Distribution and Diversity of Acyl Homoserine Lactone Producing Bacteria from Four Different Soils

The distribution and diversity of acyl homoserine lactone (AHL) producing bacteria in black, brown, red, and meadow soils were investigated using culture-dependent method. One out of seventy six and five out of fifty isolates from the black and the brown soil were AHL-biosensor active, respectively. They were affiliated to the genera Ensifer and Pseudomonas and the family Enterobacteriaceae. Thin layer chromatography showed that the most of them produced AHLs with acyl side chain of 6 or 8 carbons. No AHL-producer was found in red and meadow soils. A potential novel AHL-based quorum sensing and quenching system was identified by sequencing in the black soil isolate Ensifer adhaerens X097 that harbored two AHL synthetase-like proteins and one amidohydrolase-like protein. This is the first report of comparison of AHL-producers among different soils. Our data showed that composition of AHL-producers were niche specific and were not in proportion with community population.     

A Novel Degenerated Primer Pair Detects Diverse Genes of Acyl Homoserine Lactone Synthetase in Rhizobiaceae Family

A novel degenerated primer set was designed to amplify acyl homoserine lactone (AHL) synthetase genes from members of the family Rhizobiaceae. The primer set successfully amplified AHL synthetase genes from pure cultures of AHL producers from Rhizobiaceae, but not from AHL producers out of the Rhizobiaceae family, indicating the specificity of this primer set to the Rhizobiaceae family. An inoculation experiment showed that the minimal detectable concentration of AHL producers from the soil was around 2.5 × 10⁷ CFU/g soil. When applying to environmental samples, 7 and 14 different genotypes of AHL synthetase genes were identified in the rhizosphere of Glycine max and Vigna unguiculata, respectively, which revealed complicated and unknown AHL-based quorum-sensing networks in the rhizosphere. This is the first primer set that covers diverse AHL synthetase genes from different genera. It will be a useful culture-independent approach for better understanding of the ecological significance of QS in natural habitats.     

Anti-protease and Immunomodulatory Activities of Bacteria Associated with Caribbean Sponges

Marine sponges and their associated bacteria have been proven to be a rich source of novel secondary metabolites with therapeutic usefulness in cancer, infection, and autoimmunity. In this study, 79 strains belonging to 20 genera of the order Actinomycetales and seven strains belonging to two genera of the order Sphingomonadales were cultivated from 18 different Caribbean sponges and identified by 16S rRNA gene sequencing. Seven of these strains are likely to represent novel species. Crude extracts from selected strains were found to exhibit protease inhibition against cathepsins B and L, rhodesain, and falcipain-2 as well as immunomodulatory activities such as induction of cytokine release by human peripheral blood mononuclear cells. These results highlight the significance of marine sponge-associated bacteria to produce bioactive secondary metabolites with therapeutic potential in the treatment of infectious diseases and disorders of the immune system.     

Possible Quorum Sensing in Marine Snow Bacteria: Production of Acylated Homoserine Lactones by Roseobacter Strains Isolated from Marine Snow

We report here, for the first time, that bacteria associated with marine snow produce communication signals involved in quorum sensing in gram-negative bacteria. Four of 43 marine microorganisms isolated from marine snow were found to produce acylated homoserine lactones (AHLs) in well diffusion and thin-layer chromatographic assays based on the Agrobacterium tumefaciens reporter system. Three of the AHL-producing strains were identified by 16S ribosomal DNA gene sequence analysis as Roseobacter spp., and this is the first report of AHL production by these α-Proteobacteria. It is likely that AHLs in Roseobacter species and other marine snow bacteria govern phenotypic traits (biofilm formation, exoenzyme production, and antibiotic production) which are required mainly when the population reaches high densities, e.g., in the marine snow community.     

Production and Degradation of N-Acylhomoserine Lactone Quorum Sensing Signal Molecules in Bacteria Isolated from Activated Sludge

N-Acylhomoserine lactones (AHLs) function as quorum-sensing signaling molecules in many Gram-negative bacteria. We isolated a total of 672 bacterial strains from activated sludge obtained from seven sewage treatment plants in Tochigi Prefecture, Japan, and screened for AHL-producing and degrading strains. Isolates (n=107) stimulated AHL-mediated purple pigment production in AHL reporter strains Chromobacterium violaceum CV026 and VIR07. Based on their 16S rRNA gene sequences, most of these AHL-producing isolates were assigned to the genus Aeromonas, and they were divided into six groups. Isolates (n=46) degraded N-decanoyl-L-homoserine lactone (C10-HSL) within 24 h. Based on their 16S rRNA gene sequences, the most dominant AHL-degrading isolates were assigned to the genus Acinetobacter and divided into six groups. Strains Ooi24, Omo91, and Uzu81, which showed higher C10-HSL-degrading activity, showed putative AHL-acylase activity.     

Reaction of Acylated Homoserine Lactone Bacterial Signaling Molecules with Oxidized Halogen Antimicrobials

Oxidized halogen antimicrobials, such as hypochlorous and hypobromous acids, have been used extensively for microbial control in industrial systems. Recent discoveries have shown that acylated homoserine lactone cell-to-cell signaling molecules are important for biofilm formation in Pseudomonas aeruginosa, suggesting that biofouling can be controlled by interfering with bacterial cell-to-cell communication. This study was conducted to investigate the potential for oxidized halogens to react with acylated homoserine lactone-based signaling molecules. Acylated homoserine lactones containing a 3-oxo group were found to rapidly react with oxidized halogens, while acylated homoserine lactones lacking the 3-oxo functionality did not react. The Chromobacterium violaceum CV026 bioassay was used to determine the effects of such reactions on acylated homoserine lactone activity. The results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity. Experiments with the marine alga Laminaria digitata demonstrated that natural haloperoxidase systems are capable of mediating the deactivation of acylated homoserine lactones. This may illustrate a natural defense mechanism to prevent biofouling on the surface of this marine alga. The Chromobacterium violaceum activity assay illustrates that reactions between 3-oxo acylated homoserine lactone molecules and oxidized halogens do occur despite the presence of biofilm components at much greater concentrations. This work suggests that oxidized halogens may control biofilm not only via a cidal mechanism, but also by possibly interfering with 3-oxo acylated homoserine lactone-based cell signaling.     

Association of Thioautotrophic Bacteria with Deep-Sea Sponges

We investigated microorganisms associated with a deep-sea sponge, Characella sp. (Pachastrellidae) collected at a hydrothermal vent site (686 m depth) in the Sumisu Caldera, Ogasawara Island chain, Japan, and with two sponges, Pachastrella sp. (Pachastrellidae) and an unidentified Poecilosclerida sponge, collected at an oil seep (572 m depth) in the Gulf of Mexico, using polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) directed at bacterial 16S rRNA gene sequences. In the PCR-DGGE profiles, we detected a single clearly dominant band in each of the Characella sp. and the unidentified Poecilosclerida sponge. BLAST search of their sequences showed that they were most similar (>99% identity) to those of the gammaproteobacterial thioautotrophic symbionts of deep-sea bivalves from hydrothermal vents, Bathymodiolus spp. Phylogenetic analysis of the near-full length sequences of the 16S rRNA genes cloned from the unidentified Poecilosclerida sponge and Characella sp. confirmed that they were closely related to thioautotrophic symbionts. Although associations between sponges and methanotrophic bacteria have been reported previously, this is the first report of a possible stable association between sponges and thioautotrophic bacteria.     

Bacteria Associated with the Surface and Gut of Marine Copepods

Little is known about the nature of bacteria associated with the surface and gut of marine copepods, either in laboratory-reared animals or in the natural environment. Nor is it known whether such animals possess a gut flora. The present report deals with studies of microorganisms isolated from healthy, laboratory-reared copepods of the species Acartia tonsa Dana, from several species of wild copepods collected from a marine or estuarine environment, and from laboratory dishes containing moribund copepods. Evidence for a unique gut flora in laboratory-reared animals is presented; the predominant bacteria were represented by the genus Vibrio. Other organisms such as Pseudomonas and Cytophaga were found less abundantly associated with the copepods and not specifically associated with the gut.     

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.     

Inter- and Intraspecific Variations of Bacterial Communities Associated with Marine Sponges from San Juan Island,Washington

This study attempted to assess whether conspecific or congeneric sponges around San Juan Island, Washington, harbor specific bacterial communities. We used a combination of culture-independent DNA fingerprinting techniques (terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis [DGGE]) and culture-dependent approaches. The results indicated that the bacterial communities in the water column consisted of more diverse bacterial ribotypes than and were drastically different from those associated with the sponges. High levels of similarity in sponge-associated bacterial communities were found only in Myxilla incrustans and Haliclona rufescens, while the bacterial communities in Halichondria panicea varied substantially among sites. Certain terminal restriction fragments or DGGE bands were consistently obtained for different individuals of M. incrustans and H. rufescens collected from different sites, suggesting that there are stable or even specific associations of certain bacteria in these two sponges. However, no specific bacterial associations were found for H. panicea or for any one sponge genus. Sequencing of nine DGGE bands resulted in recovery of seven sequences that best matched the sequences of uncultured Proteobacteria. Three of these sequences fell into the sponge-specific sequence clusters previously suggested. An uncultured alphaproteobacterium and a culturable Bacillus sp. were found exclusively in all M. incrustans sponges, while an uncultured gammaproteobacterium was unique to H. rufescens. In contrast, the cultivation approach indicated that sponges contained a large proportion of Firmicutes, especially Bacillus, and revealed large variations in the culturable bacterial communities associated with congeneric and conspecific sponges. This study revealed sponge species-specific but not genus- or site-specific associations between sponges and bacterial communities and emphasized the importance of using a combination of techniques for studying microbial communities.Marine sponges (phylum Porifera) harbor a remarkable array of microorganisms, including bacteria (51, 54), unicellular algae (50), cyanobacteria (45, 48), dinoflagellates (14), zoochlorellae (58), and members of the domain Archaea (33). Of these microorganisms, bacteria are the most dominant group of microbial associates in sponges and can account for up to 40 to 50% of a sponge''s biomass (17). The density of bacteria can be up to 10⁸ to 10¹⁰ bacteria per g (wet weight) of sponge (18). The great abundance of bacteria in sponges caused workers to coin the term “bacteriosponges” (35) and has attracted much research interest in the role and specificity of the sponge-bacterium association.There are generally two pathways by which sponges may acquire their bacterial associates. The first pathway is filter feeding and selective retention of bacteria (44). Bacteria in the surrounding seawater can be captured by sponges when sponges filter the food particles out of the water column. The bacteria that resist digestion by sponge choanocytes and archaeocytes can survive and live inside the sponges. Sponge-associated bacterial communities acquired via this pathway are therefore heavily influenced by the type of bacteria in the water column. The second pathway is vertical transmission of bacterial associates from adult sponges to their progeny (10, 40, 49). Bacteria acquired through this pathway may exhibit specificity for genera or species of sponges due to coevolution (for a review, see reference 44).Sponge-bacterium symbioses are often mutualistic; while bacteria may benefit from the favorable nutritional conditions in sponges (18, 44), some bacterial associates may help their hosts eliminate metabolic waste (4), stabilize the sponge skeleton (36), and defend against pathogens, predators, or competitors via the production of bioactive secondary metabolites (5, 21, 39, 47). In addition, some cyanobacterial symbionts are a source of nutrients for their hosts because of their photosynthetic and nitrogen-fixing abilities (3, 57).There is a large body of knowledge concerning the possible roles of bacterial associates in sponges, but whether sponges harbor specific bacterial communities deserves more detailed study. Many previous investigations demonstrated that associated bacterial communities in certain species of sponges were highly similar and consistently different from the bacterial communities in the ambient environment (6, 22, 46). For instance, Hentschel et al. (16) showed that there were uniform microbial communities in the marine sponges Aplysina aerophoba, Rhopaloeides odorabile, and Theonella swinhoei from different geographic regions that were distinct from those in the water column or in sediments. In addition, Friedrich et al. (12) demonstrated that the composition of sponge-associated bacterial communities was resistant to environmental perturbations resulting from transplantation to different habitats. These studies suggest that there is a stable, specific, and perhaps mutualistic relationship between the two types of organisms (44). However, some bacterium-sponge symbioses do not appear to be consistent. For instance, Wichels et al. (56) demonstrated that the bacterial communities associated with the North Sea sponge Halichondria panicea varied substantially over time. Qian et al. (34) and Lee et al. (25) showed that the congeneric Callyspongia and Mycale sponges from different biogeographic regions had different bacterial associates. Although the discrepancies may be attributed to the methods employed in different studies, whether consistent sponge-bacterium associations occur in different species or genera of sponges remains unclear. Furthermore, most of the previous studies focused on only one sponge species or involved a few sponges from geographically separated regions. So far, there has been no large-scale study comparing the bacterial communities associated with sponges of different genera and species.In this study, we compared the bacterial communities associated with marine sponges around San Juan Island, Washington, in order to investigate the specificity of congeneric and conspecific sponge-associated bacterial communities. Most previous studies have relied on only one method to assess the bacterial communities, limiting the resolution of community assessments. In addition, different studies have used different approaches to address the same question, creating uncertainties and making generalizations difficult. Here, we employed both culture-independent and -dependent approaches to compensate for the limitations of different methods and to obtain a more reliable assessment of the associated bacterial communities. We used two DNA fingerprinting techniques, terminal restriction fragment length polymorphism (TRFLP) (27) and denaturing gradient gel electrophoresis (DGGE) (11). TRFLP is an effective, sensitive, high-throughput technique that differentiates bacterial community structures, while DGGE allows subsequent identification of bacteria of interest by excision and sequencing of specific bands. Both techniques have been successfully and widely used for characterization of bacterial communities in marine samples (13, 26, 28, 43, 56). In addition, bacteria associated with sponges were isolated using cultivation methods and identified by comparative analysis of 16S rRNA gene sequences. Phylogenetic affiliations of the isolates were determined and compared for different samples in order to study the specificity of the sponge-associated culturable bacterial communities.     

Evidence for a Methionine-controlled Homoserine Dehydrogenase in Salmonella typhimurium

Evidence is presented for the existence of a second homoserine dehydrogenase in Salmonella typhimurium. The formation, but not the activity, of this enzyme is controlled by methionine. Two distinct homoserine dehydrogenases were separated from wild-type cells by diethylaminoethyl (cellulose) column chromatography. Sucrose gradient ultracentrifugation gave molecular weight estimates for the threonine-regulated enzyme (HSD I) of 220,000 to 240,000 and for the methionine controlled enzyme (HSD II) of 130,000 to 140,000. Approximately 12% of the total HSD activity in wild-type cells was accounted for by HSD II. A threonine-requiring strain of S. typhimurium was found to lack HSD I but not HSD II. Under certain conditions, this mutant grew rapidly in minimal medium. Rapid growth in minimal medium was correlated with the appearance of an enzyme with similar characteristics to HSD I. The possible origins of this HSD I-like enzyme are presented.     

黄海繁茂膜海绵中微生物多样性的研究

构建了中国黄海繁茂膜海绵中细菌16S rDNA克隆,对其遗传多样性进行了分析,发现海绵中相关细菌16S rDNA基因主要归类于紫硫细菌门(Proteobacteria)中的α-亚门、γ-亚门,和放线菌门(Actinobacteria)等类群。所获得的16S rDNA序列与GenBank中的已知序列差异较大,反映出该海绵存在尚未发现的微生物新信息。     

Cell Turnover and Detritus Production in Marine Sponges from Tropical and Temperate Benthic Ecosystems

This study describes in vivo cell turnover (the balance between cell proliferation and cell loss) in eight marine sponge species from tropical coral reef, mangrove and temperate Mediterranean reef ecosystems. Cell proliferation was determined through the incorporation of 5-bromo-2′-deoxyuridine (BrdU) and measuring the percentage of BrdU-positive cells after 6 h of continuous labeling (10 h for Chondrosia reniformis). Apoptosis was identified using an antibody against active caspase-3. Cell loss through shedding was studied quantitatively by collecting and weighing sponge-expelled detritus and qualitatively by light microscopy of sponge tissue and detritus. All species investigated displayed substantial cell proliferation, predominantly in the choanoderm, but also in the mesohyl. The majority of coral reef species (five) showed between 16.1±15.9% and 19.0±2.0% choanocyte proliferation (mean±SD) after 6 h and the Mediterranean species, C. reniformis, showed 16.6±3.2% after 10 h BrdU-labeling. Monanchora arbuscula showed lower choanocyte proliferation (8.1±3.7%), whereas the mangrove species Mycale microsigmatosa showed relatively higher levels of choanocyte proliferation (70.5±6.6%). Choanocyte proliferation in Haliclona vansoesti was variable (2.8–73.1%). Apoptosis was negligible and not the primary mechanism of cell loss involved in cell turnover. All species investigated produced significant amounts of detritus (2.5–18% detritus bodyweight⁻¹·d⁻¹) and cell shedding was observed in seven out of eight species. The amount of shed cells observed in histological sections may be related to differences in residence time of detritus within canals. Detritus production could not be directly linked to cell shedding due to the degraded nature of expelled cellular debris. We have demonstrated that under steady-state conditions, cell turnover through cell proliferation and cell shedding are common processes to maintain tissue homeostasis in a variety of sponge species from different ecosystems. Cell turnover is hypothesized to be the main underlying mechanism producing sponge-derived detritus, a major trophic resource transferred through sponges in benthic ecosystems, such as coral reefs.     

Use of Cellulolytic Marine Bacteria for Enzymatic Pretreatment in Microalgal Biogas Production

In this study, we designed and evaluated a microalgal pretreatment method using cellulolytic bacteria that naturally degrades microalgae in their native habitat. Bacterial strains were isolated from each of two mollusk species in a medium containing 1% carboxymethyl cellulose agar. We selected nine bacterial strains that had endoglucanase activity: five strains from Mytilus chilensis, a Chilean mussel, and four strains from Mesodesma donacium, a clam found in the Southern Pacific. These strains were identified phylogenetically as belonging to the genera Aeromonas, Pseudomonas, Chryseobacterium, and Raoultella. The cellulase-producing capacities of these strains were characterized, and the degradation of cell walls in Botryococcus braunii and Nannochloropsis gaditana was tested with “whole-cell” cellulolytic experiments. Aeromonas bivalvium MA2, Raoultella ornithinolytica MA5, and Aeromonas salmonicida MC25 degraded B. braunii, and R. ornithinolytica MC3 and MA5 degraded N. gaditana. In addition, N. gaditana was pretreated with R. ornithinolytica strains MC3 and MA5 and was then subjected to an anaerobic digestion process, which increased the yield of methane by 140.32% and 158.68%, respectively, over that from nonpretreated microalgae. Therefore, a “whole-cell” cellulolytic pretreatment can increase the performance and efficiency of biogas production.     

Ferric Iron Reduction by Bacteria Associated with the Roots of Freshwater and Marine Macrophytes

In vitro assays of washed, excised roots revealed maximum potential ferric iron reduction rates of >100 μmol g (dry weight)⁻¹ day⁻¹ for three freshwater macrophytes and rates between 15 and 83 μmol (dry weight)⁻¹ day⁻¹ for two marine species. The rates varied with root morphology but not consistently (fine root activity exceeded smooth root activity in some but not all cases). Sodium molybdate added at final concentrations of 0.2 to 20 mM did not inhibit iron reduction by roots of marine macrophytes (Spartina alterniflora and Zostera marina). Roots of a freshwater macrophyte, Sparganium eurycarpum, that were incubated with an analog of humic acid precursors, anthroquinone disulfate (AQDS), reduced freshly precipitated iron oxyhydroxide contained in dialysis bags that excluded solutes with molecular weights of >1,000; no reduction occurred in the absence of AQDS. Bacterial enrichment cultures and isolates from freshwater and marine roots used a variety of carbon and energy sources (e.g., acetate, ethanol, succinate, toluene, and yeast extract) and ferric oxyhydroxide, ferric citrate, uranate, and AQDS as terminal electron acceptors. The temperature optima for a freshwater isolate and a marine isolate were equivalent (approximately 32°C). However, iron reduction by the freshwater isolate decreased with increasing salinity, while reduction by the marine isolate displayed a relatively broad optimum salinity between 20 and 35 ppt. Our results suggest that by participating in an active iron cycle and perhaps by reducing humic acids, iron reducers in the rhizoplane of aquatic macrophytes limit organic availability to other heterotrophs (including methanogens) in the rhizosphere and bulk sediments.     

Multiple N-Acyl Homoserine Lactone Signals of Rhizobium leguminosarum Are Synthesized in a Distinct Temporal Pattern

A common form of bacterial quorum sensing involves the production and release of acyl homoserine lactone (AHL) signal metabolites. The nitrogen-fixing symbiont Rhizobium leguminosarum reportedly produces at least six different AHLs, but little is known about the regulation of biosynthesis of these molecules. We used a radiolabeling protocol to quantify the relative amounts of AHLs synthesized over time by R. leguminosarum cells with and without the symbiosis plasmid pRL1JI. Cells containing pRL1JI were found to produce three predominant signals. In decreasing order of abundance, these were N-(3-oxo)octanoyl homoserine lactone [(3-O)C(8)HSL], N-octanoyl homoserine lactone, and N-hexanoyl homoserine lactone. Cells without pRL1JI produced only two major signals, N-(3-hydroxy-7-cis)tetradecanoyl homoserine lactone [(3-OH)C(14:1)HSL] and (3-O)C(8)HSL. Each AHL exhibited a distinct temporal pattern of synthesis, suggesting that each AHL is subject to unique regulatory mechanisms. While (3-O)C(8)HSL was produced in both cultures, the patterns of synthesis were different in cells with and without pRL1JI, possibly as a result of redundant gene functions that are present on both the chromosome and the symbiosis plasmid. None of the AHLs appeared to regulate its own biosynthesis, although exogenous (3-OH)C(14:1)HSL did activate synthesis of the three AHLs made by cells containing pRL1JI. These results indicate that the synthesis of multiple AHLs in R. leguminosarum is regulated by complex mechanisms that operate independently of quorum sensing itself but that (3-OH)C(14:1)HSL can supersede these controls in pRL1JI-containing cells. This work provides an important global perspective for AHL regulation that both complements and contrasts with the results of previous studies performed with isolated gene systems.