Bacteria Associated with Benthic Diatoms from Lake Constance: Phylogeny and Influences on Diatom Growth and Secretion of Extracellular Polymeric Substances

The composition of diatom-associated bacterial communities was studied with 14 different unialgal xenic diatom cultures isolated from freshwater epilithic biofilms of Lake Constance, Germany. A clear dominance of Alphaproteobacteria was observed, followed by Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia. Pure cultures of the diatom Cymbella microcephala, which was found to be dominant in epilithic biofilms in Lake Constance, were cocultivated with six associated bacterial strains. All these bacterial strains were able to grow in C. microcephala cultures in the absence of organic cosubstrates. Diatom growth was generally enhanced in the presence of bacteria, and polysaccharide secretion was generally increased in the presence of Proteobacteria. The monomer composition of extracellular polysaccharides of C. microcephala changed in relation to the presence of different bacteria, but the dominant monomers were less affected. Our results indicate that these changes were caused by the diatom itself rather than by specific bacterial degradation. One Bacteroidetes strain strongly influenced carbohydrate secretion by the alga via extracellular soluble compounds. Biofilms were formed only in the presence of bacteria. Phylogenetic analysis and coculture studies indicate an adaptation of Proteobacteria and Bacteroidetes to the microenvironment created by the diatom biofilm.     

Benzylideneacetone and other phenylethylamide bacterial metabolites induce apoptosis to kill insects

Xenorhabdus and Photorhabdus are entomopathogenic bacteria that can induce immunosuppression against target insects by suppressing eicosanoid biosynthesis, leading to fatal septicemia. These bacteria can synthesize and release secondary metabolites such as benzylideneacetone (BZA) and other phenylethylamide compounds that can inhibit phospholipase A₂ (PLA₂) and shut down eicosanoid biosynthesis. However, insecticidal activities of these bacterial metabolites remain unclear. Thus, the objective of this study was to assess cytotoxicities of BZA and seven other bacterial metabolites to insect cells. These eight bacterial metabolites exhibited significant cytotoxicities against an insect cell line Sf9 at micromolar range. Especially, BZA and cPY were highly potent at low micromolar range. When these eight bacterial metabolites were injected to hemocoels of Spodoptera exigua larvae, they significantly decreased total count of hemocytes. In Sf9 cell line and hemocytes, these bacterial metabolites induced cell membrane blebbings, apoptotic vesicles, and genomic DNA fragmentation. Terminal deoxyribonucleotidyl transferase nick end translation assay showed that these bacterial metabolites caused significant DNA breakages in cells in a dose-dependent manner. However, a pan caspase inhibitor treatment significantly rescued the cell death induced by these bacterial metabolites. Cytotoxicities of these bacterial metabolites were highly correlated with their insecticidal activities. These results indicate that the insecticidal activities of the bacterial metabolites may be induced by their apoptotic activities against hemocytes and other insect cells. Taken together, these results suggest that phenylethylamide compounds might have potential as novel insecticides.     

Assessing the diversity of bacterial communities from marine sponges and their bioactive compounds

Symbiotic bacteria play vital roles in the survival and health of marine sponges. Sponges harbor rich, diverse and species-specific microbial communities. Symbiotic marine bacteria have increasingly been reported as promising source of bioactive compounds. A culturomics-based study was undertaken to study the diversity of bacteria from marine sponges and their antimicrobial potential. We have collected three sponge samples i.e. Acanthaster carteri, Rhytisma fulvum (soft coral) and Haliclona caerulea from north region (Obhur) of Red Sea, Jeddah Saudi Arabia. Total of 144 bacterial strains were isolated from three marine sponges using culture dependent method. Screening of isolated strains showed only 37 (26%) isolates as antagonists against oomycetes pathogens (P. ultimum and P. capsici). Among 37 antagonistic bacteria, only 19 bacterial strains exhibited antibacterial activity against human pathogens (Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 8739, Enterococcus faecalis ATCC 29212). Four major classes of bacteria i.e γ-Proteobacteria, α-Proteobacteria, Firmicutes and Actinobacteria were recorded from three marine sponges where γ-Proteobacteria was dominant class. One potential bacterial strain Halomonas sp. EA423 was selected for identification of bioactive metabolites using GC and LC-MS analyses. Bioactive compounds Sulfamerazine, Metronidazole-OH and Ibuprofen are detected from culture extract of strain Halomonas sp. EA423. Overall, this study gives insight into composition and diversity of antagonistic bacterial community of marine sponges and coral from Red Sea and presence of active metabolites from potential strain. Our results showed that these diverse and potential bacterial communities further need to be studied to exploit their biotechnological significance.     

Resource partitioning of phytoplankton metabolites that support bacterial heterotrophy

The communities of bacteria that assemble around marine microphytoplankton are predictably dominated by Rhodobacterales, Flavobacteriales, and families within the Gammaproteobacteria. Yet whether this consistent ecological pattern reflects the result of resource-based niche partitioning or resource competition requires better knowledge of the metabolites linking microbial autotrophs and heterotrophs in the surface ocean. We characterized molecules targeted for uptake by three heterotrophic bacteria individually co-cultured with a marine diatom using two strategies that vetted the exometabolite pool for biological relevance by means of bacterial activity assays: expression of diagnostic genes and net drawdown of exometabolites, the latter detected with mass spectrometry and nuclear magnetic resonance using novel sample preparation approaches. Of the more than 36 organic molecules with evidence of bacterial uptake, 53% contained nitrogen (including nucleosides and amino acids), 11% were organic sulfur compounds (including dihydroxypropanesulfonate and dimethysulfoniopropionate), and 28% were components of polysaccharides (including chrysolaminarin, chitin, and alginate). Overlap in phytoplankton-derived metabolite use by bacteria in the absence of competition was low, and only guanosine, proline, and N-acetyl-d-glucosamine were predicted to be used by all three. Exometabolite uptake pattern points to a key role for ecological resource partitioning in the assembly marine bacterial communities transforming recent photosynthate.Subject terms: Microbial ecology, Bacteria     

Diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii

Aggregation of algae, mainly diatoms, is an important process in marine systems leading to the settling of particulate organic carbon predominantly in the form of marine snow. Exudation products of phytoplankton form transparent exopolymer particles (TEP), which acts as the glue for particle aggregation. Heterotrophic bacteria interacting with phytoplankton may influence TEP formation and phytoplankton aggregation. This bacterial impact has not been explored in detail. We hypothesized that bacteria attaching to Thalassiosira weissflogii might interact in a yet-to-be determined manner, which could impact TEP formation and aggregate abundance. The role of individual T. weissflogii-attaching and free-living new bacterial isolates for TEP production and diatom aggregation was investigated in vitro. T. weissflogii did not aggregate in axenic culture, and striking differences in aggregation dynamics and TEP abundance were observed when diatom cultures were inoculated with either diatom-attaching or free-living bacteria. The data indicated that free-living bacteria might not influence aggregation whereas bacteria attaching to diatom cells may increase aggregate formation. Interestingly, photosynthetically inactivated T. weissflogii cells did not aggregate regardless of the presence of bacteria. Comparison of aggregate formation, TEP production, aggregate sinking velocity and solid hydrated density revealed remarkable differences. Both, photosynthetically active T. weissflogii and specific diatom-attaching bacteria were required for aggregation. It was concluded that interactions between heterotrophic bacteria and diatoms increased aggregate formation and particle sinking and thus may enhance the efficiency of the biological pump.     

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.     

Competition-Mediated Antibiotic Induction in the Marine Bacterium Streptomyces tenjimariensis

Microbial competition for limiting natural resources within a community is thought to be the selective force that promotes biosynthesis of antimicrobial compounds The marine bacterium Streptomyces tenjimariensis produces the antibiotics istamycin A and B under select laboratory culture conditions; presumably these compounds serve an, ecological role under natural conditions. Here we report results of a novel marine microbial competion experiment that examined the impact of co-culture of marine bacteria on istamycin production by S. tenjimariensis. Twelve of the 53 bacterial species tested (i.e., 22.6%) induced Istamycin production; this antibiotic also inhibited growth of the competitor colonies. These results suggest that marine bacterial metabolites, serve an ecological role in countering competitive species.     

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.     

Antimicrobial activity of bacteria from marine sponge Suberea mollis and bioactive metabolites of Vibrio sp. EA348

Discovery of potential bioactive metabolites from sponge-associated bacteria have gained attraction in recent years. The current study explores the potential of sponge (Suberea mollis) associated bacteria against bacterial and fungal pathogens. Sponge samples were collected from Red sea in Obhur region, Jeddah, Saudi Arabia. Of 29 isolated bacteria belong to four different classes i.e. Firmicutes (62%), γ-Proteobacteria (21%), α-Proteobacteria (10%) and Actinobacteria (7%). Among them nineteen (65%) bacterial strains showed antagonistic activity against oomycetes and only 3 (10%) bacterial strains were active against human pathogenic bacteria tested. Most bioactive genera include Bacillus (55%), Pseudovibrio (13%) and Ruegeria (10%). Enzyme production (protease, lipase, amylase, cellualse) was identified in 12 (41%) bacterial strains where potential strains belonging to γ-Proteobacteria and Firmicutes groups. Production of antimicrobial metabolites and hydrolysates in these bacteria suggest their potential role in sponge against pathogens. Further bioactive metabolites from selected strain of Vibrio sp. EA348 were identified using LC-MS and GC–MS analyses. We identified many active metabolites including antibiotics such as Amifloxacin and fosfomycin. Plant growth hormones including Indoleacetic acid and Gibberellin A3 and volatile organic compound such as methyl jasmonate were also detected in this strain. Our results highlighted the importance of marine bacteria inhabiting sponges as potential source of antimicrobial compounds and plant growth hormones of pharmaceutical and agricultural significance.     

Production of Wax Esters during Aerobic Growth of Marine Bacteria on Isoprenoid Compounds

This paper describes the production of isoprenoid wax esters during the aerobic degradation of 6,10,14-trimethylpentadecan-2-one and phytol by four bacteria (Acinetobacter sp. strain PHY9, Pseudomonas nautica [IP85/617], Marinobacter sp. strain CAB [DSMZ 11874], and Marinobacter hydrocarbonoclasticus [ATCC 49840]) isolated from the marine environment. Different pathways are proposed to explain the formation of these compounds. In the case of 6,10,14-trimethylpentadecan-2-one, these esters result from the condensation of some acidic and alcoholic metabolites produced during the biodegradation, while phytol constitutes the alcohol moiety of most of the esters produced during growth on this isoprenoid alcohol. The amount of these esters formed increased considerably in N-limited cultures, in which the ammonium concentration corresponds to conditions often found in marine sediments. This suggests that the bacterial formation of isoprenoid wax esters might be favored in such environments. Although conflicting evidence exists regarding the stability of these esters in sediments, it seems likely that, under some conditions, bacterial esterification can enhance the preservation potential of labile compounds such as phytol.     

Cytotoxicity and apoptosis induction of some selected marine bacteria metabolites

AIMS: To study the potential apoptosis effects of cytotoxic marine bacterial metabolites on human HeLa cell line. METHODS AND RESULTS: After HeLa cells were routinely cultured, tetrazolium-based colorimetric assay for cytotoxicity was performed to screen the marine bacteria extracts showing 12 strains active. To find the potential active strain with apoptosis mechanism, a battery of apoptosis assays, including AO/EB staining, TUNEL assay (terminal-deoxynucleotidyl transferase mediated nick end labelling), gel electrophoresis and flow cytometry, were used to determine whether apoptosis was involved in HeLa cell cytotoxicity of marine bacterial extracts. The results indicated that four strains could induce cell shrinkage, cell membrane blebbing, formation of apoptotic body and DNA fragmentation. CONCLUSIONS: Crude extracts of 12 of 153 strains of marine bacteria showed cytotoxic effects with ID50 ranged from 77.20 to 199.84 microg ml(-1), in which eight strains of bacteria were associated bacteria. The metabolites in the strains of QD1-2, NJ6-3-1, NJ1-1-1 and SS6-4 were able to induce HeLa cells apoptosis. Furthermore, the assessment by flow cytometry indicated that the hypodiploid apoptotic cells increased in a time-dependent manner, suggesting that induced apoptosis occurred from 24 h to 48 h after the extracts treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggested that the compounds from fermentation in these four marine bacterial strains could be candidates for developing apoptosis specific anti-tumour agents with lower toxicity. This study indicated that associated marine bacteria could be good source to find cytotoxic metabolites, and some cytotoxic marine bacterial metabolites could have apoptosis mechanisms.     

Phylogenetic diversity of bacteria associated with ascidians in Eel Pond (Woods Hole, Massachusetts, USA)

Recent studies have revealed that many marine invertebrates are closely associated with diverse microorganisms, frequently resulting in the production of compounds of biomedical interest. Thus far, ascidians have not been widely examined for the presence of bacterial associations, although the production of secondary metabolites is well documented. In the present study, we examined the gonad of Molgula manhattensis and the tunic surfaces of Botryllus schlosseri, Didemnum sp., and Botrylloides violaceus for the presence of associated bacteria. These ascidians are common inhabitants of the coastal ocean of Cape Cod, Massachusetts. We used denaturing gradient gel electrophoresis (DGGE) as well as cloning and sequencing of 16S rDNA to analyze the microbial communities. There is a strong evidence that spiroplasma-like bacteria colonize the gonad of the solitary ascidian M. manhattensis. The bacteria might be vertically transmitted and may be involved in the production of secondary metabolites that deter predators of the ascidians. The bacterial communities found on the tunic surfaces of the colonial ascidians were found to be more diverse. However, in all cases the bacterial communities were predominated by alpha-proteobacteria. Alpha-proteobacteria related to the obtained sequences have been identified as symbionts in a variety of hosts, suggesting a specific role for these bacteria. However, based on our data it is difficult to differentiate between persistently and only transiently associated bacteria. Overall, this study demonstrates that ascidian species are associated with diverse bacterial populations. Future studies will aim to elucidate the precise relationships between bacteria and ascidians and to identify bioactive compounds that might be produced as a result of these relationships.     

Extracts of seaweeds as potential inhibitors of quorum sensing and bacterial growth

Macroalgae are an important source of antimicrobial compounds. However, it is unclear if these compounds are produced by the algae themselves, by their associated bacteria, or by both. The main aim of this study was to investigate the potential of macroalgae and their associated microorganisms to inhibit bacterial quorum sensing (QS) and growth. Before extraction, half of the algal specimens were treated with 30% ethanol to remove surface associated bacteria. Canistrocarpus cervicornis extracts were able to inhibit QS of the reporter Chromobacterium violaceum CV017, where extracts with associated bacteria were more efficient than those without bacteria. However, not all algal extracts that inhibited QS of CV017 were able to inhibit bacterial attachment of Pseudomonas aeruginosas PA01, showing specific activity of algal metabolites. Only 58% of the extracts showed antibacterial activity against eight marine fouling and pathogenic bacterial strains tested. Our data suggests that algae and their associated microbiota are important sources of antimicrobial compounds which potentially can be used in future biotechnological applications.