High Ratio of Bacteriochlorophyll Biosynthesis Genes to Chlorophyll Biosynthesis Genes in Bacteria of Humic Lakes

Recent studies highlight the diversity and significance of marine phototrophic microorganisms such as picocyanobacteria, phototrophic picoeukaryotes, and bacteriochlorophyll- and rhodopsin-holding phototrophic bacteria. To assess if freshwater ecosystems also harbor similar phototroph diversity, genes involved in the biosynthesis of bacteriochlorophyll and chlorophyll were targeted to explore oxygenic and aerobic anoxygenic phototroph composition in a wide range of lakes. Partial dark-operative protochlorophyllide oxidoreductase (DPOR) and chlorophyllide oxidoreductase (COR) genes in bacteria of seven lakes with contrasting trophic statuses were PCR amplified, cloned, and sequenced. Out of 61 sequences encoding the L subunit of DPOR (L-DPOR), 22 clustered with aerobic anoxygenic photosynthetic bacteria, whereas 39 L-DPOR sequences related to oxygenic phototrophs, like cyanobacteria, were observed. Phylogenetic analysis revealed clear separation of these freshwater L-DPOR genes as well as 11 COR gene sequences from their marine counterparts. Terminal restriction fragment length analysis of L-DPOR genes was used to characterize oxygenic aerobic and anoxygenic photosynthesizing populations in 20 lakes differing in physical and chemical characteristics. Significant differences in L-DPOR community composition were observed between dystrophic lakes and all other systems, where a higher proportion of genes affiliated with aerobic anoxygenic photosynthetic bacteria was observed than in other systems. Our results reveal a significant diversity of phototrophic microorganisms in lakes and suggest niche partitioning of oxygenic and aerobic anoxygenic phototrophs in these systems in response to trophic status and coupled differences in light regime.Recent studies have discovered novel phototrophic organisms and pointed us to their diversity in the oceans (1, 2, 3, 27, 39, 47, 50). Microorganisms such as picocyanobacteria, picoeukaryotes, and bacteriochlorophyll- and rhodopsin-containing bacteria use diverse photopigments to photosynthesize. These organisms represent a significant fraction of marine microbial communities and are likely to be ecologically and biogeochemically significant (1, 2, 13, 27, 28, 35, 39, 47). Several molecular studies based on genes of the puf operon, coding for the bacteriochlorophyll subunits, have shown that Roseobacter and Roseobacter-like bacteria constitute a significant portion of the aerobic anoxygenic photosynthetic bacteria (AAnPB) in marine waters (37). Microscopic counts have revealed that AAnPB contribute 1 to 16% to the total marine bacterioplankton in the euphotic zone and that there are regional and temporal differences in their abundances (see, for example, references 28, 41, and 50). Still, the global significance of this functional group and the role of, for example, AAnPB in the oceanic flow of energy and carbon are controversial (13, 20, 27, 28, 41, 48). Most AAnPB isolated so far have been described as photoorganoheterotrophs that rely primarily on organic substrates for growth but can derive a significant portion of their energy requirements from solar radiation (references 13 and 26 and references therein).AAnPB have been isolated from various freshwater habitats, ranging from cyanobacterial mats (49) to the pelagic zone of oligotrophic lakes (19, 38), but there are so far no studies of freshwater AAnPB diversity and community composition based on culture-independent techniques. A recent survey revealed some first patterns in the distribution of bacteriochlorophyll a-containing cells as well as the concentrations of the pigments in lakes ranging from oligotrophic to eutrophic. Infrared epifluorescence microscopy, fluorescence emission spectroscopy, and high-performance liquid chromatography were used to demonstrate that AAnPB may constitute up to 80% of total bacterial biomass in some low-productive lakes, implying that they are an important component of many lake ecosystems (33). In addition, genes encoding proteins for light harvesting (bacteriochlorophyll pufL and pufM gene clusters) have been identified in fosmid libraries from bacteria of the Delaware River (48) and in a functional gene survey of an Antarctic lake (24).In the present study, we used a specific primer set that amplifies the L subunit of the dark-operative protochlorophyllide oxidoreductase (L-DPOR) and its homologs (nitrogenase and chlorophyllide oxidoreductase [COR]). The dark-operative protochlorophyllide oxidoreductase (DPOR) is encoded by three genes (chlN-bchN, chlB-bchB, and chlL-bchL) and catalyzes the reductive formation of chlorophyllide from protochlorophyllide during biosynthesis of chlorophylls (chl) and bacteriochlorophylls (bch) in the dark (see reference 7 for more detail). Analysis of the deduced amino acid sequences indicated the presence of significant sequence dissimilarity in DPOR between oxygenic and anoxygenic photosynthetic organisms (5, 8, 16, 17, 18). Molecular studies have shown that AAnPB contain only DPOR (15) but that cyanobacteria, algae, and gymnosperms (nonflowering plants) contain both DPOR and a light-dependent protochlorophyllide oxidoreductase (POR) which carries one of the only two known enzymes other than photochemical reaction centers with light-driven catalysis (7).Despite the lack of POR, AAnPB are able to modify their chlorophyllide so that their absorption spectrum is broadened to span from <350 to <1,050 nm (usually 365 to 770 nm) in the UV and near-infrared ranges. This spectral characteristic allows AAnPB to utilize light at a wavelength other than that utilized by chlorophyll-containing cyanobacteria and algae. The first step in a series of modifications is performed by COR, an enzyme that is found in anoxygenic phototrophic bacteria and that transforms chlorophyllide to bacteriochlorophyll (7).In the present study, we assessed the diversity of L-DPOR, COR, and the coamplified homolog nifH genes in bacteria of seven different lakes by using PCR-based clone libraries parallel to a molecular fingerprinting technique to study L-DPOR gene composition in a larger data set (comprising 20 Swedish lakes). By using L-DPOR genes as our target, we simultaneously assessed the compositions of both AAnPB and oxygenic phototrophs in freshwater ecosystems and their distributions along trophic gradients.     

Spatiotemporal Variations in Microcystin Concentrations and in the Proportions of Microcystin-Producing Cells in Several Microcystis aeruginosa Populations

With the aim of explaining the variations in microcystin (MC) concentrations during cyanobacterial blooms, we studied several Microcystis aeruginosa populations blooming in different freshwater ecosystems located in the same geographical area. As assessed by real-time PCR, it appeared that the potentially MC-producing cells (mcyB⁺) were predominant (70 to 100%) in all of these M. aeruginosa populations, with the exception of one population in which non-MC-producing cells always dominated. Apart from the population in the Grangent Reservoir, we found that the proportions of potentially MC-producing and non-MC-producing cells varied little over time, which was consistent with the fact that according to a previous study of the same populations, the intergenic transcribed spacer (ITS) genotype composition did not change (38). In the Grangent Reservoir, the MC-RR variant was the dominant microcystin variant throughout the bloom season, despite changes in the ITS composition and in the proportions of mcyB⁺ cells. Finally, the variations in total MC concentrations (0.3 to 15 μg liter⁻¹) and in the MC cellular quotas (0.01 to 3.4 pg cell⁻¹) were high both between and within sites, and no correlation was found between the MC concentrations and the proportion of mcyB⁺ cells. All of these findings demonstrate that very different results can be found for the proportions of potentially MC-producing and non-MC-producing cells and MC concentrations, even in M. aeruginosa populations living in more or less connected ecosystems, demonstrating the importance of the effect of very local environmental conditions on these parameters and also the difficulty of predicting the potential toxicity of Microcystis blooms.Microcystins (MCs) are the most common cyanotoxins and have been involved in several animal and human poisoning episodes (8). These hepatotoxic cyclic heptapeptides are synthesized by a multifunctional enzyme complex (10, 40), and the discovery of the gene cluster encoding this complex has made it possible in recent years to develop molecular tools for studying the relative proportions of MC-producing and non-MC-producing cells in natural cyanobacterial populations. Potentially MC-producing and non-MC-producing cells can coexist in these populations, but the factors and processes governing the dynamics of these subpopulations remain unclear.Some recent papers on the Microcystis genus have shown that the proportions of potentially MC-producing cells can differ considerably from lake to lake. For example, in Lake Wannsee, Germany, this proportion was between 0 and 40% (28), as it was in Lake Oneida, United States (18), and in Lake Mikata, Japan (48). In contrast, large variations over time (6 to 93%) of potentially MC-producing cells were found in the Grangent Reservoir, France (4). Major variations (30 to 80%) were also found in a natural French population of Planktothrix agardhii (3), and the variations in the proportions of potentially MC-producing cells reflected those of the MC concentrations. However, only 54% of the variation in MC concentrations could be explained by changes in the proportion of MC-producing cells, suggesting that a considerable part of the MC concentrations was also due to variations in MC cell quotas. These findings suggest that the toxic risks during cyanobacterial proliferations are due to variations in both the proportion of MC-producing cells and the production of MC by the toxic cells.Numerous papers have already investigated the impact of various biotic and abiotic environmental factors on microcystin production by toxic cells. These studies demonstrate that MC production can be influenced by temperature (35), light (46), nutrients such as nitrogen and phosphorus (12, 32), pH (39), iron (42), xenobiotics (17, 34, 45), and predators (22, 23, 47). Despite inconsistent results, the production of microcystins by the cells does seems to be linked to their growth rate (11, 31, 33), which is itself affected by environmental conditions. On the other hand, several studies of variations in the proportions of MC-producing cells have demonstrated the potential influence of nutrient concentrations (9, 48) and light and temperature (5), and two papers (3, 5) have suggested that there is a negative correlation between the proportions of MC-producing cells and the abundance of cyanobacterial cells. These findings are consistent with the data of Kardinaal and Visser (26), showing that in Dutch lakes there is a negative relationship between the densities of cyanobacterial cells and the mean MC concentration in the cells.In an overall attempt to explain the variations of toxicity during cyanobacterial blooms, we studied the spatiotemporal variations in MC concentrations and in the proportions of MC-producing and non-MC-producing cells in several Microcystis aeruginosa populations blooming in different freshwater ecosystems located in the same geographical area. The point of this study was to analyze these variations in terms of the characteristics of these ecosystems and the population dynamics of the M. aeruginosa populations. In addition, these data were compared to the variations in the intergenic transcribed spacer (ITS) composition of the same populations recently reported by Sabart et al. (38). The proportion of potentially MC-producing cells was estimated by a real-time quantitative PCR approach, the change in threshold cycle (ΔC_T) method recently developed by Briand et al. for Planktothrix (3) and Microcystis (4) and targeting the mcyB (mcyA for Planktothrix) and phycocyanin (PC) genes.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Population Turnover in a Microcystis Bloom Results in Predominantly Nontoxigenic Variants Late in the Season

Surface samples of the 2007 Microcystis bloom occurring in Copco Reservoir on the Klamath River in Northern California were analyzed genetically by sequencing clone libraries made with amplicons at three loci: the internal transcribed spacer of the rRNA operon (ITS), cpcBA, and mcyA. Samples were taken between June and October, during which time two cell count peaks occurred, in mid-July and early September. The ITS and cpcBA loci could be classified into four or five allele groups, which provided a convenient means for describing the Microcystis population and its changes over time. Each group was numerically dominated by a single, highly represented sequence. Other members of each group varied by changes at 1 to 3 nucleotide positions, while groups were separated by up to 30 nucleotide differences. As deduced by a partial sampling of the clone libraries, there were marked population turnovers during the season, indicated by changes in allele composition at both the ITS and cpcBA loci. Different ITS and cpcBA genotypes appeared to be dominant at the two population peaks. Toxicity (amount of microcystin per cell) and toxigenic potential (mcyB copy number) were lower during the second peak, and the mcyB copy number fell further as the bloom declined.Toxic freshwater cyanobacterial blooms, commonly caused by Microcystis, are of current concern in many parts of the world because of their effects on drinking water, water-based recreation, and watershed ecology (5, 7). Microcystis cells are able to produce microcystin, a nonribosomally synthesized cyclic heptapeptide hepatotoxin with potent inhibitory activity against mammalian protein phosphatases (27) whose synthesis is directed by the 55-kb mcy gene cluster (25). The Microcystis genus exhibits worldwide occurrence, although the extent of genetic differentiation between or within geographical regions is currently uncertain due to a relatively sparse database, in spite of a growing number of studies (1, 2, 9, 11, 26, 28, 29).Only a few studies to date have used gene-specific tools to investigate the changes in the Microcystis population structure throughout the development of a bloom season. In some instances, there has been little indication of major population changes. Thus, the proportion of toxigenic (mcyB⁺) Microcystis was stable over the course of two consecutive bloom seasons in Lake Wannsee (Berlin, Germany) (17). The internal transcribed spacer of the rRNA operon (ITS) genotype, as assessed by denaturing gradient gel electrophoresis (DGGE) and sequencing, was also stable in Lake Volkerak (Netherlands) during 2001 (15). In contrast, studies of other lakes have observed changes in the Microcystis genotypes and in the proportion of potentially toxigenic cells during a bloom season (3, 15, 21, 31, 32). A better understanding of the population changes that occur during the development of toxic blooms is important in understanding their ecology and in assessing whether it might be feasible to manage Microcystis blooms in order to minimize toxicity.Copco Reservoir is a lake formed by a hydroelectric dam on the Klamath River in northern California. Beginning in 2004, highly toxic blooms dominated by Microcystis have developed between June and November (10, 13). Most studies of Microcystis blooms have been conducted in lakes with low in- and outflows. Copco Reservoir sits on a major river with normal through-flows of 1,000 to 3,000 cubic feet per second (cfs) during bloom season, although much of this flow occurs below the epilimnion, resulting in a surface water residence time of 20 to 25 days during summer (13). The consequences of toxic blooms in the reservoir may be carried to downstream reaches of the river, since elevated Microcystis levels have been present downstream of Copco Reservoir (14). We report here the results of a survey of the genotypic structure of the Microcystis population in Copco Reservoir during the 2007 bloom season. Major population shifts evident at the ITS and cpcBA loci coincided with the replacement of toxigenic with nontoxigenic strains.     

Isolation of Novel Extreme-Tolerant Cyanobacteria from a Rock-Dwelling Microbial Community by Using Exposure to Low Earth Orbit

Many cyanobacteria are known to tolerate environmental extremes. Motivated by an interest in selecting cyanobacteria for applications in space, we launched rocks from a limestone cliff in Beer, Devon, United Kingdom, containing an epilithic and endolithic rock-dwelling community of cyanobacteria into low Earth orbit (LEO) at a height of approximately 300 kilometers. The community was exposed for 10 days to isolate cyanobacteria that can survive exposure to the extreme radiation and desiccating conditions associated with space. Culture-independent (16S rRNA) and culture-dependent methods showed that the cyanobacterial community was composed of Pleurocapsales, Oscillatoriales, and Chroococcales. A single cyanobacterium, a previously uncharacterized extremophile, was isolated after exposure to LEO. We were able to isolate the cyanobacterium from the limestone cliff after exposing the rock-dwelling community to desiccation and vacuum (0.7 × 10⁻³ kPa) in the laboratory. The ability of the organism to survive the conditions in space may be linked to the formation of dense colonies. These experiments show how extreme environmental conditions, including space, can be used to select for novel microorganisms. Furthermore, it improves our knowledge of environmental tolerances of extremophilic rock-dwelling cyanobacteria.The surface and interior of rocks is a ubiquitous environment for microorganisms. Comprehensive culturing and culture-independent analyses of endolithic (interior of rocks) and epilithic (on the surface of rocks) microbial communities have been conducted. The primary producers in these environments are phototrophs, such as cyanobacteria, that are either free living or endosymbionts in lichens (16).Epilithic microorganisms are often an important part of rock-dwelling communities. The characterization of the epilithic cyanobacteria from natural environments, such as beach rock and caves, and from human-made environments, such as hypogea and buildings, has identified a variety of cyanobacteria. This includes both unicellular and filamentous forms, for example, Lyngbya-related species and Chroococcidiopsis (5, 14, 37, 47).Many microorganisms also inhabit the interior of rocks as endoliths. The endolithic environment provides protection from environmental stresses such as desiccation, extreme temperature, UV radiation, and high photosynthetically active radiation (400 to 700 nm) (6, 16, 25, 32). Endolithic communities are often the dominant form of life in extreme environments such as hot and cold deserts (15-17), savannahs, and semideserts (3, 6, 15, 48). In these extreme environments, the endolithic cyanobacteria are mainly unicellular cyanobacteria, for example, Chroococcidiopsis, Myxosarcina, and Gloeocapsa species (11, 46, 50). Conversely, in nondesert environments, such as dolomitic rocks in Switzerland (41), the limestone of the Niagara Escarpment (19, 20), and travertine deposits in Yellowstone National Park, the endolithic communities are more diverse and include both filamentous and unicellular types of cyanobacteria, such as Leptolyngbya, Nostoc, and Synechocystis (34).Although rock-dwelling cyanobacteria communities are diverse, there has been limited, if any, use of artificial extreme conditions to select for novel extremophilic cyanobacteria from these environments. Such an investigation could have implications for understanding the physiological requirements of life in extreme environments.The work described in this paper was motivated by an interest in understanding the physiological tolerance of cyanobacteria to space conditions and their potential use in space applications, such as oxygen and feedstock provision, which are crucial for extraterrestrial settlements (23, 29). In this work, we exposed samples of a coastal limestone cliff in Beer, Devon, United Kingdom, which is inhabited by a diverse cyanobacterial community, to low Earth orbit (LEO) to isolate novel extreme-tolerant cyanobacteria.     

Detection of Microcystin-Producing Cyanobacteria in Missisquoi Bay,Quebec, Canada,Using Quantitative PCR

Toxic cyanobacterial blooms, as well as their increasing global occurrence, pose a serious threat to public health, domestic animals, and livestock. In Missisquoi Bay, Lake Champlain, public health advisories have been issued from 2001 to 2009, and local microcystin concentrations found in the lake water regularly exceeded the Canadian drinking water guideline of 1.5 μg liter⁻¹. A quantitative PCR (Q-PCR) approach was developed for the detection of blooms formed by microcystin-producing cyanobacteria. Primers were designed for the β-ketoacyl synthase (mcyD_KS) and the first dehydratase domain (mcyD_DH) of the mcyD gene, involved in microcystin synthesis. The Q-PCR method was used to track the toxigenic cyanobacteria in Missisquoi Bay during the summers of 2006 and 2007. Two toxic bloom events were detected in 2006: more than 6.5 × 10⁴ copies of the mcyD_KS gene ml⁻¹ were detected in August, and an average of 4.0 × 10⁴ copies ml⁻¹ were detected in September, when microcystin concentrations were more than 4 μg liter⁻¹ and approximately 2 μg liter⁻¹, respectively. Gene copy numbers and total microcystin concentrations (determined by enzyme-linked immunosorbent assay [ELISA]) were highly correlated in the littoral (r = 0.93, P < 0.001) and the pelagic station (r = 0.87, P < 0.001) in 2006. In contrast to the situation in 2006, a cyanobacterial bloom occurred only in late summer-early fall of 2007, reaching only 3 × 10² mcyD_KS copies ml⁻¹, while the microcystin concentration was barely detectable. The Q-PCR method allowed the detection of microcystin-producing cyanobacteria when toxins and toxigenic cyanobacterial abundance were still below the limit of detection by high-pressure liquid chromatography (HPLC) and microscopy. Toxin gene copy numbers grew exponentially at a steady rate over a period of 7 weeks. Onshore winds selected for cells with a higher cell quota of microcystin. This technique could be an effective approach for the routine monitoring of the most at-risk water bodies.Toxic cyanobacterial blooms, as well as their increasing global occurrence, pose a serious threat to human health, domestic animals, and livestock. The frequency and severity of bloom events continue to rise, most probably as a direct result of increased nutrient loading of water systems worldwide. The number of lakes in Quebec, Canada, affected by blooms of 2 × 10⁴ cells ml⁻¹ or more has been increasing from 21 (2004) to 28 (2005), 62 (2006), 157 (2007), 138 (2008), and 150 (2009). Government agencies are under tremendous pressure to cope with escalating demands for water analysis, specifically, for cyanotoxins.In Missisquoi Bay, Lake Champlain, public health advisories have been issued from 2001 to 2009 resulting in the closure of several beaches and periodic no-drinking warnings for the water. These advisories forbid any direct contact with the lake water by both people and animals because of the presence of cyanotoxins. The economic impact around the lake has been substantial, with revenues sometimes falling by 40 to 80% (20).For the past 8 years, microcystin (MCYST)-producing cyanobacterial genera have composed a major part of the bacterial community in Missisquoi Bay during both the summer and the fall. To date, five species known to produce toxins based on the literature (2) have been identified in the lake, including species of Microcystis and Anabaena (27).All species with microcystin-producing strains also include related strains that lack the ability to produce this toxin. The nonproducing strains cannot be differentiated by traditional microscopy or ribosomal gene sequences (15). Reliable tools to detect and characterize toxin-producing cyanobacteria are required. Enzyme-linked immunosorbent assay (ELISA) and high-pressure liquid chromatography (HPLC) are currently the most widely used techniques to evaluate whether toxins are present in water samples. The risk assessment response to the increasing occurrence of cyanotoxins has been seriously constrained due to the limited number of available standards and the limited analytical capability of some laboratories. At least 89 microcystin analogues have been characterized (35), but fewer than 10 reference standards are currently available.The development and validation of increasingly sensitive, specific, and reliable molecular tools will contribute to the next generation of monitoring approaches. The detection and quantitation of specific target genes, such as those involved in the synthesis of toxins in cyanobacteria, are the cornerstone of new techniques to identify, monitor, or profile specific targets in environmental samples. These approaches are in general less expensive and faster than the currently available chemical assays and do not rely on reference standards.The structure of the microcystin biosynthesis cluster of two strains of Microcystis aeruginosa (17, 18, 31), Planktothrix agardhii NIVA CYA 126/8 (3), and Anabaena sp. strain 90 (28) which encodes the nonribosomal peptide synthetase-polyketide synthase enzyme complex has been elucidated. The mcy gene cluster is located on the chromosome and contains 10 genes (mcyA to mcyJ). The two polyketide synthase modules of mcyG and mcyE, together with the two polyketide synthase modules of mcyD, are responsible for the synthesis of the unique Adda moiety of microcystins. The Adda side chain is largely responsible for the toxicity through protein phosphatase inhibition (7, 8, 12).The number of water bodies affected by cyanobacterial blooms has been increasing worldwide, and scientists have designed primers for the various genes involved in the biosynthesis of microcystins. The mcyD gene has been used in conventional PCR for phylogenetic studies (16, 22, 29) and as a target to characterize cyanobacterial blooms in Lakes Ontario (11) and Erie (19). The mcyE gene has also been targeted to design genus-specific primers (23, 24, 33) and universal primers encoding the aminotransferase domain of various genera of cyanobacteria (4, 13). All of these primers generated PCR fragments that were larger than the recommended size for quantitative PCR (Q-PCR) (100 to 200 bp).A Q-PCR technique, the Taq nuclease assay, was developed by Rinta-Kanto and colleagues (25, 26) to study the distribution and abundance of toxic Microcystis blooms in western Lake Erie. In their first study, the mcyD probe was highly specific to Microcystis species but failed to detect the mcyD gene in one of the samples that had a detectable concentration of microcystin in the water. Other microcystin-producers, such as Anabaena and Planktothrix, were identified in that sample and were likely responsible for toxin production.The number of gene sequences related to microcystin biosynthesis in the databases has been increasing rapidly since the beginning of 2000. The objectives of this study were to develop a rapid, Q-PCR-based technique for detecting and monitoring the dynamics of microcystin-producing cyanobacteria and to determine the correlation between toxigenic cells and toxin concentration. The mcyD gene was selected as the specific target for characterizing cyanobacterial blooms and applied in Missisquoi Bay, Lake Champlain, during the summers of 2006 and 2007. Oligonucleotide primers were designed based on the alignments of all 50 mcyD nucleotide sequences available in GenBank as of March 2006 and were verified in December 2009. The alignments revealed that the polyketide synthase sequences were divided into three major clusters: some of the submitted sequences encoded the first dehydratase domain, others were from one of the β-ketoacyl synthases and the third group of sequences encoded part of both the ketoacetyl synthase and the acetyltransferase domains. Primers were designed to create PCR fragments from two different regions of the mcyD gene. These fragments were cloned to create a standard curve for absolute quantification. This strategy was chosen to ensure that the quantification of cells carrying the target gene would be performed with a standard curve that originated from a single gene copy.     

Highly Diverse Cyanobactins in Strains of the Genus Anabaena

Cyanobactins are small, cyclic peptides recently found in cyanobacteria. They are formed through proteolytic cleavage and posttranslational modification of short precursor proteins and exhibit antitumor, cytotoxic, or multi-drug-reversing activities. Using genome project data, bioinformatics, stable isotope labeling, and mass spectrometry, we discovered novel cyclic peptides, anacyclamides, in 27 Anabaena strains. The lengths of the anacylamides varied greatly, from 7 to 20 amino acids. Pronounced sequence variation was also detected, and only one amino acid, proline, was present in all anacyclamides. The anacyclamides identified included unmodified proteinogenic or prenylated amino acids. We identified an 11-kb gene cluster in the genome of Anabaena sp. 90, and heterologous expression in Escherichia coli confirmed that this cluster was responsible for anacyclamide production. The discovery of anacyclamides greatly increases the structural diversity of cyanobactins.Cyanobacteria are a prolific source of secondary metabolites with potential as drug leads or useful probes for cell biology studies (23). They include biomedically interesting compounds, such as the anticancer drug lead cryptophycin (15), and environmentally problematic hepatotoxic peptides, such as microcystins and nodularins produced by bloom-forming cyanobacteria (23). Many of these compounds contain nonproteinogenic amino acids and modified peptides and are produced by nonribosomal peptide synthesis (23, 26).The cyanobactins are a new group of cyclic peptides recently found in cyanobacteria (4). They are assembled through posttranslational proteolytic cleavage and head-to-tail macrocyclization of short precursor proteins. The cyanobactins are low-molecular-weight cyclic peptides that contain heterocyclized amino acids and can be prenylated or contain d-amino acids (3, 4). The cyanobactins that contain heterocyclized amino acids include patellamides, ulithiacyclamides, trichamide, tenuecyclamides, trunkamides, patellins, and microcyclamides and are synthesized in this manner (3, 4, 20, 24, 28). They possess antitumor, cytotoxic, and multi-drug-reversing activities and have potential as drug leads (4, 18, 20).Cyanobactins containing heterocyclized amino acids are found in a variety of cyanobacteria (4). A recent study demonstrated that the cyanobactin biosynthetic pathway is prevalent in planktonic bloom-forming cyanobacteria (14). However, the products of these gene clusters encoding new cyanobactins are unknown. Here we report discovery of a novel family of low-molecular-weight cyanobactins and show that these compounds are common in strains of the genus Anabaena. These anacyclamides exhibit pronounced length and sequence variation and contain unmodified or prenylated amino acids.     

Host-Interactive Genes in Amerindian Helicobacter pylori Diverge from Their Old World Homologs and Mediate Inflammatory Responses

Helicobacter pylori is the dominant member of the gastric microbiota and has been associated with an increased risk of gastric cancer and peptic ulcers in adults. H. pylori populations have migrated and diverged with human populations, and health effects vary. Here, we describe the whole genome of the cag-positive strain V225d, cultured from a Venezuelan Piaroa Amerindian subject. To gain insight into the evolution and host adaptation of this bacterium, we undertook comparative H. pylori genomic analyses. A robust multiprotein phylogenetic tree reflects the major human migration out of Africa, across Europe, through Asia, and into the New World, placing Amerindian H. pylori as a particularly close sister group to East Asian H. pylori. In contrast, phylogenetic analysis of the host-interactive genes vacA and cagA shows substantial divergence of Amerindian from Old World forms and indicates new genotypes (e.g., VacA m3) involving these loci. Despite deletions in CagA EPIYA and CRPIA domains, V225d stimulates interleukin-8 secretion and the hummingbird phenotype in AGS cells. However, following a 33-week passage in the mouse stomach, these phenotypes were lost in isolate V225-RE, which had a 15-kb deletion in the cag pathogenicity island that truncated CagA and eliminated some of the type IV secretion system genes. Thus, the unusual V225d cag architecture was fully functional via conserved elements, but the natural deletion of 13 cag pathogenicity island genes and the truncation of CagA impaired the ability to induce inflammation.Helicobacter pylori is a microaerophilic bacterium of the Epsilonproteobacteria that has colonized the stomach since early in human evolution (45) and diverged with ancient human migrations (24, 45, 92). Thus, several major H. pylori populations, such as hpAfrica1, hpEurope, hspEAsia, and hspAmerind, whose names indicate their original geographic associations (45, 51), have been defined. In particular, similarities between the hspAmerind and hspEAsia populations suggest that the first colonizers of the New World brought H. pylori with them (24, 28). With recent mixing of human groups, H. pylori populations are also mixing and competing, with an apparent dominance by the hpEurope population at least in Latin America (19).H. pylori usually does not cause illness, but colonization with strains bearing the cag (cytotoxin-associated gene) pathogenicity island (cag PAI) (3, 7, 25, 52, 57, 61, 63) is associated with an increased risk of noncardia gastric adenocarcinoma and peptic ulcer disease (56, 64). Nonetheless, a high prevalence of cag-positive H. pylori strains occurs concurrently with low gastric cancer rates in Africa (40) and some regions in Latin America, such as the Venezuelan savannas and Amazonas (29, 53). Moreover, clinical and epidemiological data provide evidence for an inverse relationship between H. pylori colonization and the prevalence of certain metabolic disorders, esophageal diseases, asthma and allergic disorders, and acute infectious diseases, as well as a direct relationship with improved nutritional status of rural children (3, 14, 34, 37, 49, 68). That the host interaction with an indigenous gastric microbe provides some health benefits to the host is not unexpected given the well-established role of gastrointestinal microflora in maintaining gastroenteric homeostasis (8).The most thoroughly studied H. pylori proteins that interact with human cells are CagA and VacA. CagA is an effector protein injected into gastric epithelial cells by a type IV secretion system encoded by the cag PAI (10, 12, 15, 83). VacA is initially secreted from the bacterial cell by an autotransporter mechanism (16). Both proteins have multiple effects on host cells. Inside the host cell, phosphorylation of CagA on EPIYA repeats in the phosphotyrosine (PY) region (73) induces cellular elongation known as the hummingbird phenotype (72). CagA may also induce secretion of interleukin-8 (IL-8) (11), a process commonly attributed to NF-κB, and disrupt the barrier function of the tight junctions in polarized epithelial cells, leading to a loss of adhesion (1, 5). Other motifs in the PY region promote phosphorylation-independent effects (79). In addition, cagA may be considered an oncogene (60), since transgenic expression of cagA in mice leads to gastric epithelial hyperplasia through aberrant epithelial cell signaling and gastric carcinogenesis (60, 62). In contrast, VacA is a multifunctional protein with several activities in epithelial and immune cells (16). VacA induces cell vacuolation (43), alters mitochondrial membrane permeability (27, 41, 90), and increases epithelial monolayer permeability. VacA also activates several signal transduction pathways that are important in immune and epithelial cells, including the mitogen-activated protein (MAP) kinase and p38/ATF-2-mediated signal pathways (9, 55).Genomic analysis provides insights into the evolution of H. pylori strains and their relation with their human hosts and may be useful for the development of diagnostic tools and novel therapies. To date, there are six published complete H. pylori genomes, mostly from the hpEurope population (see Table SA1 in the supplemental material). Here, we report the whole genome of a newly characterized hspAmerind strain, V225d, and assess its genetic structure in comparison to those of Old World H. pylori strains through a comprehensive multiprotein phylogenetic analysis, as well as through single-gene examination of cagA and vacA, revealing clues to the evolution and migration of this strain into the New World and the implications for human health. We also present the results of functional and genomic studies using gastric epithelial cells demonstrating that V225d can induce an inflammatory host response, an effect that was lost following passage through the mouse stomach.     

Abundance of Novel and Diverse tfdA-Like Genes,Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases,in Soil

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAα). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 × 10⁶ to 65 × 10⁶ per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.Phenoxyalkanoic acid (PAA) herbicides such as MCPA (4-chloro-2-methyl-phenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are widely used to control broad-leaf weeds in agricultural as well as nonagricultural areas (19, 77). Degradation occurs primarily under oxic conditions in soil, and microorganisms play a key role in the degradation of such herbicides in soil (62, 64). Although relatively rapidly degraded in soil (32, 45), both MCPA and 2,4-D are potential groundwater contaminants (10, 56, 70), accentuating the importance of bacterial PAA herbicide-degrading bacteria in soils (e.g., references 3, 5, 6, 20, 41, 59, and 78).Degradation can occur cometabolically or be associated with energy conservation (15, 54). The first step in the degradation of 2,4-D and MCPA is initiated by the product of cadAB or tfdA-like genes (29, 30, 35, 67), which constitutes an α-ketoglutarate (α-KG)- and Fe²⁺-dependent dioxygenase. TfdA removes the acetate side chain of 2,4-D and MCPA to produce 2,4-dichlorophenol and 4-chloro-2-methylphenol, respectively, and glyoxylate while oxidizing α-ketoglutarate to CO₂ and succinate (16, 17).Organisms capable of PAA herbicide degradation are phylogenetically diverse and belong to the Alpha-, Beta-, and Gammproteobacteria and the Bacteroidetes/Chlorobi group (e.g., references 2, 14, 29-34, 39, 60, 68, and 71). These bacteria harbor tfdA-like genes (i.e., tfdA or tfdAα) and are categorized into three groups on an evolutionary and physiological basis (34). The first group consists of beta- and gammaproteobacteria and can be further divided into three distinct classes based on their tfdA genes (30, 46). Class I tfdA genes are closely related to those of Cupriavidus necator JMP134 (formerly Ralstonia eutropha). Class II tfdA genes consist of those of Burkholderia sp. strain RASC and a few strains that are 76% identical to class I tfdA genes. Class III tfdA genes are 77% identical to class I and 80% identical to class II tfdA genes and linked to MCPA degradation in soil (3). The second group consists of alphaproteobacteria, which are closely related to Bradyrhizobium spp. with tfdAα genes having 60% identity to tfdA of group 1 (18, 29, 34). The third group also harbors the tfdAα genes and consists of Sphingomonas spp. within the alphaproteobacteria (30).Diverse PAA herbicide degraders of all three groups were identified in soil by cultivation-dependent studies (32, 34, 41, 78). Besides CadAB, TfdA and certain TfdAα proteins catalyze the conversion of PAA herbicides (29, 30, 35). All groups of tfdA-like genes are potentially linked to the degradation of PAA herbicides, although alternative primary functions of group 2 and 3 TfdAs have been proposed (30, 35). However, recent cultivation-independent studies focused on 16S rRNA genes or solely on group 1 tfdA sequences in soil (e.g., references 3-5, 13, and 41). Whether group 2 and 3 tfdA-like genes are also quantitatively linked to the degradation of PAA herbicides in soils is unknown. Thus, tools to target a broad range of tfdA-like genes are needed to resolve such an issue. Primers used to assess the diversity of tfdA-like sequences used in previous studies were based on the alignment of approximately 50% or less of available sequences to date (3, 20, 29, 32, 39, 47, 58, 73). Primers specifically targeting all major groups of tfdA-like genes to assess and quantify a broad diversity of potential PAA degraders in soil are unavailable. Thus, the objectives of this study were (i) to develop primers specific for all three groups of tfdA-like genes, (ii) to establish quantitative kinetic PCR (qPCR) assays based on such primers for different soil samples, and (iii) to assess the diversity and abundance of tfdA-like genes in soil.     

Discovery of [NiFe] Hydrogenase Genes in Metagenomic DNA: Cloning and Heterologous Expression in Thiocapsa roseopersicina

Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a Thiocapsa roseopersicina O₂-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the hyaA and hyaB genes of an Alteromonas macleodii hydrogenase, indicating that it belongs to the Alteromonas clade. We were able to express our new Alteromonas hydrogenase in T. roseopersicina. Expression was accomplished by coexpressing only two accessory genes, hyaD and hupH, without the need to express any of the hyp accessory genes (hypABCDEF). These results suggest that the native accessory proteins in T. roseopersicina could substitute for the Alteromonas counterparts that are absent in the host to facilitate the assembly of a functional Alteromonas hydrogenase. To further compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed complementation experiments by introducing the new Alteromonas hyaD gene into the T. roseopersicina hynD mutant. Interestingly, Alteromonas endopeptidase HyaD could complement T. roseopersicina HynD to cleave endoproteolytically the C-terminal end of the T. roseopersicina HynL hydrogenase large subunit and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe] hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes from environmental microbes in a culture-independent manner.Hydrogen is a promising energy carrier for the future (10). Photosynthetic microbes such as cyanobacteria have attracted considerable attention, because they can split water photolytically to produce H₂. However, one major drawback of the processes is that their H₂-evolving hydrogenases are extremely sensitive to O₂, which is an inherent by-product of oxygenic photosynthesis. Thus, transfer of O₂-tolerant [NiFe] hydrogenases into cyanobacteria might be one approach to overcome this O₂ sensitivity issue. A small number of O₂-tolerant hydrogenases has been identified (9, 21, 47). However, they tend to favor H₂ uptake over evolution. Searching for novel O₂-tolerant [NiFe] hydrogenases from environmental microbes therefore becomes an important part of the effort to construct such biophotolytic systems.The oceans harbor an abundance of microorganisms with H₂ production capability. Traditionally, new hydrogenases have been screened only from culturable organisms. However, since only a few microbes can be cultured (14), many of them have not been identified, and their functions remain unknown. Metagenomics is a rapidly growing field, which allows us to obtain information about uncultured microbes and to understand the true diversity of microbes in their natural environments. Metagenomics analysis provides a completely new approach for identifying novel [NiFe] hydrogenases from the oceans in a culture-independent manner. The Global Ocean Sampling (GOS) expedition has produced the largest metagenomic data set to date, providing a rich catalog of proteins and protein families, including those enzymes involved in hydrogen metabolism (45, 52, 56-58). Putative novel [NiFe] hydrogenase enzymes that were identified from marine microbial metagenomic data in these expeditions can be examined to find potentially important new hydrogenases. Because source organisms for metagenomic sequences are not typically known, these hydrogenases have to be heterologously expressed in culturable foreign hosts for protein and functional analyses.Unlike most proteins, hydrogenases have a complex architecture and must be assembled and matured through a multiple-step process (7, 11). Hydrogenases are divided into three distinct groups based on their metal contents (54): Fe-S cluster-free hydrogenases (22, 23, 48), [FeFe] hydrogenases (1, 12, 25), and [NiFe] hydrogenases (2, 3, 55). [NiFe] hydrogenases are heterodimers composed of a large subunit and a small subunit, and their NiFe catalytic centers are located in the large subunits (2, 15, 19, 40). A whole set of accessory proteins are required to properly assemble the catalytic centers (7). The accessory protein HypE first interacts with HypF to form a HypF-HypE complex, and the carbamyl group linked to HypF is then dehydrated by HypE in the presence of ATP to release the CN group that is transferred to iron through a HypC-HypD-HypE complex (6). The origin of the CO ligand that is also bound to the iron is not clear, and possibly it comes from formate, formyl-tetrahydrofolate, or acetate. The liganded Fe atom is inserted into the immature large subunit, in which HypC proteins function as chaperones to facilitate the metal insertion (5, 34, 36). Ni is delivered to the catalytic center by the zinc-metalloenzyme HypA that interacts with HypB, a nickel-binding and GTP-hydrolyzing protein. The final step in the maturation process is endoproteolytic cleavage. Once the nickel is transferred to the active site, the endopeptidase, such as HyaD or HynD, cleaves the C-terminal end of the large subunit (33, 43), which triggers a conformational change of the protein so that the Ni-Fe catalytic center can be internalized.Heterologous expression of functional [NiFe] hydrogenases has been demonstrated in several studies (4, 18, 31, 39, 44, 50), suggesting that it could be a feasible approach to express novel hydrogenases from the environment for functional analysis. In this study, we sought to prove the concept that metagenomically derived environmental DNA can give rise to a functional [NiFe] hydrogenase through expression in a foreign host and that novel [NiFe] hydrogenases from environmental microbes can be studied in a culture-independent manner. We cloned environmental DNA that harbors the genes of a putative novel hydrogenase that shows strong homology to a known O₂-tolerant hydrogenase, HynSL, from the phototrophic purple sulfur bacterium Thiocapsa roseopersicina (21, 28, 41, 59). We heterologously expressed the two structural genes (hyaA and hyaB) and two accessory genes (hupH and hyaD) of this novel environmental hydrogenase in T. roseopersicina, a foreign host that may already have the necessary machinery required to process the environmental hydrogenase since it carries the homologous hydrogenase HynSL. We analyzed the new hydrogenase protein and its functions. In addition, we compared the maturation mechanisms between the two homolog hydrogenases by performing complementation experiments.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Diversity of Dissimilatory Sulfite Reductase Genes (dsrAB) in a Salt Marsh Impacted by Long-Term Acid Mine Drainage

Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered dsrAB sequences from three sites sampled along an AMD flow path indicated the dominance of a single Desulfovibrio species. Other major sequence clades were related most closely to Desulfosarcina, Desulfococcus, Desulfobulbus, and Desulfosporosinus species. The presence of metal sulfides with low δ³⁴S values relative to δ³⁴S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of ∼2), although possibly within less acidic microenvironments. Interestingly, δ³⁴S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments.Salt marshes exhibit high primary production rates (1, 101) and form biogeochemical “transition zones” for nutrient production, transport, and cycling between terrestrial and coastal marine environments (41, 66, 100). These zones also serve to reduce the flux of potentially toxic metals in contaminated groundwater to estuaries (12, 99, 106). Both functions depend strongly on microbial activity, especially that of sulfate-reducing bacteria (SRB) (42, 62, 67). SRB recycle much of the sedimentary organic carbon pool in marsh sediments (42-44) and indirectly inhibit production of the greenhouse gas methane (37, 71). They can restrict the mobility of dissolved contaminant metals by inducing precipitation of poorly soluble metal sulfides, and studies have examined their use in constructed wetlands to bioremediate acid mine drainage (AMD) and other metalliferous waste streams (11, 35, 40, 46, 50, 76, 90, 94, 104). However, the high acidity and metal concentrations inherent to AMD can inhibit SRB growth (15, 88, 98), and preferential growth of iron- and sulfur-oxidizing bacteria over SRB has been observed in some treatment wetlands (39).For natural salt marshes, 16S ribosomal nucleic acid- and phospholipid fatty acid (PLFA)-based analyses have shown that SRB commonly comprise a significant fraction of the microbial community (13, 24, 31, 34, 51, 58). Studies of salt marsh dissimilatory sulfite reductase genes (dsrAB), a highly conserved functional phylogenetic marker of prokaryotic sulfate reducers (49, 57, 102, 103, 107), have revealed both novel and deeply branching clades (3). Studies of mining-impacted sites at pH 2.0 to 7.8 (5, 7, 39, 70, 72, 77, 84), of soils and geothermal settings at a pH of ∼4 (55, 68), of metal-contaminated estuaries at pH 6.8 to 7.2 (65), and of hypersaline lakes at pH 7.5 (56) further outline the distribution and tolerance of specific groups and species of SRB under geochemically stringent conditions. Other findings point toward the existence of deltaproteobacteria in environments at a pH of ∼1 (10), although it is unknown if these include SRB. SRB diversity in salt marshes under long-term contamination by AMD has not been well investigated. Such studies may provide useful information for bioremediation projects in estuarine environments, as well as general insights into relationships between SRB physiology and the geochemistry of AMD.We studied the diversity of SRB, based on phylogenetic analysis of recovered DsrAB gene sequences (∼1.9 kb), in natural salt marsh sediments of the San Francisco Bay impacted by AMD for over 100 years. Sulfur isotope ratio and concentration measurements of pore water sulfate and metal sulfide minerals provided information about the spatial and temporal extent of active bacterial sulfate reduction (BSR) in sediment cores taken from specific sites along an AMD flow path. Collectively, the results revealed a tidal marsh system characterized by rapidly cycling bacterial sulfate reduction and sulfide reoxidation associated with oscillating tidal inundation and groundwater infiltration.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.