective lipopolysaccharides with 1% acetic acid. Based on thin-layer chromatography data, the lipids A studied had low heterogeneity and generated family-specific patterns varying in numbers of bands and their chromatographic mobility. Total chemical analysis of the compounds showed that they contained glucosamine, phosphate, and fatty acids with decanoate (I. zobellii KMM 231^T lipid A) or dodecanoate (lipids A of the other bacteria) and 3-hydroxy alkanoates as the major fatty acid components. Unlike terrestrial bacterial lipids A, lipids A of marine Proteobacteria had basically monophosphoryl (except V. fluvialis AQ 0002B lipid A with its two phosphate groups) and pentaacyl (except S. alga 48055 and V. fluvialis AQ 0002B lipids A which were found to have six residues of fatty acids per molecule of glucosamine disaccharide) structural types, low toxicity, and may be useful as potential endotoxin antagonists.     

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.     

Isolation and Characterization of Two Groups of Novel Marine Bacteria Producing Violacein

Thirteen strains of novel marine bacteria producing a purple pigment were isolated from the Pacific coast of Japan. They were divided into two groups based on their 16S ribosomal RNA gene sequences, and both groups of bacteria belonged to the genus Pseudoalteromonas. The UV-visible spectrum of the pigment was identical to those of violacein, a pigment produced by several species of bacteria including Chromobacterium violaceum, an opportunistic pathogen. Further analysis of the chemical structure of the pigment by mass spectroscopy and nuclear magnetic resonance spectroscopy showed that the pigment was violacein. The high purity of violacein in the crude extract enabled us to employ simple and nonpolluting procedures to purify the pigment. Isolated bacteria may be useful as a C. violaceum substitute for the safe production of violacein.     

北黄海沉积物可培养产蛋白酶细菌分离鉴定

【目的】揭示北黄海沉积物中可培养产胞外蛋白酶细菌及蛋白酶多样性,增加人们对北黄海生态系统中产蛋白酶菌多样性的认识,为海洋产蛋白酶微生物的挖掘提供菌群资源。【方法】分别将5个北黄海沉积物样品梯度稀释涂布至酪蛋白明胶筛选平板,选择性分离产蛋白酶细菌;并通过分析基于16S rRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;分别测定胞外蛋白酶活性并对酶活较高的39株菌进行基于苯甲基磺酰氟(PMSF,丝氨酸蛋白酶抑制剂)、邻菲罗啉(o-phenanthroline,O-P,金属蛋白酶抑制剂)、E-64(半胱氨酸蛋白酶抑制剂)和pepstatin A(天冬氨酸蛋白酶抑制剂)4种抑制剂的酶活抑制实验以及所有菌株对3种底物(酪蛋白、明胶、弹性蛋白)的水解能力;分析这些细菌所产胞外蛋白酶的特性及多样性。【结果】从5个北黄海沉积物样品中分离获得66株产蛋白酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria、Actinobacteria和Firmicutes 4个门的7个属,其中Pseudoalteromonas(69.9%)、Sulfitobacter(12.1%)和Salegentibacter(10.6%)是优势菌群;沉积物中可培养的产蛋白酶细菌的丰度为104 CFU/g;蛋白酶酶活抑制实验表明所有测定菌株产生的胞外蛋白酶属于丝氨酸蛋白酶和/或金属蛋白酶,仅有少数菌株所产蛋白酶具有半胱氨酸蛋白酶或天冬氨酸蛋白酶活性。【结论】北黄海沉积物中可培养产蛋白酶细菌类群较为丰富,Pseudoalteromonas、Sulfitobacter和Salegentibacter菌株是优势菌群,测定菌株所产胞外蛋白酶主要是丝氨酸蛋白酶和/或金属蛋白酶。     

Illuminating microbial species-specific effects on organic matter remineralization in marine sediments

Marine microorganisms play a fundamental role in the global carbon cycle by mediating the sequestration of organic matter in ocean waters and sediments. A better understanding of how biological factors, such as microbial community composition, influence the lability and fate of organic matter is needed. Here, we explored the extent to which organic matter remineralization is influenced by species-specific metabolic capabilities. We carried out aerobic time-series incubations of Guaymas Basin sediments to quantify the dynamics of carbon utilization by two different heterotrophic marine isolates (Vibrio splendidus 1A01; Pseudoalteromonas sp. 3D05). Continuous measurement of respiratory CO₂ production and its carbon isotopic compositions (¹³C and ¹⁴C) shows species-specific differences in the rate, quantity and type of organic matter remineralized. Each species was incubated with hydrothermally-influenced versus unimpacted sediments, resulting in a ~2-fold difference in respiratory CO₂ yield across the experiments. Genomic analysis indicated that the observed carbon utilization patterns may be attributed in part to the number of gene copies encoding for extracellular hydrolytic enzymes. Our results demonstrate that the lability and remineralization of organic matter in marine environments is not only a function of chemical composition and/or environmental conditions, but also a function of the microorganisms that are present and active.     

Extracellular enzymes of cold-adapted bacteria from Arctic sea ice,Canada Basin

A total of 338 aerobic heterotrophic bacterial strains were isolated from Arctic sea ice, Canada Basin (77°30′N–80°12′N). The capability of the isolates to produce protease, lipase, amylase, chitinase, β-galactosidase, cellulase and/or agarase was investigated. Isolates that were able to degrade tributyrin, skim milk, starch, lactose and chitin accounted for 71.6, 65.7, 38.5, 31.6 and 16.9% of sea ice strains, respectively. Lipase producers and/or protease producers were phylogenetically widespread among the isolated strains. Starch and/or lactose hydrolytic strains were mainly distributed among Colwellia, Marinomonas, Pseudoalteromonas, Pseudomonas and Shewanella isolates. Pseudoalteromonas tetraodonis, Pseudoalteromonas elyakovii, Bacillus firmus and Janibacter melonis isolates all have the ability to degrade chitin. Only some strains belonging to Pseudoalteromonas genus scored positive for agarase (6) and cellulose (9). The temperature dependences for lipase activities were determined for five psychrophilic and six psychrotolerant bacteria. At low temperatures, the psychrophilic bacterial lipase activity was not significantly higher than psychrotolerant bacterial lipase, though all lipases showed remarkably high activity with 10–36% residual activity at 0°C.