Short-Term Responses of the Natural Planktonic Bacterial Community to the Changing Water Properties in an Estuarine Environment: Ectoenzymatic Activity,Glucose Incorporation,and BiomassProduction

The possibility that two principlal bacterial communities expressing different levels of heterotrophic activity might coexist in an estuarine ecosystem (Ria de Aveiro, Portugal) and could quickly respond to tidal fluctuations of environmental factors was experimentally tested in diffusion chambers by swapping the dissolved components of the natural water between the two communities and comparing their reactivity against the unaltered controls. The results for ectoenzymatic activity (Leuaminopeptidase and β-glucosidase), glucose incorporation and biomass production after transference of the marine bacterial community to brackish water showed maxima in the range of 241–384% of the control values. The opposite transference of the brackish-water bacterial community to marine water produced maximal decreases to 0.14–0.58% of the control values. In a reverse experiment, designed as the return to the initial conditions after 2 hours of the first exposure, the marine community rapidly re-acquired the characteristic low profile of activity. Contrastingly, the negative effects of 2 hours of exposure to marine water on the activity of the brackish water bacteria persisted, at least for 4 hours, after return to their own water. The apparent short-term irreversibility of the decline in activity of the brackish water bacteria when exposed to marine water, in parallel with the quick and reversible positive response of the marine water bacteria to the brackish water, suggests the development of two distinct bacterioplankton communities adapted to the environmental conditions prevailing at distinct sections of the estuary. The reactivity to environmental changes demonstrated by the two communities allows the prediction of estuarine profiles of bacterial activity steeper than those expected from the conservative transport of bacterial cells associated with tidal currents.     

Soil Bacteria are Differentially Affected by the Resin of the Medicinal Plant Pseudognaphalium vira vira and Its Main Component Kaurenoic Acid

The diterpenoid kaurenoic acid is the main component of the resin from the medicinal plant Pseudognaphalium vira vira. As some diterpenoids have antimicrobial properties, the effect of this resin and the kaurenoic acid on soil bacteria was studied. The resin of P. vira vira and purified kaurenoic acid were two to four times more effective as antibacterial agents with Gram-positive than with Gram-negative soil isolates. The chemical stability of kaurenoic acid and the antibacterial activity of both the resin and the diterpenoid were studied in microcosms containing plant-associated soil. After 15 days of incubation, the diterpenoid was stable, as determined by ¹H nuclear magnetic resonance and thin-layer chromatography, and soil extracts still exhibited antibacterial activity. However, after 30 days of incubation, loss of antibacterial activity of soil extracts correlated with removal or chemical modification of kaurenoic acid. The effect of the resin or this diterpenoid on the soil bacteria community was analyzed by the terminal restriction fragment length polymorphisms technique. After 15 days of incubation, the resin and the pure compound caused significant changes in the soil bacterial community. The relative abundance of specific bacterial groups was differentially affected by the resin components, being the effects with the resin stronger than with the kaurenoic acid. After 30 days of incubation, these changes mostly reverted. These results indicate that a plant resin containing diterpenoid compounds plays a significant role controlling specific groups of microorganisms in the soil associated with the plant.     

Culturable heterotrophic bacteria from the marine sponge <Emphasis Type="Italic">Dendrilla</Emphasis> <Emphasis Type="Italic">nigra</Emphasis>: isolation and phylogenetic diversity of actinobacteria

Culturable heterotrophic bacterial composition of marine sponge Dendrilla nigra was analysed using different enrichments. Five media compositions including without enrichment (control), enriched with sponge extract, with growth regulator (antibiotics), with autoinducers, and complete enrichment containing sponge extract, antibiotics, and autoinducers were developed. DNA hybridization assay was performed to explore host specific bacteria and ecotypes of culturable sponge-associated bacteria. Enrichment with selective inducers (AHLs and sponge extract) and regulators (antibiotics) considerably enhanced the cultivation potential of sponge-associated bacteria. It was found that Marinobacter (MSI032), Micromonospora (MSI033), Streptomyces (MSI051), and Pseudomonas (MSI057) were sponge-associated obligate symbionts. The present findings envisaged that “Micromonospora–Saccharomonospora–Streptomyces” group was the major culturable actinobacteria in the marine sponge D. nigra. The DNA hybridization assay was a reliable method for the analysis of culturable bacterial community in marine sponges. Based on the culturable community structure, the sponge-associated bacteria can be grouped (ecotypes) as general symbionts, specific symbionts, habitat flora, and antagonists.     

Impact of <Emphasis Type="Italic">cry1AC</Emphasis>-carrying <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> on leaf bacterial community

The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of trans-gene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.     

Zooplankton and aggregates as refuge for aquatic bacteria: protection from UV,heat and ozone stresses used for water treatment

Aggregates and zooplankton may provide refuge for aquatic bacteria against external hazards. The ability of attached bacteria to survive and recover from stressors commonly used for water treatment was tested in the laboratory. Without zooplankton or aggregates, both UV and ozone significantly reduced abundance of free‐living bacteria in both freshwater and marine medium. The presence of zooplankton carcasses and aggregates, however, allowed some of the attached bacteria to survive and recover quickly within 3 days. Heat exposure was the least effective as both free‐living and attached bacteria were able to recover quickly. Selective survival of bacterial phylotypes led to large changes in bacterial community composition after stress exposures, and some of the bacteria that recovered belonged to groups with known pathogens. This study demonstrates that zooplankton and aggregates protected various aquatic bacteria from external stressors, and organic remains generated from zooplankton and aggregates after stress exposure even enabled the surviving bacteria to quickly regrow and subsequently be released into the surrounding water. Hence, water disinfection treatments that overlooked the potential persistence of bacteria associated with organisms and aggregates may not be effective in preventing the spread of undesirable bacteria.     

Natural assemblages of marine bacteria exhibiting high-speed motility and large accelerations.

Natural communities of marine bacteria, an isolate (FMB-Bf3) from one marine community, and Escherichia coli were examined by video microscopy for the magnitude and uniformity of their speed. Natural communities formed tight microswarms that showed higher speeds (mean = 230 microns s-1) than did E. coli (15 microns s-1) or FMB-Bf3 (mean = 62 microns s-1). Outside the microswarms, the marine bacteria slowed to 45 microns s-1. Between turns, in mid run, and while travelling in straight lines, the natural-community bacteria accelerated up to 1,450 microns s-2 while the cultured bacteria showed maximum accelerations of 70 and 166 microns s-2. The frequency distribution of speed change for the marine bacteria was skewed towards a few large negative accelerations and a range of positive accelerations. The general pattern was one of relatively slow increases in speed followed by abrupt declines. The results indicate that the mechanical generation and energetic maintenance, as well as the environmental function, of bacterial motility need reappraisal. We conclude that the standard bacterial motility parameters of low and uniform speed, derived from culture-based studies, are not necessarily applicable to marine bacterial communities.     

Release and degradation of amnesic shellfish poison from decaying Pseudo-nitzschia multiseries in presence of bacteria and organic matter

Domoic acid (DA), the neurotoxin produced by diatoms such as Pseudo-nitzschia multiseries is water-soluble and can bioaccumulate, causing mass death of birds and marine mammals worldwide. Humans eating contaminated shellfish most commonly suffer from memory loss but mortalities have been recorded. The fate of particulate and dissolved DA released from the cells or added as standards was studied when incubated with different bacterial abundances, copepod faecal pellets, mussel pseudo-faeces and bottom sediment. Strains of P. multiseries from Canada and Brazil were grown in non-axenic continuous monocultures with different nutrient conditions, or in a follow-up mesocosm experiment. Incubation lasted up to 75 days in the dark under quiescent conditions after the cells had been killed. Release of DA from decaying cells did not depend on bacterial abundance when the bacterial source was cultures of P. multiseries, and the dissolved toxin was stable with bacteria from P. multiseries cultures (at least 20 days with 1× or 4× bacterial concentration), or with a naturally occurring density of bacteria from surface waters of a known P. multiseries bloom area (35 days). However, four-fold concentration of the natural bacterial consortium from the bloom site reduced the onset of DA degradation to 16 days. Thus, this study suggests that when testing toxin degradation by bacteria, it is important to use bacterial consortia from known bloom areas of Pseudo-nitzschia. Copepod faecal pellets did not affect DA degradation, whereas the presence of mussel pseudo-faeces and bottom sediment rapidly removed most of the toxin. We believe that the rapid removal of DA in the two latter treatments was due to higher bacterial abundance and the presence of enzymes from the mussels and/or associated bacteria that are important for the degradation process. The mechanisms underlying the observed effects on DA degradation with mussel pseudo-faeces and sediment require further research, but suggest interesting possibilities as a potential future mitigation technique.     

Different Marine Heterotrophic Nanoflagellates Affect Differentially the Composition of Enriched Bacterial Communities

We studied the effects of predation on the cytometric and phylogenetic features of two enriched bacterial communities obtained from two cultures of marine heterotrophic nanoflagellates: Jakoba libera and a mixed culture of Cafeteria sp. and Monosiga sp. Protists were harvested by flow cytometric cell sorting and eight different treatments were prepared. Each bacterial community was incubated with and without protists, and we added two treatments with protists and the bacteria present after the sorting procedure (cosorted bacteria). The bacterial community derived from the culture of Jakoba libera had higher green fluorescence per cell (FL1) than that derived from the mixed culture of Cafeteria sp. and Monosiga sp. When the experiment began all treatments presented bacterial communities that increase in fluorescence per bacterium (FL1); after that the FL1 decreased when bacteria attained maximal concentrations; and, finally, there was a new increase in FL1 toward the end of the experiment. Cosorted bacteria of Jakoba libera had the same fluorescence as the bacterial community derived from this protist, while the bacteria derived from the mixed culture of Cafeteria sp. and Monosiga sp. was nearly twice as fluorescent than that of the parental community. All treatments presented a general decline of SSC along the incubation. Therefore, there was a small influence of protists on the cytometric signature of each bacterial community. However, each bacterial community preyed by Jakoba libera or the mixed culture of Cafeteria sp. and Monosiga sp. led to four different phylogenetic fingerprint. Besides, the final Communities were different from the fingerprint of controls without protists, and most of them diverge from the fingerprint of cosorted bacteria. Our results confirm that changes in the phylogenetic composition of marine bacterial communities may depend on the initial communities of both bacteria and protists.     

松墨天牛成虫室内外种群肠道细菌的多样性及功能分析

[目的]为明确松墨天牛Monochamus alternatus成虫中、后肠细菌群落结构,探索肠道细菌的潜在功能.[方法]分别提取室外和室内饲养的松墨天牛成虫各15头(室内和室外各15个中肠、15个后肠)的肠道DNA,利用二代测序技术对松墨天牛成虫肠道细菌的16S rDNA V3-V4区序列进行测序,统计操作分类单元(...     

舟山群岛不同功能区划海域细菌群落结构分析

浮游细菌在海洋生态系统中不可或缺,在海洋生物地球化学循环过程中起着关键性作用。【目的】为了解舟山群岛不同功能区划海域细菌群落结构及丰度变化,探索海洋生态因子对细菌群落结构的影响。【方法】于2016年夏季(8月)在舟山群岛不同功能区划海域共设置8个典型站位采集表层海水,基于细菌16S rRNA基因进行高通量测序;利用流式细胞术揭示各海域细菌丰度;利用典范对应分析(Canonical correspondence analysis,CCA)探讨海洋生态因子与细菌多样性之间的关系。【结果】共获取到305487条原始序列,基于97%相似性水平进行OTU(Operational Taxonomic Units)聚类分析,共得到1088个OTUs,包括29个门、62个纲、138个目、239个科、416个属。细菌群落组成在各个站位之间不尽相同,但都主要包括Flavobacteria、Alphaproteobacteria、Gammaproteobacteria三大优势菌纲。CCA结果表明细菌群落结构和多样性情况与站位分布和所在站位的环境因子息息相关,Cyanobacteria受硝酸盐影响显著,Parcubacteria受温度影响最大,而磷酸盐对本实验海域菌群影响甚微。对海洋菌群潜在功能进行预测的结果显示,各海域菌群在氨基酸代谢、碳水化合物代谢、膜运输等方面功能较为突出,为今后舟山海洋微生物研究提供了新的方向。【结论】高通量测序分析可以更精确地揭示海洋菌群的群落结构信息。该研究为细菌群落结构与环境因素的关联提供参考。本研究所取得的大量数据既可以作为对舟山市海洋功能区划施行情况的响应,又将为舟山及其邻近海域浮游细菌群落结构的进一步研究奠定基础。     

The Gut Bacterial Community of Mammals from Marine and Terrestrial Habitats

After birth, mammals acquire a community of bacteria in their gastro-intestinal tract, which harvests energy and provides nutrients for the host. Comparative studies of numerous terrestrial mammal hosts have identified host phylogeny, diet and gut morphology as primary drivers of the gut bacterial community composition. To date, marine mammals have been excluded from these comparative studies, yet they represent distinct examples of evolutionary history, diet and lifestyle traits. To provide an updated understanding of the gut bacterial community of mammals, we compared bacterial 16S rRNA gene sequence data generated from faecal material of 151 marine and terrestrial mammal hosts. This included 42 hosts from a marine habitat. When compared to terrestrial mammals, marine mammals clustered separately and displayed a significantly greater average relative abundance of the phylum Fusobacteria. The marine carnivores (Antarctic and Arctic seals) and the marine herbivore (dugong) possessed significantly richer gut bacterial community than terrestrial carnivores and terrestrial herbivores, respectively. This suggests that evolutionary history and dietary items specific to the marine environment may have resulted in a gut bacterial community distinct to that identified in terrestrial mammals. Finally we hypothesize that reduced marine trophic webs, whereby marine carnivores (and herbivores) feed directly on lower trophic levels, may expose this group to high levels of secondary metabolites and influence gut microbial community richness.     

A culture-dependent bacterial community structure analysis based on liquid cultivation and its application to a marine environment

A method for analyzing culture-dependent bacterial community structure by liquid cultivation was established using 96-well microplates. Using 96-well microplates, this method can easily provide accurate enumeration of viable microorganisms and simultaneous separation of bacteria, which allowed us to analyze the bacterial community. Bacteria in diluted surface seawater were separated using 96-well microplates and cultivated with 1/5 ZoBell 2216E liquid medium. The 98 cultures obtained were subsequently applied to phylogenetic analysis based on 16S rRNA gene sequences. The bacterial diversity, evaluated by the Shannon–Weaver index, was relatively small but comparable to previously reported bacterial communities of several environments. The most abundant group was the family Rhodobacteraceae , which has been frequently detected in marine environments. Most bacteria were phylogenetically related to bacteria or uncultured clones detected in marine environments, but distant from published species. The analysis of bacterial community structure by liquid cultivation would be useful as an alternative culture-dependent approach.     

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.     

Effects of Bacteriophages on the Population Dynamics of Four Strains of Pelagic Marine Bacteria

Viral lysis of specific bacterial populations has been suggested to be an important factor for structuring marine bacterioplankton communities. In the present study, the influence of bacteriophages on the diversity and population dynamics of four marine bacterial phage-host systems was studied experimentally in continuous cultures and theoretically by a mathematical model. By use of whole genome DNA hybridization toward community DNA, we analyzed the dynamics of individual bacterial host populations in response to the addition of their specific phage in continuous cultures of mixed bacterial assemblages. In these experiments, viral lysis had only temporary effects on the dynamics and diversity of the individual bacterial host species. Following the initial lysis of sensitive host cells, growth of phage-resistant clones of the added bacteria resulted in a distribution of bacterial strains in the phage-enriched culture that was similar to that in the control culture without phages after about 50-60 h incubation. Consequently, after a time frame of 5-10 generations after lysis, it was the interspecies competition rather than viral lysis of specific bacterial strains that was the driving force in the regulation of bacterial species composition in these experiments. The clonal diversity, on the other hand, was strongly influenced by viral activity, since the clonal composition of the four species in the phage-enriched culture changed completely from phage-sensitive to phage-resistant clones. The model simulation predicted that viral lysis had a strong impact on the population dynamics, the species composition, and the clonal composition of the bacterial community over longer time scales (weeks). However, according to the model, the overall density of bacteria in the system was not affected by phages, since resistant clones complemented the fluctuations caused by viral lysis. Based on the model analysis, we therefore suggest that viral lysis can have a strong influence on the dynamics of bacterial populations in planktonic marine systems.     

Relationship between the Intracellular Integrity and the Morphology of the Capsular Envelope in Attached and Free-Living Marine Bacteria

The integrity of the intracellular structures and the presence and dimension of the capsular envelope were investigated in marine snow-associated and marine free-living bacteria by transmission electron microscopy and special fixation techniques. Three categories depending on the presence of internal structures were differentiated. In marine snow, 51% of the marine snow-associated bacterial community was considered intact, 26% had a partly degraded internal structure, and 23% were empty with only the cell wall remaining. For the free-living bacterial community, 34% were intact cells, 42% exhibited damage, and 24% of the cells were lacking any internal structure. We also investigated the morphology and the extent of the bacterial capsular envelope. More than 95% of all intact marine snow-associated bacteria were surrounded by a capsule while (apprx=)55% of empty marine snow-associated bacteria had no capsule. For free-living bacteria, (apprx=)65% of the intact cells had a capsule while (apprx=)80% of the empty free-living bacteria lacked a capsule. Thus there is a clear trend from intact cells which are commonly surrounded by a capsular envelope to empty bacteria for which only the cell wall is remaining. Since bacterioplankton represent the largest living surface in the ocean, it is concluded that the release of intracellular material from bacteria into the environment as well as the release of extracellular capsular material might fuel the dissolved organic matter pool of the ocean.     

南海冷泉区深海沉积物中细菌的分离培养及多样性分析

为了认识南海深海冷泉区沉积物中可培养微生物的多样性,本文以冷泉区与非冷泉区两个站点的深海沉积物为样品,通过两种培养基(R2A海水培养基和2216E培养基)直接涂布或富集后平板分离纯化,从9个样品中共得到395株菌株,并通过16SrRNA基因鉴定,分属10个属。发现产芽胞细菌分布最广、丰度最大,包括3个属、15个种。其中芽胞杆菌(Bacillus)无论是在数量还是在种类上都分布最多。并且,随着水深和沉积物深度的增加,分离到的可培养微生物丰富度降低。本研究表明,即使在冷泉区,南海深海沉积物中产芽胞细菌也比较丰富。     

Repulsion of bacteria from marine surfaces.

Organic compounds are capable of repelling motile bacteria from marine surfaces. The most effective compounds were acrylamide and benzoic and tannic acids. These were active at concentrations that were not toxic to the bacteria. Repellents were incorporated in nontoxic paints and applied to metal panels. Treated panels immersed in seawater developed a bacterial film of only 10(6) bacteria per cm6 after 12 days compared with untreated panels, which had 5 times 10(12) bacteria per cm2 after the same period. Field studies confirmed the effectiveness of these repellents. The use of biological repellents provides a new approach to the control of marine fouling.     

Indicator bacteria in freshwater and marine molluscs

The freshwater mussel Anodonta cygnea and four marine shellfish (mussels, Mytilus edulis; cockles, Cerastoderma edule; clams, Mya arenaria; Scrobicularia plana) from a total of six sites were surveyed for Escherichia coli, Clostridium perfringens, faecal streptococci, 25 and 37 °C coliforms, 25 °C and 37 °C total viable numbers and fluorescent pseudomonads. The A. cygnea from an urban lake contained greater numbers of the faecal indicator bacteria than animals from a rural lake. There were also differences in the other bacterial counts and these were discussed with respect to bacterial parameter and animal characteristics. When freshwater mussels were transferred from the city site to the rural site for 24 h the load of faecal indicator bacteria was eliminated or significantly reduced. Other bacterial types took longer to become stabilised. Loss of indicator bacteria from Anodonta was also demonstrated using cleansing in the laboratory. Very high bacterial numbers were found in some marine molluscs notably Scrobicularia plana and most shellfish contained significant numbers of the three faecal indicator bacteria at every sampling occasion. The relationship between bacterial types was discussed and it was concluded that in both freshwater and marine animals the bacterial numbers were determined more by sampling site than by species of shellfish.     

Response of Bacterial Metabolic Activity to Riverine Dissolved Organic Carbon and Exogenous Viruses in Estuarine and Coastal Waters: Implications for CO2 Emission

A cross-transplant experiment between estuarine water and seawater was conducted to examine the response of bacterial metabolic activity to riverine dissolved organic carbon (DOC) input under virus-rich and virus-free conditions, as well as to exogenous viruses. Riverine DOC input increased bacterial production significantly, but not bacterial respiration (BR) because of its high lability. The bioavailable riverine DOC influenced bulk bacterial respiration in two contrasting ways; it enhanced the bulk BR by stimulating bacterial growth, but simultaneously reduced the cell-specific BR due to its high lability. As a result, there was little stimulation of the bulk BR by riverine DOC. This might be partly responsible for lower CO₂ degassing fluxes in estuaries receiving high sewage-DOC that is highly labile. Viruses restricted microbial decomposition of riverine DOC dramatically by repressing the growth of metabolically active bacteria. Bacterial carbon demand in the presence of viruses only accounted for 7–12% of that in the absence of viruses. Consequently, a large fraction of riverine DOC was likely transported offshore to the shelf. In addition, marine bacteria and estuarine bacteria responded distinctly to exogenous viruses. Marine viruses were able to infect estuarine bacteria, but not as efficiently as estuarine viruses, while estuarine viruses infected marine bacteria as efficiently as marine viruses. We speculate that the rapid changes in the viral community due to freshwater input destroyed the existing bacteria-virus relationship, which would change the bacterial community composition and affect the bacterial metabolic activity and carbon cycling in this estuary.