Distribution of Viruses and Dissolved DNA along a Coastal Trophic Gradient in the Northern Adriatic Sea

The distribution of viral and other microbial abundances as well as the concentrations of dissolved DNA (D-DNA) along a trophic gradient in the northern Adriatic Sea were determined. Virus abundances, covering a range of 1.2 × 10⁹ to 8.7 × 10¹⁰ liter^-1 were on average 2.5-fold higher in eutrophic than in mesotrophic stations. A 2.5-fold enrichment was also measured for chlorophyll a concentrations, whereas the densities of bacteria and heterotrophic nanoflagellates were only approximately 1.5-fold higher. The frequency of bacteria containing mature phage increased linearly with bacterial abundance. Assuming that mature phage is only visible during the last 14 to 27% of the latent period (L. M. Proctor, A. Okubo, and J. A. Fuhrman, Microb. Ecol. 25:161-182, 1993), we estimated that between 3.5 and 7.3% of the bacterial population was infected at mesotrophic stations versus between 7.0 and 19.5% at eutrophic stations, indicating that the bacterial mortality due to viral lysis might increase with the degree of eutrophication. The frequency of bacteria with mature phage and the burst size varied significantly with the bacterial morphotype; rod-shape cells, the most abundant morphotype, showed low infection rates but a high burst size. Concentrations of D-DNA varied significantly with season but not with trophic conditions. The estimated percentage of viral DNA on total D-DNA concentrations averaged 17.1% (range, 0.7 to 88.3%). Some kind of interaction between heterotrophic nanoflagellates and viruses is proposed. We conclude (i) that the significance of viruses varies with changing trophic conditions and (ii) that viral activity may play a significant role in food web structure under changing trophic conditions.     

A microautoradiographic study of the activity of attached and free-living bacteria

Microautoradiography, combined with epifluorescent microscopy, was used to evaluate the uptake of tritiated amino acids by a marine Pseudomonas sp. A comparison was made between the activity of bacteria free-living in the medium and bacteria which were attached to glass, polyethylene or polystyrene substrata. The proportion of active bacteria was lower for free-living cells (53–82%) and those attached to polystyrene (53–76%) than for those attached to glass (77–99%) or polyethylene (73–96%). For bacteria attached to glass, assimilated labelled substrate was retained within the cell over 3 h, whereas with polyethylene, labelled material was released from the cells and adsorbed on the surrounding substratum. Hence the physiological activity of attached bacteria depended on the chemical composition of the substratum.     

Polar lipid biomarkers of free-living bacteria from oligotrophic marine waters

Free-living marine bacteria isolated from oligotrophic Mediterranean waters were enriched in culture to characterize their phospholipid fatty acids (PLFAs). Odd chain iso- and anteiso-FAMEs and n–16:0 were the predominant structural PLFAs, together with a homologous series identified as mid-chain methoxy FAMEs. The dominant methoxy fatty acids identified were 9-CH₃O-15:0, 9-CH₃-16:0 and 11-CH₃O-17:0, occuring as pairs of stereoimers. Methoxy fatty acids accounted for up to 37% of PLFAs of free-living bacteria, which sets them as promising new biomarkers for bacteria of oligotrophic waters. Although similar homologues have already been characterized in a variety of eukaryotes and prokaryotes, methoxy fatty acids are identified here for the first time in marine bacteria. Analytical difficulties that may hinder the characterization of these biomarkers are presented, and structural elucidation keys by gas chromotography coupled to mass spectrometry are discussed. Whilst bacterial branched fatty acids were transferred to storage lipids of bacterivorous flagellates methoxy acids were not transferred to higher trophic levels in the studied conditions.     

Characterization of the bacterial flora of mass cultivated Brachionus plicatilis

Bacterial density and composition in association of mass cultivated rotifers (Brachionus plicatilis, SINTEF-strain) was investigated, during experimental conditions identical to the procedures used for preparing rotifers as live food for marine cold water fish larvae. These procedures include cultivation, enrichment with squid meal and acclimation to low temperature by storage of the rotifer culture at 6 °C. Large variations were observed in the number of rotifer associated (1.8–7.6 · 10³ colony forming units per rotifer^–1) and free-living (0.6–25 10⁷ cells·ml^–1) bacteria. An increase of 50–150% in the bacterial number was normally observed after feeding the rotifer with squid meal, but after three days of acclimation at 6 °C, the bacterial numbers decreased to the initial level.After enrichment of the cultures with squid meal, the similarity in the composition of the bacterial flora between the rotifers and water was reduced. However, acclimation of the culture at 6 °C resulted in better agreement of the rotifer associated flora and that in water. Enrichment of the cultures induced a shift in the bacterial composition from Cytophaga/Flavobacterium dominance to Pseudomonas/Alcaligenes dominance. The bacterial flora of the rotifer cultures are dominated by presumably opportunistic species after enrichment, which may have detrimental effects when rotifers are fed as live food to marine fish larvae.     

The ecological significance of sulfur in the energy dynamics of salt marsh and coastal marine sediments

Sulfur is an important element in the metabolism of salt marshes and subtidal, coastal marine sediments because of its role as an electron acceptor, carrier, and donor. Sulfate is the major electron acceptor for respiration in anoxic marine sediments. Anoxic respiration becomes increasingly important in sediments as total respiration increases, and so sulfate reduction accounts for a higher percentage of total sediment respiration in sediments where total respiration is greater. Thus, sulfate accounts for 25% of total sediment respiration in nearshore sediments (200 m water depth or less) where total respiration rates are 0.1 to 0.3gCm^–1 day^–1 , for 50% to 70% in nearshore sediments with higher rates of total respiration (0.3 to 3gCm^–2 day^–1), and for 70% to 90% in salt marsh sediments where total sediment respiration rates are 2.5 to 5.5gcm^–2 day^–1 .During sulfate reduction, large amounts of energy from the respired organic matter are conserved in inorganic reduced sulfur compounds such as soluble sulfides, thiosulfate, elemental sulfur, iron monosulfides, and pyrite. Only a small percentage of the reduced sulfur formed during sulfate reduction is accreted in marine sediments and salt marshes. When these reduced sulfur compounds are oxidized, energy is released. Chemolithoautotrophic bacteria which catalyze these oxidations can use the energy of oxidation with efficiencies (the ratio of energy fixed in organic biomass to energy released in sulfur oxidation) of up to 21–37% to fix CO₂ and produce new organic biomass.Chemolithoautotrophic bacterial production may represent a significant new formation of organic matter in some marine sediments. In some sediments, chemolithoautotrophic bacterial production may even equal or exceed organoheterotrophic bacterial production. The combined cycle of anaerobic decomposition through sulfate reduction, energy conservation as reduced sulfur compounds; and chemolithoautotrophic production of new organic carbon serves to take relatively low-quality organic matter from throughout the sediments and concentrate the energy as living biomass in a discrete zone near the sediment surface where it can be readily grazed by animals.Contribution from a symposium on the role of sulfur in ecosystem processes held August 10, 1983, at the annual meeting of the A.I.B.S., Grand Forks, ND; Myron Mitchell, convenor.     

Bacterial Production and Growth Rate Estimation from [3H]Thymidine Incorporation for Attached and Free-Living Bacteria in Aquatic Systems

Production and specific growth rates of attached and free-living bacteria were estimated in an oligotrophic marine system, La Salvaje Beach, Vizcaya, Spain, and in a freshwater system having a higher nutrient concentration, Butron River, Vizcaya, Spain. Production was calculated from [methyl-³H]thymidine incorporation by estimating specific conversion factors (cells or micrograms of C produced per mole of thymidine incorporated) for attached and free-living bacteria, respectively, in each system. Conversion factors were not statistically different between attached and free-living bacteria: 6.812 × 10¹¹ and 8.678 × 10¹¹ μg of C mol⁻¹ for free-living and attached bacteria in the freshwater system, and 1.276 × 10¹¹ and 1.354 × 10¹¹ μg of C mol⁻¹ for free-living and attached bacteria in the marine system. Therefore, use of a unique conversion factor for the mixed bacterial population is well founded. However, conversion factors were higher in the freshwater system than in the marine system. This could be due to the different trophic conditions of the two systems. Free-living bacteria contributed the most to production in the two systems (85% in the marine system and 67% in the freshwater system) because of their greater contribution to total biomass. Specific growth rates calculated from production data and biomass data were similar for attached and free-living bacteria.     

Experimental Examination of Bacteriophage Latent-Period Evolution as a Response to Bacterial Availability

For obligately lytic bacteriophage (phage) a trade-off exists between fecundity (burst size) and latent period (a component of generation time). This trade-off occurs because release of phage progeny from infected bacteria coincides with destruction of the machinery necessary to produce more phage progeny. Here we employ phage mutants to explore issues of phage latent-period evolution as a function of the density of phage-susceptible bacteria. Theory suggests that higher bacterial densities should select for shorter phage latent periods. Consistently, we have found that higher host densities (≥~10⁷ bacteria/ml) can enrich stocks of phage RB69 for variants that display shorter latent periods than the wild type. One such variant, dubbed sta5, displays a latent period that is ~70 to 80% of that of the wild type—which is nearly as short as the RB69 eclipse period—and which has a corresponding burst size that is ~30% of that of the wild type. We show that at higher host densities (≥~10⁷ bacteria/ml) the sta5 phage can outcompete the RB69 wild type, though only under conditions of direct (same-culture) competition. We interpret this advantage as corresponding to slightly faster sta5 population growth, resulting in multifold increases in mutant frequency during same-culture growth. The sta5 advantage is lost, however, given indirect (different-culture) competition between the wild type and mutant or given same-culture competition but at lower densities of phage-susceptible bacteria (≤~10⁶ bacteria/ml). From these observations we suggest that phage displaying very short latent periods may be viewed as specialists for propagation when bacteria within cultures are highly prevalent and transmission between cultures is easily accomplished.     

Seasonal dynamics of particle-associated and free-living marine Proteobacteria in a salt marsh tidal creek as determined using fluorescence in situ hybridization

The seasonal distributions of salt marsh free-living and particle-associated bacteria belonging to three subdivisions of the Proteobacteria were determined by fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). More than 66% (median = 78%) of total bacterial cells that were stainable with the fluorescent DNA stain Yo-Pro-1 were also detected using the bacterial probe EUB338. The alpha-Proteobacteria, especially those from the marine Rhodobacter group, were abundant on suspended particles and as free-living cells all year round. The marine Rhodobacter group constituted more than 25% of the particle-associated bacteria and more than 18% of the free-living bacteria. Probes specific for three subgroups within the marine Rhodobacter group detected more than 49% of the total marine Rhodobacter group cells. These subgroups displayed different seasonal dynamics. The marine Rhodobacter group is clearly a widespread, diverse and important bacterial lineage in bacterioplankton and particle-associated assemblages in south-eastern United States salt marshes at all times of the year.     

Growth characteristics of small and large free-living and attached bacteria in Lake Constance

The growth characteristics of small (0.2–1.0m) and large (1.0–3.0 (m) free-living and attached bacteria were studied in Lake Constance by comparing the spatial and seasonal dynamics of their biomass turnover time (ratio of biomass/production). The biomass of small free-living bacteria usually turned over significantly faster than that of large free-living bacteria throughout the water column. The turnover of attached bacterial biomass was characterized by large fluctuations. Occasionally, in aphotic water layers, it was as long as that of large free-living bacteria, but when large amounts of decaying organic particles were present, it was shorter than that of small free-living cells. Biomass turnover times of free-living bacteria were in the same range as their generation times, which were estimated from the increase in bacterial abundance in 3m prefiltered samples. The biomass turnover time of actively metabolizing bacteria was comparable to the generation time of actively metabolizing cells. These results indicate that the biomass turnover time is a useful indicator of the growth of different bacterial fractions, as it reflects their different amounts of participation in microbial processes of aquatic ecosystems.     

Bacterial bioluminescence as an early indication of marine fish spoilage

Summary The relationships between different microbiological and biochemical parameters and the development of bacterial luminescence associated with the spoilage of marine fish from the Mediterranean-Sea was studied during storage at different temperatures. The bioluminescence level of the bacterial suspensions that were taken from the fish skin increased during the storage; at 20°–25°C the growth and luminescence of the luminuous bacteria correlated well with the total bacterial count while at 5°C the bacterial proliferation was not accompanied by a parallel increase in luminescence.The shift in storage temperature from 25°C to 5°C stabilized the level of the luminescence of bacterial suspension taken from the winter fish which were comprised mainly by Photobacterium phosphoreum, and caused a drop in the luminescence of bacterial suspension taken from the fish caught in the summer which were comprised mainly by Beneckea barveyi. The increase in the bioluminescence level appeared earlier than the increase in trimethylamine level and occured approximately at the same time as the increase in the hypoxanthine concentration. The potential value of the use of bacterial bioluminescence as an early indication for marine fish spoilage is discussed.     

Effects of Furazolidone on the Infection of Vibrio cholerae by Bacteriophage φ149

Furazolidone in concentrations which had little effect on the growth of host organisms greatly reduced the yield of phage 149 from the host Vibrio cholerae OGAWA 154. This phage was resistant to the in vitro action of the drug. The phage yield of infected bacteria depended significantly on the time of addition or withdrawal of the drug. The average burst size of the drug-treated and infected bacteria decreased exponentially with increase in drug concentration. The latent period of phage multiplication and also the eclipse period did not change significantly from the control values. A concentration of 0.05 μg of furazolidone per ml inhibited DNA synthesis by about 50% in phage-infected cells and only by about 18% in noninfected ones, relative to the respective controls. RNA and protein synthesis were affected by a much smaller degree both in infected and noninfected cells. Quantitative deduction of the length of furazolidone-treated cells from their phage adsorption characteristics and its agreement with previous electron microscopy data indicated that furazolidone did not affect the phage receptors.     

Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria

Myxobacteria are common in terrestrial habitats and well known for their formation of fruiting bodies and production of secondary metabolites. We studied a cluster of myxobacteria consisting only of sequences of marine origin (marine myxobacteria cluster, MMC) in sediments of the North Sea. Using a specific PCR, MMC sequences were detected in North Sea sediments down to 2.2 m depth, but not in the limnetic section of the Weser estuary and other freshwater habitats. In the water column, this cluster was only detected on aggregates up to a few meters above the sediment surface, but never in the fraction of free-living bacteria. A quantitative real-time PCR approach revealed that the MMC constituted up to 13% of total bacterial 16S rRNA genes in surface sediments of the North Sea. In a global survey, including sediments from the Mediterranean Sea, the Atlantic, Pacific and Indian Ocean and various climatic regions, the MMC was detected in most samples and to a water depth of 4300 m. Two fosmids of a library from sediment of the southern North Sea containing 16S rRNA genes affiliated with the MMC were sequenced. Both fosmids have a single unlinked 16S rRNA gene and no complete rRNA operon as found in most bacteria. No synteny to other myxobacterial genomes was found. The highest numbers of orthologues for both fosmids were assigned to Sorangium cellulosum and Haliangium ochraceum. Our results show that the MMC is an important and widely distributed but largely unknown component of marine sediment-associated bacterial communities.     

Organic sedimentation and macrofauna as forcing factors in marine benthic nanofagellate communities

We investigated how benthic nanoflagellate communities in marine sediments respond to sedimentation of organic material and to the presence of macrofaunal organisms in controlled boxcosms. An input of 24 g C m^–2 resulted in a sharp increase in densities, from 93 to 477 × 10³ flagellates cm^–3 within 11 days. At the onset, this increase was paralleled by enhanced bacterial production and bacterial numbers. When bacterial production collapsed, flagellate ingestion rates, varying from 17 to 67 bact flag^–1 h^–1, were sufficient to control bacterial abundance. The presence of macrofauna accelerated the burst in flagellate densities. With macrofauna the same maximum densities were reached, but later densities dropped to relatively low levels. Macrofaunal bioturbation resulted in higher flagellate densities deeper in the sediment (up to 1200% at 3 cm and up to 460% at 6 cm deep). Correspondence to: R.P.M. Bak.     

Elevated Lytic Phage Production as a Consequence of Particle Colonization by a Marine <Emphasis Type="Italic">Flavobacterium</Emphasis> (<Emphasis Type="Italic">Cellulophaga</Emphasis> sp.)

Bacteria growing on marine particles generally have higher densities and cell-specific activities than free-living bacteria. Since rapidity of phage adsorption is dependent on host density, while infection productivity is a function of host physiological status, we hypothesized that marine particles are sites of elevated phage production. In the present study, organic-matter-rich agarose beads and a marine phage-host pair (Cellulophaga sp., PhiS(M)) were used as a model system to examine whether bacterial colonization of particles increases phage production. While no production of phages was observed in plain seawater, the presence of beads enhanced attachment and growth of bacteria, as well as phage production. This was observed because of extensive lysis of bacteria in the presence of beads and a subsequent increase in phage abundance both on beads and in the surrounding water. After 12 h, extensive phage lysis reduced the density of attached bacteria; however, after 32 h, bacterial abundance increased again. Reexposure to phages and analyses of bacterial isolates suggested that this regrowth on particles was by phage-resistant clones. The present demonstration of elevated lytic phage production associated with model particles illustrates not only that a marine phage has the ability to successfully infect and lyse surface-attached bacteria but also that acquisition of resistance may affect temporal phage-host dynamics on particles. These findings from a model system may have relevance to the distribution of phage production in environments rich in particulate matter (e.g., in coastal areas or during phytoplankton blooms) where a significant part of phage production may be directly linked to these nutrient-rich "hot spots."     

Laboratory investigations on the survival of marine bacteriophages in raw and treated seawater

Laboratory investigations were performed to gain insight into the mechanisms which govern the survival of marine bacteriophages in nature. Samples collected in 1988 to 1990 at station “Kabeltonne” near Helgoland were used raw, membrane-filtered (0.15μm), and/or after inverse filtration through 10 μm-mesh gauze to reduce or increase live and dead particles. The development of natural or artificial bacterial populations and the survival of 2 to 10 distinguishable strains of test phage were followed during incubation at 20°C. The results obtained with most test phages point to the predominant role of indigenous bacteria for marine phage inactivation which was generally enhanced by sample managements leading to improved growth of bacteria. The virucidal properties of the samples differed greatly in total strength as well as in the changes taking place during incubation, the latter resulting in conspicuously differing inactivation curves. Generally, phage inactivation was slow during the first 2 to 3 days of incubation, followed by a period of very rapid inactivation which usually coincided with the die-away of colony-forming bacteria. This period lasted either only a few days or until the concentration of test phage was reduced to (near) zero. While the inactivation of most test phage is assumedly caused by proteolytic enzymes released during the die-away of bacteria, the survivability of one test phage (H7/2) was also markedly influenced by the bacteria sensitive to it. Survival rates of the test phages in the laboratory tests were generally of the same order of magnitude as those recently observed with natural phage populations.     

Significance of Bacteriophages for Controlling Bacterioplankton Growth in a Mesotrophic Lake

Bacterium-specific viruses have attracted much interest in aquatic microbial ecology because they have been shown to be about 10 times more abundant than planktonic bacteria. So far most of the studies of interactions of planktonic bacteria and viruses have been done in marine environments, and very little is known about these interactions in lakes. Therefore, we studied phage proliferation in Lake Constance, a large mesotrophic lake in Germany. We enumerated bacteria and quantified the fraction of bacteria with mature intracellular phage particles and the number of free viruses by transmission electron microscopy. Between the end of March and early August 1992, peaks of bacterial abundance were followed in 1 to 2 weeks by peaks in the fraction of bacteria containing visible phage particles (0 to 1.7%) and in the number of free viruses (1 x 10(sup7) to 4 x 10(sup7) ml(sup-1)). We estimated that 1 to 17% +/- 12% of all bacteria were phage infected, implying that phage-induced mortality was <34% +/- 24% of total mortality. A direct comparison between phage-induced mortality, the net decrease of bacterial numbers, and bacterial growth rates indicated that phage-induced mortality accounted for <11% of total bacterial mortality during the phytoplankton spring bloom and 18 to 21% following the bloom. Estimated burst sizes ranged from 21 to 121 phages. Phage production rates of 0.5 x 10(sup6) to 2.5 x 10(sup6) ml(sup-1) day(sup-1) accounted for 70 to 380% of the observed net increase rates of free phages, implying high rates of simultaneous phage decay. The cyclic dynamics between bacteria and phages and the varying size structure of the intracellular mature phage particles suggested that phage infection was important in structuring the bacterial host assemblage during the study period.     

Stimulation of marine free-living and epibiotic bacterial activity by copepod excretions

Abstract The influence of marine copepod exudates (amino acids and NH₄⁺) on activity by free-living bacteria, and the relative activity of epibiotic bacteria living on copepods were examined. Samples that contained copepods (100 copepods 1⁻¹) were estimated to have leucine concentrations (K+S) that were higher by factor of 4.4, and leucine V_max's that were higher by a factor of 1.8 relative to samples without copepods. NH₄⁺ additions stimulated bacterial activity in samples that did not contain copepods, but had no effect in samples that contained copepods, suggesting that free-living bacteria were N-limited. Epibiotic bacteria accounted for about 20% of total bacterial activity. These results suggested that high-density copepod aggregations may liberate nutrients in sufficient quantity to significantly stimulate bacterial activity, and that epibiotic bacteria may be optimally positioned to exploit these resources.     

Distribution and biology of marine cladocerans in the coastal waters of southern China

Geographical and seasonal distributions of marine cladocerans in the coastal waters of southern China were studied. Penilia avirostris was the most common species, followed by Evadne tergestina and Podon schmackeri. P. avirostris and E. tergestina were most common during summer. P. schmackeri, found only in a small bay northeast of Hong Kong, showed no clear seasonal pattern of occurrence. P. avirostris and E. tergestina were found at temperatures ranging from 16–32°C and salinity ranging from 7.3–37.2. P. schmackeri was restricted to a temperature range of 17–29°C and a salinity range of 31.0–37.2. No significant relationships between marine cladoceran abundance and chlorophyll a concentration were found in samples taken from Tolo Harbour. Parthenogenetic brood size of P. avirostris and E. tergestina ranged from 1 to 14, while P. schmackeri was found to carry up to 19 embryos per brood. No geographical trend in fecundity patterns was observed. No correlation was found between body length and brood size. The occurrence of females with resting eggs was rare.     

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.     

Induction of luciferase synthesis in Beneckea harveyi by other marine bacteria

It has been previously demonstrated that luciferase synthesis in the luminous marine bacteria, Beneckea harveyi and Photobacterium fischeri is induced only when sufficient concentrations of metabolic products (autoinducers) of these bacteria accumulate in growth media. Thus, when cells are cultured in liquid medium there is a lag in luciferase synthesis. A quantitative bioassay for B. harveyi autoinducer was developed and it was shown that many marine bacteria produce a substance that mimics its action, but in different amounts, (20–130% of the activity produced by B. harveyi) depending on the species and strain. This is referred to as alloinduction. None of the bacteria tested produced detectable quantities of inducer for P. fischeri luciferase synthesis. These findings may have significance with respect to the ecology of B. harveyi and P. fischeri.Non-Standard Abbreviation AB medium autoinducer bioassay medium