Interactions between the unicellular red alga Rhodella reticulata (Rhodophyta) and contaminated bacteria

AIMS: To define the role of the bacterial strains LR1 and LR3 in the Rhodella cell destruction caused by Cytophaga sp.LR2. METHODS AND RESULTS: The bacteria were obtained from algal culture with destruction. They were isolated in pure culture and tested for biochemical activities using Polymicrotest. The ability of bacteria to degrade and utilize the algal polysaccharide was investigated. The bacteria were grown in a media containing Rhodella polysaccharide as a sole carbon source. The level of the reducing sugars in the culture media was determined. Scanning electron microscopy (SEM) was used to define the location of bacteria in extensively and intensively cultivated Rhodella reticulata previously infected by Cytophaga sp. LR2. CONCLUSIONS: The lysis of Rhodella reticulata cells is due to the joint action of the three bacterial strains with the former pathogen Cytophaga sp. LR2 playing the main role. The accumulation of the polysaccharide and the excreted metabolites of the strains LR1 and LR3 stimulated the development of Cytophaga sp. LR2. The adaptation of the strain to particular conditions of alga cultivation and the utilization of polysaccharide as a sole carbon source supported its stable growth in alga suspension and destruction of Rhodella cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The predominance of Cytophaga sp. LR2 over the two other contaminants and the lysis of Rhodella reticulata cells resulted from the ability of the bacterium to attach to the algal polysaccharide sheath. The formation of slime and extrusions facilitated the phenomenon of bacterial adhesion to the algal surface as well as the formation of colonial alga - bacterial spherules. The sedimentation of these aggregates decreased the ability of the algal strain to photosynthesize, led to the lysis of the cells and finally caused the death of Rhodella.     

Extra- and intra-cellular lytic effects of Cytophaga sp. LR2 on the red microalgae Rhodella reticulata

AIMS: To evaluate the lytic activities of crude enzymes from Cytophaga sp. LR2 on Rhodella reticulata cells and isolated algal polysaccharide. METHODS AND RESULTS: The Cytophaga compartment was separated after centrifugation in a cell suspension for 30 min at 18,000 g. The extracellular enzyme was obtained from the supernatant and the intracellular from the pelleted cells after sonication and removal of debris. Algal cells were incubated with extra- or intracellular preparations and sowed onto agar medium. The suppressive effect of the extracellular enzyme on colony-forming units was found to be almost twice as high. The result was still more pronounced when treated cells had been shocked osmotically before seeding. Saccharolytic activity was evaluated by changes in the reducing sugars in the media. Concerning isolated algal polysaccharide, the reducing power of the two bacterial preparates was relatively low. A combined fraction showed the highest lytic activity. Using native and SDS electrophoresis some relation between the prevalence of the extra and intracellular protein patterns was registered. Two of the common components' molecular weight masses of 50 and 21 kDa were found to be reproducible in native- and SDS-containing gel. CONCLUSIONS: Cytophaga sp. LR2 produce extra- and intracellular enzymes active in destroying Rhodella cultures. The agents excreted in the medium are more effective.We suppose that two or three different classes of enzymes are involved in the lysis process. The comparative electrophoresis in this case shows the protein components with predictable functions. SIGNIFICANCE AND IMPACT OF THE STUDY: Combining different simple and reproducible approaches to identify the lytic capability of Cytophaga sp. LR2 on R. reticulata.     

Responses of the coastal bacterial community to viral infection of the algae Phaeocystis globosa

The release of organic material upon algal cell lyses has a key role in structuring bacterial communities and affects the cycling of biolimiting elements in the marine environment. Here we show that already before cell lysis the leakage or excretion of organic matter by infected yet intact algal cells shaped North Sea bacterial community composition and enhanced bacterial substrate assimilation. Infected algal cultures of Phaeocystis globosa grown in coastal North Sea water contained gamma- and alphaproteobacterial phylotypes that were distinct from those in the non-infected control cultures 5 h after infection. The gammaproteobacterial population at this time mainly consisted of Alteromonas sp. cells that were attached to the infected but still intact host cells. Nano-scale secondary-ion mass spectrometry (nanoSIMS) showed ∼20% transfer of organic matter derived from the infected ¹³C- and ¹⁵N-labelled P. globosa cells to Alteromonas sp. cells. Subsequent, viral lysis of P. globosa resulted in the formation of aggregates that were densely colonised by bacteria. Aggregate dissolution was observed after 2 days, which we attribute to bacteriophage-induced lysis of the attached bacteria. Isotope mass spectrometry analysis showed that 40% of the particulate ¹³C-organic carbon from the infected P. globosa culture was remineralized to dissolved inorganic carbon after 7 days. These findings reveal a novel role of viruses in the leakage or excretion of algal biomass upon infection, which provides an additional ecological niche for specific bacterial populations and potentially redirects carbon availability.     

Lysis of a red-tide causing alga,Alexandrium tamarense,caused by bacteria from its phycosphere

There are bacteria coexisting in xenic cultures of Alexandrium tamarense, a red-tide causing alga. However little is known concerning the interactions between the alga and the bacteria in its phycosphere. The objective of the current study was to determine the effect of the bacteria in its phycosphere on the growth of the alga. We added one percent (v/v) Zobell 2216E medium to A. tamarense culture to alter bacterial growth and the results showed that algal cells were all lysed within 14 h. After adding the medium, both the abundance and the extracellular enzyme activity of the bacteria increased by 50–100 times from the 4th to the 10th hour which resulted in lysis of the algae. The 16S rRNA gene fragments of the bacteria were amplified from the DNA extracted from A. tamarense cultures and analyzed by denaturing gradient gel electrophoresis and sequencing. The structure of the bacterial community in phycosphere changed significantly during algal lysis. Two bacterial genera, Alteromonas sp. and Thalassobius aestuarii sp. are key factors that caused the lysis, and the β-glucosidase and chitinase produced by the bacteria in the phycosphere could directly cause the lysis. These experiments provide evidence that bacteria in its phycosphere play a key role in the culture of A. tamarense, and may provide insights into the future biocontrol of red-tides.     

Effect of Bacteria Associated with the Green Alga Ulva reticulata on Marine Micro- and Macrofouling

The green alga Ulva reticulata (Forsskal) is often free from biofouling in Hong Kong waters. An early study indicated that bioactive substances from this alga inhibit settlement of the polychaete Hydroides elegans (Haswell). It is also predicted that epibiotic bacteria protect this alga from micro- and macrofouling. In this study, bacterial strains from the surface of U. reticulata were isolated and their inhibitive activities on micro- and macrofouling assayed. The strains were identified by 16S rRNA analysis as belonging to the genera Alteromonas , Pseudoalteromonas and Vibrio . There was no significant effect of these strains or their extracts (aqueous and ethanol) on the growth of five Vibrio strains isolated from natural biofilm. Two bacterial strains ( Alteromonas sp. and Vibrio sp. 3) were non-toxic to the benthic diatom Nitzschia paleacea (Grunow) while the other five strains caused a low level of mortality. No one bacterial strain was toxic to the larvae of H. elegans . Aqueous extract of one of the isolated bacterial species, i.e. Vibrio sp. 2, significantly ( p <0.00001) inhibited the settlement and metamorphosis of H. elegans larvae. The putative antifouling compounds have a molecular weight of >100 kD. On the other hand, biofilm of Pseudoalteromonas sp. 2 and aqueous extract of Vibrio sp. 2 suppressed the settlement of larvae induced by 3-isobutyl-1-methylxanthine (IBMX). Other epibiotic bacteria and their extracts had neither inhibitive nor inductive effects on larval settlement of H. elegans . The results indicate that the antifouling mechanism of U. reticulata may be dependent not only on materials from the macroalga itself but also on the epibiotic bacteria on the algal surface.     

Growth of bacteria on chitin, fungal cell walls and fungal biomass, and the effect of extracellular enzymes produced by these cultures on the antifungal activity of amphotericin B

Vibrio alginolyticus, Streptomyces griseus, Arthrobacter G12, Bacillus sp. and Cytophaga sp. were grown on solid and liquid media containing soluble and insoluble carbon sources. Arthrobacter G12, Bacillus sp. and Cytophaga sp. grew well on media which contained fungal cell walls or fungal biomass as the main carbon source. All bacteria produced extracellular proteases and all bacteria except Arthrobacter G12 produced extracellular chitinases. Growth of Cytophaga sp. on colloidal chitin was paralleled by the accumulated chitinase activity in the culture filtrate, and growth of Cytophaga sp. and Arthrobacter G12 on cell walls of Geotrichum candidum and cell walls of Candida pseudotropicalis was paralleled by the accumulation of laminarinase activity in the culture filtrate, but little or no extracellular chitinase activity was observed in these cultures. Mycolases purified from the culture filtrates of Cytophaga sp. grown on colloidal chitin on cell walls of C. pseudotropicalis potentiated the antifungal activity of amphotericin B.     

Increased extracellular production of a cholinesterase-solubilising factor by Cytophaga NCMB 1314 during magnesium starvation

A Cytophaga sp. with the property of liberating a cholinesterase which is found in body muscle of plaice was studied. The liberation was caused by a factor of which more than 90% was found outside the bacterial cell and might possibly be associated within the slime material surrounding the bacteria. Magnesium limitation during growth of Cytophaga sp. in batch cultures resulted in an about 10-fold increase in extracellular factor activity. The increase could be immediately stopped by addition of magnesium ions or chloramphenicol to the medium. The effect of the latter might indicate that the increase in factor activity is dependent on protein synthesis under magnesium-limiting conditions.     

Do detached root-cap cells influence bacteria associated with maize roots?

Abstract. A model rhizosphere has been used which consisted of detached root-cap cells of maize in their surrounding root-cap mucilage on the surface of Noble agar. These cells were co-cultured for periods up to 32 d with eight different bacterial isolates from soil-grown roots and surrounding soil and two laboratory cultures. Cap cells were unaffected by the bacteria. There were five different type-specific responses of the bacteria in proximity to the cap cells. There were, strong growth inhibition ( Rhizobium sp. and Escherichia coli ), strong stimulation ( Pseudomonas fluorescens , laboratory strain), mixed weak inhibition or stimulation ( Pseudomonas fluorescens , field isolate), early inhibition followed by strong stimulation then spore formation ( Bacillus spp.), no effect ( Streptomyces sp. and Cytophaga sp.). It is concluded that detached root-cap cells are actively involved in the establishment of characteristic rhizosphere bacterial microflora.     

溶微囊藻细菌的富集筛选及其菌群结构特征

利用铜绿微囊藻(Microcystis aeruginosa)作为溶藻对象富集、筛选, 获得一个稳定的溶藻菌群。采用叶绿素、PCR和变性梯度凝胶电泳(DGGE)方法研究溶藻过程及其细菌种群结构的变化。结果显示, 富集的溶藻菌经1×10-5稀释后仍有显著溶藻效果。Rubritepida菌C1、假单胞菌C2和鞘氨醇单胞菌C3是存在于铜绿微囊藻中的3种伴生细菌。加入富集的溶藻菌群后, 菌群结构发生明显的变化, Rubritepida菌C1、假单胞菌C2消失, 混合菌群包含未培养黄杆菌A2、鞘氨醇单胞菌C3和噬氢     

Characteristics of adhesion of epiphytic bacteria on leaves of the seagrass Zostera marina and on abiotic surfaces

A comparative study of the adhesion of epiphytic bacteria and marine free-living, saprophytic, and pathogenic bacteria on seagrass leaves and abiotic surfaces was performed to prove the occurrence of true epiphytes of Zostera marina and to elucidate the bacterium-plant symbiotrophic relationships. It was shown that in the course of adhesion to the seagrass leaves of two taxonomically different bacteria, Cytophaga sp. KMM 3552 and Pseudoalteromonas citrea KMM 461, isolated from the seagrass surface, the number of viable cells increased 3-7-fold after 60 h of incubation, reaching 1.0-2.0 x 10(5) cells/cm2; however, in the case of adhesion of these bacteria to abiotic surfaces, such as glass or metal, virtually no viable cells were observed after 60 h of incubation. Such selectivity of cell adhesion was not observed in the case of three other bacterial species studied, viz., Vibrio alginolyticus KMM 3551, Bacillus subtilis KMM 430, and Pseudomonas aeruginosa KMM 433. The amount of viable cells of V. alginolyticus KMM 3551 adsorbed on glass and metal surfaces increased twofold after 40 h of incubation. The cells of saprophytic B. subtilis KMM 430 and pathogenic P. aeruginosa KMM 433 adsorbed on three studied substrata remained viable for 36 h and died by the 60th hour of incubation.     

Evaluation of candidate probiotic strains for gilthead sea bream larvae (Sparus aurata) using an in vivo approach

AIMS: The aim of this study was to evaluate the effect of six bacterial strains on gilthead sea bream larvae (Sparus aurata). METHODS AND RESULTS: Six bacterial strains isolated from well-performing live food cultures were identified by sequencing fragments of their 16s rDNA genome to the genus level as Cytophaga sp., Roseobacter sp., Ruergeria sp., Paracoccus sp., Aeromonas sp. and Shewanella sp. Survival rates of gilthead sea bream larvae transferred to seawater added these bacterial strains at concentrations of 6 +/- 0.3 x 10(5) bacteria ml(-1) were similar to those of larvae transferred to sterilized seawater and showed an average of 86% at 9 days after hatching, whereas, survival rates of larvae transferred to filtered seawater were lower (P < 0.05), and showed an average of 39%, 9 days after hatching. CONCLUSION: Several bacterial strains isolated from well-performing live food cultures showed a positive effect for sea bream larvae when compared with filtered seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach used in this study could be applied as an in vivo evaluation method of candidate probiotic strains used in the rearing of marine fish larvae.     

Algal growth enhancement by bacteria: Is consumption of photosynthetic oxygen involved?

Abstract: Pseudomonas diminuta and P. vesicularis , two obligate aerobes isolated from laboratory algal cultures, stimulated the growth of the green microalgae Scenedesmus bicellularis and Chlorella sp., without releasing any growth promoting substance. An intimate contact between both microorganisms was necessary for significant algal growth enhancement. The possibility of algal growth stimulation by bacterial attenuation of photosynthetic oxygen tension was indirectly examined by simulating the effect of bacteria through a physical removal of oxygen (air suction). Vacuum-treated cultures showed an increase in growth rate and photosynthetic activity as compared to the control, a result which cannot be explained by differences in CO₂/HCO₃⁻ pump activity. In the presence of P. diminuta , the photosynthetic activity of S. bicellularis was more strongly stimulated under a limited concentration of inorganic carbon. It is suggested that, apart from a CO₂ supply, aerobic bacteria can promote algal growth by reducing the photosynthetic oxygen tension within the microenvironment of the algal cells, thereby creating more favorable conditions for optimal photosynthetic algal growth.     

The impact of associated bacteria on morphology and physiology of the dinoflagellate Alexandrium tamarense

Despite their potential impact on phytoplankton dynamics and biogeochemical cycles, biological associations between algae and bacteria are still poorly understood. The aim of the present work was to characterize the influence of bacteria on the growth and function of the dinoflagellate Alexandrium tamarense. Axenic microalgal cultures were inoculated with a microbial community and the resulting cultures were monitored over a 15-month period, in order to allow for the establishment of specific algal–bacterial associations. Algal cells maintained in these new mixed cultures first experienced a period of growth inhibition. After several months, algal growth and cell volume increased, and indicators of photosynthetic function also improved. Our results suggest that community assembly processes facilitated the development of mutualistic relationships between A. tamarense cells and bacteria. These interactions had beneficial effects on the alga that may be only partly explained by mixotrophy of A. tamarense cells. The potential role of organic exudates in the establishment of these algal–bacterial associations is discussed. The present results do not support a role for algal–bacterial interactions in dinoflagellate toxin synthesis. However, variations observed in the toxin profile of A. tamarense cells during culture experiments give new clues for the understanding of biosynthetic pathways of saxitoxin, a potent phycotoxin.     

Different co-occurring bacteria enhance or decrease the growth of the microalga Nannochloropsis sp. CCAP211/78

Marine photosynthetic microalgae are ubiquitously associated with bacteria in nature. However, the influence of these bacteria on algal cultures in bioreactors is still largely unknown. In this study, eighteen different bacterial strains were isolated from cultures of Nannochloropsis sp. CCAP211/78 in two outdoor pilot-scale tubular photobioreactors. The majority of isolates was affiliated with the classes Alphaproteobacteria and Flavobacteriia. To assess the impact of the eighteen strains on the growth of Nannochloropsis sp. CCAP211/78, 24-well plates coupled with custom-made LED boxes were used to simultaneously compare replicate axenic microalgal cultures with addition of individual bacterial isolates. Co-culturing of Nannochloropsis sp. CCAP211/78 with these strains demonstrated distinct responses, which shows that the technique we developed is an efficient method for screening the influence of harmful/beneficial bacteria. Two of the tested strains, namely a strain of Maritalea porphyrae (DMSP31) and a Labrenzia aggregata strain (YP26), significantly enhanced microalgal growth with a 14% and 12% increase of the chlorophyll concentration, respectively, whereas flavobacterial strain YP206 greatly inhibited the growth of the microalga with 28% reduction of the chlorophyll concentration. Our study suggests that algal production systems represent a ‘natural’ source to isolate and study microorganisms that can either benefit or harm algal cultures.     

Antifungal compounds produced by bacterial populations of the Sea SpongeVerongia sp.

The taxonomic structure and some metabolic features of the bacterial populations of the sea spongeVerongia sp were studied. Twenty-three symbiotrophic strains were isolated into axenic cultures and identified. These wereProteobacteria of the generaPseudoalteromonas andVibrio (about 52%), firmicutes with low G+C content (up to 22%), and bacteria of the generaFlavobacterium/Cytophaga/Bacteroides (13% of the strains studied). The strain KMM 3427, identified asBacillus subtilis, synthesized the secondary metabolite 2-p-hydroxyphenylalcohol (tyrosol), which is known to be an antifungal compound and may perform the function of chemical defence inVerongia sp.     

Bacterial influence on the production of paralytic shellfish toxins by dinoflagellated algae

This study investigated the role of intracellular and extracellular bacteria in the production ofparalytic shellfish toxins by dinoflagellated algal cells. Three strains of the toxic dinoflagellatespecies, Alexandrium tamarense , were purified by external bacteria using penicillin G (Pen.G) at levels of 500 and 1000 p.p.m. Levels of toxicity of the resulting purified dinoflagellatecultures were similar to those of the original strains contaminated with external bacteria,indicating that the external bacteria had no influence on toxicity. No bacterial colony formingunits (cfu) arose from disruption of algal cells derived from penicillin-treated cultures, indicatingthat intracellular bacteria were not responsible for the toxicity of cultures.     

一株赤潮藻溶藻菌的筛选鉴定及溶藻特性分析

为缓解赤潮微藻对海洋生态环境的危害性问题,从潮间带泥样中筛选溶藻菌进行生物学特征分析,通过稀释涂布平板法及平板划线分离法从浙江舟山桃花岛潮间带泥样分离筛选菌株,以中肋骨条藻(Skeletonema costatum)及东海原甲藻(Prorocentrum donghaiense)为受试对象,通过丙酮法提取、测定叶绿素含量变化从中筛选高效抑制赤潮微藻生长的菌株。经形态学观察、生理生化特征检测及16S rDNA序列分析比对初步确定菌株的分类地位。通过不同pH、发酵时间、添加比例等单因素实验,对菌株溶藻特性和溶藻活性物质特性进行研究。从潮间带泥样中共获得43株菌,以菌株2-1-2抑制效果最佳,经鉴定属于芽孢杆菌属,初步命名为Bacillus sp. 2-1-2。溶藻菌Bacillus sp. 2-1-2以胞外分泌溶藻物质的方式进行间接溶藻,最适生长pH为7.0±1.0,菌液最佳发酵时间2—4d、最佳添加比例20%;溶藻活性物质耐热性好,对酸碱耐受性佳,但不耐强酸,在pH=10、80℃处理后溶藻率分别可达(90.57±0.43)%和(89.52±0.96)%。对赤潮藻细胞ROS水平与MDA含量...     

Interactions between the diatom Thallasiosira pseudonanna and an associated pseudomonad in a mariculture system.

The marine diatom Thallasiosira pseudonanna (3H) and several bacteria associated with it were isolated from batch cultures at the University of Delaware mariculture facility. The interaction between the algae and each of the bacteria was investigated. One of the isolates, T827/2B (Pseudomonas sp.), was incapable of surviving in f/2 culture medium unless the algae were present. When the algae and T827/2B were grown together in the f/2 medium, the bacterial growth was stimulated and the algal growth was inhibited. Bacterial filtrate had a similar effect on the algae, indicating that the bacterial effect is an indirect one most likely resulting from the excretion of a harmful compound into the medium. Preliminary characterization of the material excreted by the bacteria indicates that it s proteinaceous in nature. The interactions observed does not fit into any single category of interactions but can be explained as a combination of competition and indirect parasitism.     

Interactions between the diatom Thallasiosira pseudonanna and an associated pseudomonad in a mariculture system

The marine diatom Thallasiosira pseudonanna (3H) and several bacteria associated with it were isolated from batch cultures at the University of Delaware mariculture facility. The interaction between the algae and each of the bacteria was investigated. One of the isolates, T827/2B (Pseudomonas sp.), was incapable of surviving in f/2 culture medium unless the algae were present. When the algae and T827/2B were grown together in the f/2 medium, the bacterial growth was stimulated and the algal growth was inhibited. Bacterial filtrate had a similar effect on the algae, indicating that the bacterial effect is an indirect one most likely resulting from the excretion of a harmful compound into the medium. Preliminary characterization of the material excreted by the bacteria indicates that it s proteinaceous in nature. The interactions observed does not fit into any single category of interactions but can be explained as a combination of competition and indirect parasitism.     

Identification and characterization of potentially algal-lytic marine bacteria strongly associated with the toxic dinoflagellate Alexandrium catenella

The toxic dinoflagellate Alexandrium catenella isolated from fjords in Southern Chile produces several analogues of saxitoxin and has been associated with outbreaks of paralytic shellfish poisoning. Three bacterial strains, which remained in close association with this dinoflagellate in culture, were isolated by inoculating the dinoflagellate onto marine agar. The phenotypically different cultivable bacterial colonies were purified. Their genetic identification was done by polymerase chain reaction amplification of the 16S rRNA genes. Partial sequence analysis suggested that the most probable affiliations were to two bacterial phyla: Proteobacteria and the Cytophaga group. The molecular identification was complemented by morphological data and biochemical profiling. The three bacterial species, when grown separately from phytoplankton cells in high-nutrient media, released algal-lytic compounds together with aminopeptidase, lipase, glucosaminidase, and alkaline phosphatase. When the same bacteria, free of organic nutrients, were added back to the algal culture they displayed no detrimental effects on the dinoflagellate cells and recovered their symbiotic characteristics. This observation is consistent with phylogenetic analysis that reveals that these bacteria correspond to species distinct from other bacterial strains previously classified as algicidal bacteria. Thus, bacterial-derived lytic activities are expressed only in the presence of high-nutrient culture media and it is likely that in situ environmental conditions may modulate their expression.