Strong seasonality and interannual recurrence in marine myovirus communities.

The temporal community dynamics and persistence of different viral types in the marine environment are still mostly obscure. Polymorphism of the major capsid protein gene, g23, was used to investigate the community composition dynamics of T4-like myoviruses in a North Atlantic fjord for a period of 2 years. A total of 160 unique operational taxonomic units (OTUs) were identified by terminal restriction fragment length polymorphism (TRFLP) of the gene g23. Three major community profiles were identified (winter-spring, summer, and autumn), which resulted in a clear seasonal succession pattern. These seasonal transitions were recurrent over the 2 years and significantly correlated with progression of seawater temperature, Synechococcus abundance, and turbidity. The appearance of the autumn viral communities was concomitant with the occurrence of prominent Synechococcus blooms. As a whole, we found a highly dynamic T4-like viral community with strong seasonality and recurrence patterns. These communities were unexpectedly dominated by a group of persistently abundant viruses.

Viriobenthos production and virioplankton sorptive scavenging by suspended sediment particles in coastal and pelagic waters.

Virus production in oxic surface sediments and virioplankton sorption to suspended particles was estimated across three stations in the Southern California region (33.4 degrees N, 118.6 degrees W). Viriobenthos production was estimated using a sterile sediment and filtered porewater dilution technique that targeted production from both attached bacteria and bacteria living free in the porewater, and attached bacteria alone. Potential virus production rates by bacteria free in the porewater ranged from 1.7 to 4.6 x 10(8) VLP cm(-3) h(-1), while attached bacteria had slower potential production rates of between 0.4 and 1.1 x 10(8) VLP cm(-3) h(-1), suggesting turnover rates of viruses in sediments (1-5 h) which are significantly higher than those of virioplankton (approximately 24-48 h). Virioplankton adsorbed to small (<150 microm) suspended sediments at stations with high ambient suspended solid concentrations. Virioplankton scavenging rates combined with published sedimentation rates demonstrate that this mechanism of virus arrival could only account for 0.01% of daily benthic virus production. Calculated mortality rates of benthic bacteria (4-14% h(-1)) suggest viruses may play an important role in sediment carbon cycling.

Bacterial diversity in shallow oligotrophic marine benthos and overlying waters: effects of virus infection, containment, and nutrient enrichment.

Little is known of the factors shaping sediment bacterial communities, despite their high abundance and reports of high diversity. Two factors hypothesized to shape bacterial communities in the water column are nutrient (resource) availability and virus infection. The role these factors play in benthic bacterial diversity was assessed in oligotrophic carbonate-based sediments of Florida Bay (USA). Sediment-water mesocosm enclosures were made from 1-m diameter clear polycarbonate cylinders which were pushed into sediments to approximately 201 cm sediment depth enclosing approximately 80 L of water. Mesocosms were amended each day for 14 d with 10 microM NH4+ and 1 microM PO4(3-). In a second experiment, viruses from a benthic flocculent layer were concentrated and added back to flocculent layer samples which were collected near the mesocosm enclosures. Photosynthesis by microalgae in virus-amended incubations was monitored by pulse-amplitude modulated (PAM) fluorescence. In both experiments, bacterial diversity was estimated using automated rRNA intergenic spacer analysis (ARISA), a high-resolution fingerprinting approach. Initial sediment bacterial operational taxonomic unit (OTU) richness (236 +/- 3) was higher than in the water column (148 +/- 9), where an OTU was detectable when its amplified DNA represented >0.09% of the total amplified DNA. Effects on bacterial diversity and operational taxonomic unit (OTU) richness in nutrient-amended mesocosms may have been masked by the effects of containment, which stimulated OTU richness in the water column, but depressed OTU richness and diversity in sediments. Nutrient addition significantly elevated virus abundance and the ratio of viruses to bacteria (p < 0.05 for both) in the sediments, concomitant with elevated bacterial diversity. However, water column bacterial diversity (in unamended controls) was not affected by nutrient amendments, which may be due to rapid nutrient uptake by sediment organisms or adsorption of P to carbonate sediments. Addition of live viruses to benthic flocculent layer samples increased bacterial OTU diversity and richness compared with heat-killed controls; however, cluster analyses showed that the community structure in the virus-amended mesocosms varied greatly between replicates. Despite the strong effects upon eubacterial communities, photosynthesis of co-occurring protists and cyanobacteria was not significantly altered by the presence of virus concentrates. This study supports the hypothesis that nutrient availability plays a key role in shaping sediment bacterial communities, and also that viruses may regulate the abundance of the dominant competitors and allow less dominant organisms to maintain or increase their abundance in a community due to decreased competition for resources.

The vertical distribution and diversity of marine bacteriophage at a station off Southern California.

Sixty-two bacteriophages were isolated on eight indigenous bacteria from a Pacific Ocean station spanning 887-m vertical depth, on two occasions between 1999 and 2000. On the basis of 16S rRNA sequences, six hosts were tentatively identified to be in the genus Vibrio and the other two were closely related to Altermonas macleodii (W9a) and Pseudoalteromonas spp. (W13a). Restriction fragment length polymorphism (RFLP) analysis of phage genomes using AccI and HapI showed that 16 phages infecting host C4a (Vibrio) displayed 14 unique RFLP patterns. However, identical phages infecting host C4b, C6a, and C6b (all Vibrio) were obtained from both the surface layer and the hypoxic zone at 850 m. Most phage isolates from the second year had a different RFLP pattern but shared genetic similarity to the phages infecting the same host from the previous year based on a hybridization study using phage genome probes. Cluster analysis of RFLP patterns and hybridization results also indicated that phages infecting the same or genetically related hosts, in general, shared higher degrees of homology in spite of the diverse RFLP patterns. Pulsed field gel electrophoresis (PFGE) analysis of native viral genomes indicated a range in genome size from less than 40 to 200 kb, and the dominant band shifted up by about 5-10 kb in the deep samples compared to the shallow ones. Hybridization of phage genome probes with total viral community DNA from various depths suggests these isolates, or at least some of their genes, represent a detectable portion of the natural viral community and were distributed throughout the water column. Thus, the results of this study demonstrated that the genetic diversity of bacteriophage in the ocean is far greater than that of their bacterial hosts. However, host range may have contributed to the evolution of the diverse phage population in the marine environment.

Viral influence on aquatic bacterial communities.

Bacterial viruses, or bacteriophages, have numerous roles in marine systems. Although they are now considered important agents of mortality of bacteria, a second possible role of regulating bacterial community composition is less well known. The effect on community composition derives from the presumed species-specificity and density-dependence of infection. Although models have described the "kill the winner" hypothesis of such control, there are few observational or experimental demonstrations of this effect in complex natural communities. We report here on some experiments that demonstrate that viruses can influence community composition in natural marine communities. Although the effect is subtle over the time frame suitable for field experiments (days), the cumulative effect over months or years would be substantial. Other virus roles, such as in genetic exchange or microbial evolution, have the potential to be extremely important, but we know very little about them.

Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae.

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.

Marine planktonic archaea take up amino acids.

Archaea are traditionally thought of as "extremophiles," but recent studies have shown that marine planktonic Archaea make up a surprisingly large percentage of ocean midwater microbial communities, up to 60% of the total prokaryotes. However, the basic physiology and contribution of Archaea to community microbial activity remain unknown. We have studied Archaea from 200-m depths of the northwest Mediterranean Sea and the Pacific Ocean near California, measuring the archaeal activity under simulated natural conditions (8 to 17 degrees C, dark and aerobic [corrected]) by means of a method called substrate tracking autoradiography fluorescence in situ hybridization (STARFISH) that simultaneously detects specific cell types by 16S rRNA probe binding and activity by microautoradiography. In the 200-m-deep Mediterranean and Pacific samples, cells binding the archaeal probes made up about 43 and 14% of the total countable cells, respectively. Our results showed that the Archaea are active in the uptake of dissolved amino acids from natural concentrations (nanomolar) with about 60% of the individuals in the archaeal communities showing measurable uptake. Bacteria showed a similar proportion of active cells. We concluded that a portion of these Archaea is heterotrophic and also appears to coexist successfully with Bacteria in the same water.

Rapid virus production and removal as measured with fluorescently labeled viruses as tracers.

Pelagic marine viruses have been shown to cause significant mortality of heterotrophic bacteria, cyanobacteria, and phytoplankton. It was previously demonstrated, in nearshore California waters, that viruses contributed to up to 50% of bacterial mortality, comparable to protists. However, in less productive waters, rates of virus production and removal and estimates of virus-mediated bacterial mortality have been difficult to determine. We have measured rates of virus production and removal, in nearshore and offshore California waters, by using fluorescently labeled viruses (FLV) as tracers. Our approach is mathematically similar to the isotope dilution technique, employed in the past to simultaneously measure the release and uptake of ammonia and amino acids. The results indicated overall virus removal rates in the dark ranging from 1.8 to 6.2% h(-1) and production rates in the dark ranging from 1.9 to 6.1% h(-1), corresponding to turnover times of virus populations of 1 to 2 days, even in oligotrophic offshore waters. Virus removal rates determined by the FLV tracer method were compared to rates of virus degradation, determined at the same locations by radiolabeling methods, and were similar even though the current FLV method is suitable for only dark incubations. Our results support previous findings that virus impacts on bacterial populations may be more important in some environments and less so in others. This new method can be used to determine rates of virus degradation, production, and turnover in eutrophic, mesotrophic, and oligotrophic waters and will provide important inputs for future investigations of microbial food webs.

Brugia malayi: localization of nitric oxide synthase in a lymphatic filariid.

Nitric oxide synthase converts L-arginine to citrulline and nitric oxide, a gaseous signaling molecule critical to multiple physiological responses. Nitric oxide synthase was detected by Western blot analysis of Brugia malayi extracts using an antibody raised against a peptide from murine brain nitric oxide synthase. Using NADPH diaphorase staining and immunohistochemistry, nitric oxide synthase was localized in the parasitic nematode B. malayi. As in Ascaris suum, nitric oxide synthase was detected in the body wall muscles of adult B. malayi. This localization pattern is in agreement with the role of nitric oxide in the control of muscle tone in other invertebrates and in vertebrates. A novel finding was the localization of nitric oxide synthase in the oocytes, in developing embryos, and in spermatozoa. B. malayi nitric oxide synthase may play a role in developmental signaling, as has been suggested for Drosophila and Ilyanassa, a marine mud snail.

Chitin synthase in the filarial parasite, Brugia malayi.

Fragments of putative chitin synthase (chs) genes from two filarial species (Brugia malayi and Dirofilaria immitis) were amplified by PCR using degenerate primers. The full genomic and cDNA sequences were obtained for the B. malayi chs gene (Bm-chs-1); the predicted amino acid sequence is highly similar, over a large region, to two CHS sequences of the nematode Caenorhabditis elegans and also to two insect CHS sequences. Bm-chs-1 is abundantly transcribed in B. malayi adult females, independent of their fertilization status, but is also expressed in males and microfilariae. Oocytes and early embryos contain large amounts of Bm-chs-1 transcript by in situ hybridization, but later stage embryos within the maternal uterus show little or no Bm-chs-1 transcript. No specific hybridization could be demonstrated in maternal somatic tissues. Polyclonal antibodies were raised against a peptide expressed from a recombinant cDNA fragment of Bm-chs-1; immunostaining detected CHS protein in oocytes and early to midstage embryos. These studies characterize a gene that is likely to be essential to oogenesis and embryonic development in a parasitic nematode. Because chitin synthesis and eggshell formation begin after fertilization, the presence of CHS protein in early oocytes suggests that the enzyme must be activated as a result of fertilization. These studies also demonstrate that chitin synthesis may not be restricted to eggshell formation in nematodes, as the Bm-chs-1 gene is transcribed in life cycle stages other than adult females.

Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria.

Total bacterial abundances estimated with different epifluorescence microscopy methods (4',6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dead) and with flow cytometry (Syto13) showed good correspondence throughout two microcosm experiments with coastal Mediterranean water. In the Syto13-stained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, "live" bacteria (determined as such with the Molecular Probes Live/Dead BacLight bacterial viability kit), and nucleoid-containing bacteria (NuCC) comprised similar fractions of the total bacterial community. Similarly, LDNA bacteria and "dead" bacteria (determined with the kit) comprised a similar fraction of the total bacterial community in one of the experiments. The rates of change of each type of bacteria during the microcosm experiments were also positively correlated between methods. In various experiments where predator pressure on bacteria had been reduced, we detected growth of the HDNA bacteria without concomitant growth of the LDNA bacteria, such that the percentage contribution of HDNA bacteria to total bacterial numbers (%HDNA) increased. This indicates that the HDNA bacteria are the dynamic members of the bacterial assemblage. Given how quickly and easily the numbers of HDNA and LDNA bacteria can be obtained, and given the similarity to the numbers of "live" cells and NuCC, the %HDNA is suggested as a reference value for the percentage of actively growing bacteria in marine planktonic environments.

Marine viruses and their biogeochemical and ecological effects.

Viruses are the most common biological agents in the sea, typically numbering ten billion per litre. They probably infect all organisms, can undergo rapid decay and replenishment, and influence many biogeochemical and ecological processes, including nutrient cycling, system respiration, particle size-distributions and sinking rates, bacterial and algal biodiversity and species distributions, algal bloom control, dimethyl sulphide formation and genetic transfer. Newly developed fluorescence and molecular techniques leave the field poised to make significant advances towards evaluating and quantifying such effects.

Combined microautoradiography-16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ.

We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial "black box" should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines 3H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the alpha subdivision of the class Proteobacteria (alpha-Proteobacteria) and of the Cytophaga-Flavobacterium group obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined ( approximately 60% of the universal probe-labeled cells and approximately 50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the alpha-Proteobacteria and Cytophaga-Flavobacterium groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the "dissection" of microbial communities by type and function.

Increase in Fluorescence Intensity of 16S rRNA In Situ Hybridization in Natural Samples Treated with Chloramphenicol.

Despite the numerous advantages of fluorescent in situ hybridization for the identification of single prokaryotic cells with 16S rRNA probes, use of the technique with natural samples, especially those from the marine environment, is still problematic. The low percentage of fluorescently labeled cells constitutes the primary problem for in situ hybridization of natural samples, probably due to low cellular rRNA content. This study represents an attempt to improve detection of marine prokaryotes by increasing cellular rRNA content without changing the species composition. Cells from three California coastal sites were treated with chloramphenicol, an inhibitor of protein synthesis and rRNA degradation, at 100 (mu)g/ml and then were probed with a "universal" 16S rRNA fluorescent probe and viewed by image-intensified video microscopy. Counts of fluorescent cells increased from ca. 75% for untreated samples to ca. 93 to 99% for chloramphenicol-treated samples, compared to counts produced by DAPI (4(prm1),6-diamidino-2-phenylindole) staining, after at least 45 min of exposure to the drug (these percentages include autofluorescent cells, which averaged 6%). This suggests that most cells in these samples were active. We hypothesize that the low fluorescent-cell counts previously reported were probably often due to the fluorescence intensity of labeled cells being below the detection level rather than to high levels of dead cells in marine environments. This method may aid in the characterization of bacterioplankton with fluorescent probes.

Determination of Active Marine Bacterioplankton: a Comparison of Universal 16S rRNA Probes, Autoradiography, and Nucleoid Staining.

We compared several currently discussed methods for the assessment of bacterial numbers and activity in marine waters, using samples from a variety of marine environments, from aged offshore seawater to rich harbor water. Samples were simultaneously tested for binding to a fluorescently labeled universal 16S rRNA probe; (sup3)H-labeled amino acid uptake via autoradiography; nucleoid-containing bacterial numbers by modified DAPI (4(prm1),6-diamidino-2-phenylindole) staining; staining with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), a compound supposed to indicate oxidative cell metabolism; and total bacterial counts (classical DAPI staining), taken as a reference. For the universal-probe counts, we used an image intensifying and processing system coupled to the epifluorescence microscope. All of the above-mentioned methods yielded lower cell counts than DAPI total counts. Universal-probe counts averaged about half of the corresponding DAPI count and were highly correlated to autoradiography counts (r(sup2) = 0.943; n = 7). Nucleoid-containing cell counts could be lower than DAPI counts by as much as 1 order of magnitude but sometimes matched autoradiography or probe counts. CTC counts were 2 orders of magnitude below DAPI counts. Universal 16S rRNA probe counts correlated well with autoradiography results, indicating a population with at least minimal metabolic activity. The greater variability of the nucleoid-containing cell counts calls for further investigation of the processes involved, and CTC counts were well below the range of the other methods tested.

Virus decay and its causes in coastal waters.

Recent evidence suggests that viruses play an influential role within the marine microbial food web. To understand this role, it is important to determine rates and mechanisms of virus removal and degradation. We used plaque assays to examine the decay of infectivity in lab-grown viruses seeded into natural seawater. The rates of loss of infectivity of native viruses from Santa Monica Bay and of nonnative viruses from the North Sea in the coastal seawater of Santa Monica Bay were determined. Viruses were seeded into fresh seawater that had been pretreated in various ways: filtration with a 0.2-(mu)m-pore-size filter to remove organisms, heat to denature enzymes, and dissolved organic matter enrichment to reconstitute enzyme activity. Seawater samples were then incubated in full sunlight, in the dark, or under glass to allow partitioning of causative agents of virus decay. Solar radiation always resulted in increased rates of loss of virus infectivity. Virus isolates which are native to Santa Monica Bay consistently degraded more slowly in full sunlight in untreated seawater (decay ranged from 4.1 to 7.2% h(sup-1)) than nonnative marine bacteriophages which were isolated from the North Sea (decay ranged from 6.6 to 11.1% h(sup-1)). All phages demonstrated susceptibility to degradation by heat-labile substances, as heat treatment reduced the decay rates to about 0.5 to 2.0% h(sup-1) in the dark. Filtration reduced decay rates by various amounts, averaging 20%. Heat-labile, high-molecular-weight dissolved material (>30 kDa, probably enzymes) appeared responsible for about 1/5 of the maximal decay. Solar radiation was responsible for about 1/3 to 2/3 of the maximal decay of nonnative viruses and about 1/4 to 1/3 of that of the native viruses, suggesting evolutionary adaptation to local light levels. Our results suggest that sunlight is an important contributing factor to virus decay but also point to the significance of particles and dissolved substances in seawater.

Gene cloning and production of active recombinant Brugia malayi microfilarial chitinase.

Canlas and coworkers [Canlas et al. (1984) Am. J. Trop. Med. Hyg. 33, 420-424] isolated a monoclonal antibody (MF1) which, upon passive transfer, led to the clearance of Brugia malayi (Bm) microfilariae (mf) from infected jirds. The target of MF1 is a developmentally regulated mf chitinase (Cht) (Fuhrman et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1548-1552). This paper describes the production of enzymatically active Bm Cht in Escherichia coli. Standard expression conditions resulted in production of an insoluble maltose-binding protein (MBP)::Cht fusion protein, but by optimizing expression conditions, the amount of soluble MBP::Cht was increased 25-fold. The specific activity of the soluble MBP::Cht isolated from the E. coli cytoplasm was low. Exporting MBP::Cht into the E. coli periplasmic space increased the specific activity by 12-fold. This suggests that secretion through the membrane and/or the environment of the periplasmic space results in improved folding of recombinant Bm Cht.

Discrete transcripts encode multiple chitinase isoforms in Brugian microfilariae.

The blood-borne microfilariae of the Brugian nematodes produce multiple isoforms of chitinase, whose expression is coincident with the onset of microfilarial infectivity for mosquitoes. A single cDNA sequence was previously obtained by screening a Brugia malayi microfilarial cDNA library, yet two chitinase isozymes are readily distinguished in this species. In this paper, we present evidence for the existence of multiple transcripts encoding Brugian microfilarial chitinases. Using primers based on the previously-sequenced cDNA clone, we amplified and sequenced two discrete products from B. malayi microfilarial RNA by RT-PCR. While the shorter fragment was nearly identical to the previously sequenced cDNA, the larger fragment contained an extra copy of a serine/threonine-rich repeat. RNAse protection assays were used to demonstrate that both sequences represent true transcripts, and not PCR artifacts. Using primers based on the B.malayi sequence, two novel sequences were generated by RT-PCR from B. pahangi microfilariae. Homologous and cross-species RNAse protection assays verified that multiple transcripts also encode chitinase isozymes in B. pahangi microfilariae.

Expression of recombinant microfilarial chitinase and analysis of domain function.

A family of chitinase isozymes was previously characterized from the microfilariae of Brugia malayi and Brugia pahangi. The expression of these enzymes correlates with the onset of microfilarial infectivity for the mosquito vector. To study the role of chitinase activity in filarial transmission, the p70 chitinase from Brugia malayi was cloned and expressed in two forms: a full-length product of approximately 62 kDa and a truncated product of 43 kDa containing only the N-terminal catalytic domain. Two epitopes defined by monoclonal antibodies were preserved only in the full-length recombinant enzyme. It was found that deletion of the cysteine-rich C-terminal domain increased the yield of the recombinant expression product, and did not affect the K(m) for di- or trisaccharide substrates. However, affinity for high molecular weight chitin was specific to the full-length molecule, and is apparently mediated by the cysteine-rich domain, suggesting a role for this part of the protein in targeting the secreted enzyme to its substrate.