High abundance of virulence gene homologues in marine bacteria

Marine bacteria can cause harm to single-celled and multicellular eukaryotes. However, relatively little is known about the underlying genetic basis for marine bacterial interactions with higher organisms. We examined whole-genome sequences from a large number of marine bacteria for the prevalence of homologues to virulence genes and pathogenicity islands known from bacteria that are pathogenic to terrestrial animals and plants. As many as 60 out of 119 genomes of marine bacteria, with no known association to infectious disease, harboured genes of virulence-associated types III, IV, V and VI protein secretion systems. Type III secretion was relatively uncommon, while type IV was widespread among alphaproteobacteria (particularly among roseobacters) and type VI was primarily found among gammaproteobacteria. Other examples included homologues of the Yersinia murine toxin and a phage-related 'antifeeding' island. Analysis of the Global Ocean Sampling metagenomic data indicated that virulence genes were present in up to 8% of the planktonic bacteria, with highest values in productive waters. From a marine ecology perspective, expression of these widely distributed genes would indicate that some bacteria infect or even consume live cells, that is, generate a previously unrecognized flow of organic matter and nutrients directly from eukaryotes to bacteria.     

Isolation and characterization of a high molecular weight antibiotic produced by a marine bacterium

Many marine bacteria demonstrate antibiotic activity against organisms of terrestrial origin. Low molecular weight antibiotics have been extracted and, in some cases, purified, but few attempts have been made to isolate high molecular weight antibiotics produced by marine bacteria. In the study reported here, a high molecular weight antibiotic was extracted from whole cells ofAlteromonas strain P18 (NCMB 1890) grown on 2216E medium. Purification included ammonium sulfate precipitation, ultracentrifugation, chromatography on DEAE cellulose, and gel filtration on Ultrogel. A rapid method for measuring specific activity of the antibiotic was developed.     

Abundant and diverse bacteria involved in DMSP degradation in marine surface waters

An expanded analysis of oceanic metagenomic data indicates that the majority of prokaryotic cells in marine surface waters have the genetic capability to demethylate dimethylsulfoniopropionate (DMSP). The 1701 homologues of the DMSP demethylase gene, dmdA , identified in the (2007) Global Ocean Sampling (GOS) metagenome, are sufficient for 58% (±9%) of sampled cells to participate in this critical step in the marine sulfur cycle. This remarkable frequency of DMSP-demethylating cells is in accordance with biogeochemical data indicating that marine phytoplankton direct up to 10% of fixed carbon to DMSP synthesis, and that most of this DMSP is subsequently degraded by bacteria via demethylation. The GOS metagenomic data also revealed a new cluster of dmdA sequences (designated Clade E) that implicates marine gammaproteobacteria in DMSP demethylation, along with previously recognized alphaproteobacterial groups Roseobacter and SAR11. Analyses of G+C content and gene order indicate that lateral gene transfer is likely responsible for the wide distribution of dmdA among diverse taxa, contributing to the homogenization of biogeochemical roles among heterotrophic marine bacterioplankton. Candidate genes for the competing bacterial degradation process that converts DMSP to the climate-active gas dimethylsulfide (DMS) ( dddD and dddL ) occur infrequently in the (2007) GOS metagenome, suggesting either that the key DMS-producing bacterial genes are yet to be identified or that DMS formation by free-living bacterioplankton is insignificant relative to their demethylation activity.     

Purification and characterization of a natural agglutinin in the hemolymph of the prawn Penaeus indicus H. Milne Edwards.

A natural agglutinin in the hemolymph of the marine prawn Penaeus indicus was isolated by gel filtration chromatography, purified using polyacrylamide gel electrophoresis, and characterized. Prawn agglutinin has a native molecular mass of 181 kDa and consists of two monomeric units (97 and 84 kDa), maintains some agglutinating activity over a wide pH range (7-9), and is inactivated at 85 degrees C. The agglutinin was denatured upon mixing with trichloroacetic acid, phenol, chloroform, and 45% ammonium sulfate. It was also sensitive to trypsin digestion. The results indicate that prawn agglutinin is proteinaceous in nature, with agglutinating, hemolytic, and antibacterial properties against marine bacteria and erythrocytes with carbohydrate binding sites.     

Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I

Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20-200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes-Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-microm aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously.     

Lectin, a possible basis for symbiosis between bacteria and sponges.

From the marine sponge Halichondria panicea a lectin was isolated and characterized. The homogeneous lectin (composed of protein to 80.7% and of neutral carbohydrates to 14.1%) had a molecular weight of 78,000 (determined by gel filtration) and consisted of four subunits with a molecular weight of 21,000 each (determined by gel electrophoresis in the presence of sodium dodecyl sulfate). The hemagglutinating activity was only slightly dependent upon ionic strength and incubation temperature and did not require divalent cations, but it was inhibited by reagents for thiol groups. The Halichondria lectin was completely inhibited in hemagglutination competition experiments in the presence of fetuin, D-galacturonic acid, D-glucuronic acid, polygalacturonic acid, or L-fucose. The purified Halichondria lectin did not cause reaggregation of dissociated H. panicea cells. From the same sponge species bacteria were isolated and identified as Pseudomonas insolita. These bacteria were cultivated in marine broth 2216. Under these culture conditions the bacteria grew only in the presence of the homologous lectin; the lectin-caused effect was not abolished by D-glucuronic acid or D-galacturonic acid. However, after addition of a polysaccharide-containing fraction isolated from P. insolita, the lectin-caused, growth-promoting effect was abolished. Other lectins were found to exhibit no growth-promoting effect. On the basis of colony counts, P. insolita was the predominant bacterial species in the sponge extract; 1.9 X 10(6) Pseudomonas colonies were measured in extracts isolated from 1 g of sponge. The assumption of an interrelationship between the sponge and the bacterium is supported by the results indicating that the Halichondria lectin has no effect on the growth of such bacteria isolated from six other marine sponge species. Evidence is presented which indicates that the Halichondria lectin is not utilized during growth of the Pseudomonas species. Lectin activity was detected on the surface of mucoid cells from H. panicea. From the data obtained the possibility is discussed that the Halichondria lectin is a basis for a symbiotic relationship between the sponge and the bacterium.     

Isolation and characterization of filterable marine bacteria

Anderson, J. I. W. (Northeast Shellfish Sanitation Research Center, Narragansett, R.I.), and W. P. Heffernan. Isolation and characterization of filterable marine bacteria. J. Bacteriol 90:1713-1718. 1965.-By a process of double filtration of seawater, first through a membrane filter with a pore diameter of 0.45 mu and then through a membrane filter with a pore diameter of 0.22 mu, it was possible to isolate on the surface of the latter membrane a group of marine organisms not usually encountered by conventional techniques of pour plates or one-stage filtration. Many of the isolates could not be identified, but the largest single group belonged to the genus Spirillum; other isolates were placed in the genera Leucothrix, Flavobacterium, Cytophaga, and Vibrio. A group of four organisms which was not identified was characterized by the formation of large, club-shaped cells, 20 to 30 mu long. Of the 25 strains studied in detail, 22 required seawater for growth and 8 retained their filterable property after cultivation. No filterable bacteria were isolated from terrestrial samples.     

Occurrence and patterns of antibiotic resistance in vertebrates off the Northeastern United States coast

The prevalence of antibiotic-resistant bacteria in the marine environment is a growing concern, but the degree to which marine mammals, seabirds and fish harbor these organisms is not well documented. This project sought to identify the occurrence and patterns of antibiotic resistance in bacteria isolated from vertebrates of coastal waters in the northeastern United States. Four hundred and seventy-two isolates of clinical interest were tested for resistance to a suite of 16 antibiotics. Fifty-eight percent were resistant to at least one antibiotic, while 43% were resistant to multiple antibiotics. A multiple antibiotic resistance index value ≥0.2 was observed in 38% of the resistant pathogens, suggesting exposure of the animals to bacteria from significantly contaminated sites. Groups of antibiotics were identified for which bacterial resistance commonly co-occurred. Antibiotic resistance was more widespread in bacteria isolated from seabirds than marine mammals, and was more widespread in stranded or bycaught marine mammals than live marine mammals. Structuring of resistance patterns based on sample type (live/stranded/bycaught) but not animal group (mammal/bird/fish) was observed. These data indicate that antibiotic resistance is widespread in marine vertebrates, and they may be important reservoirs of antibiotic-resistant bacteria in the marine environment.     

Bacterial community associated with Pfiesteria-like dinoflagellate cultures

Dinoflagellates (Eukaryota; Alveolata; Dinophyceae) are single-cell eukaryotic microorganisms implicated in many toxic outbreaks in the marine and estuarine environment. Co-existing with dinoflagellate communities are bacterial assemblages that undergo changes in species composition, compete for nutrients and produce bioactive compounds, including toxins. As part of an investigation to understand the role of the bacteria in dinoflagellate physiology and toxigenesis, we have characterized the bacterial community associated with laboratory cultures of four ' Pfiesteria -like' dinoflagellates isolated from 1997 fish killing events in Chesapeake Bay. A polymerase chain reaction with oligonucleotide primers specific to prokaryotic 16S rDNA gene sequences was used to characterize the total bacterial population, including culturable and non-culturable species, as well as possible endosymbiotic bacteria. The results indicate a diverse group of over 30 bacteria species co-existing in the dinoflagellate cultures. The broad phylogenetic types of dinoflagellate-associated bacteria were generally similar, although not identical, to those bacterial types found in association with other harmful algal species. Dinoflagellates were made axenic, and the culturable bacteria were added back to determine the contribution of the bacteria to dinoflagellate growth. Confocal scanning laser fluorescence microscopy with 16S rDNA probes was used to demonstrate a physical association of a subset of the bacteria and the dinoflagellate cells. These data point to a key component in the bacterial community being species in the marine alpha-proteobacteria group, most closely associated with the α-3 or SAR83 cluster.     

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.     

Construction of a metagenomic library for the marine sponge Halichondria okadai

Symbionts of the marine sponge Halichondria okadai are promising as a source of natural products. Metagenomic technology is a powerful tool for accessing the genetic and biochemical potential of bacteria. Hence, we established a method of recovering bacterial-enriched metagenomic DNA by stepwise centrifugation. The metagenomic DNA was analyzed by ultrafast 454-pyrosequencing technology, and the results suggested that more than three types of bacterial DNA, Alphaproteobacteria, Actinobacteria, and Cyanobacteria, had been recovered, and that eukaryotic genes comprised only 0.02% of the metagenomic DNA. These results indicate that stepwise centrifugation and real-time quantitative PCR were effective for separating sponge cells and symbiotic bacteria, and that we constructed a bacteria-enriched metagenomic library from a marine sponge, H. okadai, selectively for the first time.     

Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum

In many bivalve molluscs, lectins are present in the hemolymph and are thought to be important for internal host defense mechanisms. For this study, we purified a novel isoform of the Manila clam lectin (designated MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum, using affinity chromatography and gel filtration. Native PAGE results showed that the MCL-4 consisted of 70 kDa protein. MCL-4 was found to be composed of 58-kDa and 43-kDa bands when examined using SDS-PAGE under reducing and non-reducing conditions. The native MCL-4 was revealed as a 147 kDa molecular mass protein by gel filtration. The purified MCL-4 agglutinates calcium-dependently in the erythrocytes of sheep and rabbit, but not in cells of the three species of marine bacteria tested. However, the phagocytic ability of the R. philippinarum hemocytes for the MCL-4-opsonized Vibrio tubiashii cells was significantly greater than that for the BSS-treated bacterial cells. Addition of purified MCL-4 markedly suppressed Alteromonas haloplanktis growth. These results suggest that MCL-4, because of its opsonizing and bacteriostatic properties, might contribute to the host defense mechanisms against invading microorganisms in R. philippinarum.     

Inhibition of larval barnacle attachment to bacterial films: An investigation of physical properties

The effects of films of two strains of a marine bacterium, Deleya marina (ATCC 25374 and 27129) on the attachment response of cypris larvae of the balanomorph barnacle, Balanus amphitrite, were examined in the laboratory. Tests showed that the cell-surface hydrophobicities of the two bacteria in suspension were different. In contrast, films derived from these cells were both highly wettable (i.e., displayed high surface free energy). Assays (22 hours) compared permanent attachment of larval barnacles to films derived from exponential and stationary phase cells for both bacteria. These films either had no effect or inhibited attachment of both 0-day- and 4-day-old cypris larvae when compared with unfilmed controls. Our data indicate that inhibition of larval barnacle attachment by films of the two bacteria is the result of factors other than surface free energy. Production of chemical barnacle settlement inhibitors by the bacteria is hypothesized.Offprint requests to: J. S. Maki.     

Effects of Ultraviolet Radiation on the Gram-Positive Marine Bacterium <Emphasis Type="Italic">Microbacterium maritypicum</Emphasis>

Although extensive information is available on the effect ultraviolet (UV) radiation has on Gram-negative marine bacteria, there is a scarcity of data concerning UV radiation and Gram-positive marine bacteria. The focus of this paper is on Microbacterium maritypicum, with the Gram-negative Vibrio natriegens being used as a standard of comparison. M. maritypicum exhibited growth over a NaCl range of 0–1000 mM, with optimum growth occurring between 0 and 400 mM NaCl. In contrast, V. natriegens grew over a NaCl span of 250–1000 mM, with best growth being observed between 250 and 600 mM NaCl. UV radiation experiments were done using the medium with 250 mM NaCl. For solar (UV-A and B) radiation and log-phase cells, M. maritypicum was determined to be three times more resistant than V. natriegens. For germicidal (UV-C) radiation, the pattern of resistance of the log-phase cells to the lethal effects of the radiation was even more pronounced, with the Gram-positive bacterium being more than 12 to 13 times more resistant. Similar data to the solar and germicidal log-phase UV kill curves were obtained for stationary-phase cells of both organisms. Photoreactivation was observed for both types of cells exposed to UV-C but none for cells treated with UV-A and B. When log phase cells of M.maritypicum were grown at 0.0 and 0.6 M NaCl and exposed to UV-C radiation, no difference in survivorship patterns was noted from that of 0.25 M NaCl grown cells. Although this study has only focused on two marine bacteria, our results indicate that the Gram-positive M. maritypicum could have a built-in advantage for survival in some marine ecosystems.     

Resolution of viable and membrane-compromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining

The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214-218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.     

Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining

The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214–218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.     

Blooms of sequence-specific culturable bacteria in the sea

Abstract Using specific deoxyoligonucleotide probes we have discovered seasonally strong (up to ∼ 100%) dominance of bacteria hybridizing to a single probe, in near shore waters off Scripps pier (32°53'N; 117°15'W). The probes were designed from partially sequenced 16S rRNA (V3 domain) of isolated marine bacteria. The results indicate that this approach may be used for studies of bacterial populations in the marine environment. We have shown that a number of genotypes that at times are dominant in the natural assemblages are culturable (and not, 'viable-but-unculturable'). Additionally, our data suggests that the discrepancy between viable counts and direct counts in sea water samples can be explained by low plating efficiency.     

Detection of Horizontal Gene Transfer by Natural Transformation in Native and Introduced Species of Bacteria in Marine and Synthetic Sediments

Both naturally occurring marine sediments and artificial sediments were used as supports for natural transformation of marine bacteria. While transformation of Pseudomonas stutzeri ZoBell suspended in artificial seawater was not detected when recipient cells and rifampin resistance DNA were loaded onto sterile sediment columns, transformation could be detected at frequencies 4 to 20 times that of spontaneous resistance when recipient cells and rifampin resistance DNA were loaded onto sterile sediment columns. Treatment of these columns with DNase I reduced transformation frequencies to levels comparable to those of spontaneous-resistance frequencies. Sediments with higher organic contents supported higher frequencies of transformation than did those with lower amounts of organic matter. Transformation was also detected when recipient cells and DNA were loaded on columns prepared from nonsterile sediments, although the frequencies of transformation were lower than when sterile sediments were used. Finally, nonsterilized sediments that were not supplemented with laboratory strains did not support detectable levels of transformation in sediment columns, but when these same sediments were transferred to filters and placed on complex media, transformation was detected at a frequency three times that for spontaneous resistance. This transformation frequency was partially reduced to levels near that for spontaneous resistance by the addition of DNase I to sediment filters. These results indicate that marine sediments facilitate the uptake and expression of exogenous DNA by transformable marine bacteria and that sediments are a more likely niche for natural transformation than the water column in the marine environment.     

Biomarker evidence for widespread anaerobic methane oxidation in Mediterranean sediments by a consortium of methanogenic archaea and bacteria. The Medinaut Shipboard Scientific Party

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible Nautile. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in (13)C (delta(13)C values are as low as -95 per thousand). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of (13)C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.     

Investigation of 0.2 µm filterable bacteria from the Western Mediterranean Sea using a molecular approach: dominance of potential starvation forms

Although the existence of 0.2 μm filterable bacteria has been known since the early 80's, they are not taken into consideration when modeling microbial food webs, due to an overall lack of information concerning this specific size class. According to physiological studies on starvation forms and investigations on small bacterial cells in marine ecosystems, a 0.2 μm filtrate may consist of different phenotypes: starvation forms of typical marine bacteria, ultramicrobacteria or bacterial cells, even larger than 0.2 μm, but flexible enough to pass the nominal filter pore-size. In this pilot study we examined three filtered seawater fractions from the Western Mediterranean Sea (Bay of Calvi, Corsica/France) - the total bacterial population, the bacterial fraction above 0.2 μm and the 0.2 μm filtrate - to investigate the bacterial community structure of each of those fractions by the molecular approach of denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments. The analysis of the resulting DGGE profiles revealed different patterns of dominant bands for the 0.2 μm filterable and the total bacterial populations within the samples. Additionally the 0.2 μm filterable bacterial compartment exhibited obvious differences in band patterns for winter and summer samples, which were not observed for the total bacterial fraction. According to the current knowledge concerning the status of 0.2 μm filterable bacteria, DGGE patterns indicate that most of the fragments representing 0.2 μm filterable bacteria were rather starvation forms of marine bacteria than ultramicrobacteria. The sequencing of excised and cloned DNA bands of the DGGE profiles characterized the phylogenetic affiliation of the corresponding 0.2 μm filterable bacteria, clustering mainly with known, typical marine isolates of both alpha-subclass and gamma-subclass of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides branch.