In vivo examination of pathogenicity and virulence in environmentally isolated Vibrio vulnificus.

Human exposure to Vibrio vulnificus, a gram-negative, halophilic environmental pathogen, is increasing. Despite this, the mechanisms of its pathogenicity and virulence remain largely unknown. Each year, hundreds of infections related to V. vulnificus occur, leading to hospitalization in 92% of cases and a mortality rate of 35%. The infection is severe, typically contracted through the consumption of contaminated food or exposure of an open wound to contaminated water. This can result in necrotizing fasciitis and the need for amputation of the infected tissue. Although several genes (rtxA1, vvpE, and vvhA) have been implicated in the pathogenicity of this organism, a defined mechanism has not been discovered. In this study, we examine environmentally isolated V. vulnificus strains using a zebrafish model (Danio rerio) to investigate their virulence capabilities. We found significant variation in virulence between individual strains. The commonly used marker gene of disease-causing strains, vcgC, did not accurately predict the more virulent strains. Notably, the least virulent strain in the study, V. vulnificus Sept WR1-BW6, which tested positive for vcgC, vvhA, and rtxA1, did not cause severe disease in the fish and was the only strain that did not result in any mortality. Our study demonstrates that virulence varies greatly among different environmental strains and cannot be accurately predicted based solely on genotype.

Occurrence and significance of pathogenicity and fitness islands in environmental vibrios.

Pathogenicity islands (PAIs) are large genomic regions that contain virulence genes, which aid pathogens in establishing infections. While PAIs in clinical strains (strains isolated from a human infection) are well-studied, less is known about the occurrence of PAIs in strains isolated from the environment. In this study we describe three PAIs found in environmental Vibrio vulnificus and Vibrio parahaemolyticus strains, as well as a genomic fitness island found in a Vibrio diabolicus strain. All four islands had markedly different GC profiles than the rest of the genome, indicating that all of these islands were acquired via lateral gene transfer. Genes on the PAIs and fitness island were characterized. The PAI found in V. parahaemolyticus contained the tdh gene, a collagenase gene, and genes involved in the type 3 secretion system II (T3SS2). A V. vulnificus environmental strain contained two PAIs, a small 25 kbp PAI and a larger 143 kbp PAI. Both PAIs contained virulence genes. Toxin-antitoxin (TA) genes were found in all three species: on the V. diabolicus fitness island, and on the V. parahaemolyticus and V. vulnificus PAIs.

Responses of Salt Marsh Plant Rhizosphere Diazotroph Assemblages to Drought.

Drought has many consequences in the tidally dominated Spartina sp. salt marshes of the southeastern US; including major dieback events, changes in sediment chemistry and obvious changes in the landscape. These coastal systems tend to be highly productive, yet many salt marshes are also nitrogen limited and depend on plant associated diazotrophs as their source of 'new' nitrogen. A 4-year study was conducted to investigate the structure and composition of the rhizosphere diazotroph assemblages associated with 5 distinct plant zones in one such salt marsh. A period of greatly restricted tidal inundation and precipitation, as well as two periods of drought (June-July 2004, and May 2007) occurred during the study. DGGE of nifH PCR amplicons from rhizosphere samples, Principal Components Analysis of the resulting banding patterns, and unconstrained ordination analysis of taxonomic data and environmental parameters were conducted. Diazotroph assemblages were organized into 5 distinct groups (R² = 0.41, p value < 0.001) whose presence varied with the environmental conditions of the marsh. Diazotroph assemblage group detection differed during and after the drought event, indicating that persistent diazotrophs maintained populations that provided reduced supplies of new nitrogen for vegetation during the periods of drought.

Ecological fitness and virulence features of Vibrio parahaemolyticus in estuarine environments.

Vibrio parahaemolyticus is a commonly encountered and highly successful organism in marine ecosystems. It is a fast-growing, extremely versatile copiotroph that is active over a very broad range of conditions. It frequently occurs suspended in the water column (often attached to particles or zooplankton), and is a proficient colonist of submerged surfaces. This organism is an important pathogen of animals ranging from microcrustaceans to humans and is a causative agent of seafood-associated food poisoning. This review examines specific ecological adaptations of V. parahaemolyticus, including its broad tolerances to temperature and salinity, its utilization of a wide variety of organic carbon and energy sources, and its pervasive colonization of suspended and stationary materials that contribute to its success and ubiquity in temperate and tropical estuarine ecosystems. Several virulence-related features are examined, in particular the thermostable direct hemolysin (TDH), the TDH-related hemolysin (TRH), and the type 3 secretion system, and the possible importance of these features in V. parahaemolyticus pathogenicity is explored. The impact of new and much more effective PCR primers on V. parahaemolyticus detection and our views of virulent strain abundance are also described. It is clear that strains carrying the canonical virulence genes are far more common than previously thought, which opens questions regarding the role of these genes in pathogenesis. It is also clear that virulence is an evolving feature of V. parahaemolyticus and that novel combinations of virulence factors can lead to emergent virulence in which a strain that is markedly more pathogenic evolves and propagates to produce an outbreak. The effects of global climate change on the frequency of epidemic disease, the geographic distribution of outbreaks, and the human impacts of V. parahaemolyticus are increasing and this review provides information on why this ubiquitous human pathogen has increased its footprint and its significance so dramatically.

The hot oyster: levels of virulent Vibrio parahaemolyticus strains in individual oysters.

Vibrio parahaemolyticus is the leading cause of seafood-associated gastroenteritis and is most commonly transmitted by raw oysters. Consequently, detection of virulent strains of this organism in oysters is a primary concern for seafood safety. Vibrio parahaemolyticus levels were determined in 110 individual oysters harvested from two sampling sites in SC, USA. The majority of oysters (98%) contained low levels of presumptive V. parahaemolyticus However, two healthy oysters contained presumptive V. parahaemolyticus numbers that were unusually high. These two 'hot' oysters contained levels of presumptive V. parahaemolyticus within the gills that were ∼100-fold higher than the average for other oysters collected at the same date and location. Current V. parahaemolyticus detection practices require homogenizing a dozen oysters pooled together to determine V. parahaemolyticus numbers, a procedure that would dilute out V. parahaemolyticus in these 'hot' oysters. This study demonstrates the variability of V. parahaemolyticus densities taken from healthy, neighboring individual oysters in the environment. Additionally, environmental V parahaemolyticus isolates were screened for the virulence-related genes, tdh and trh, using improved polymerase chain reaction primers and protocols. We detected these genes, previously thought to be rare in environmental isolates, in approximately half of the oyster isolates.

Microbial Surface Colonization and Biofilm Development in Marine Environments.

Biotic and abiotic surfaces in marine waters are rapidly colonized by microorganisms. Surface colonization and subsequent biofilm formation and development provide numerous advantages to these organisms and support critical ecological and biogeochemical functions in the changing marine environment. Microbial surface association also contributes to deleterious effects such as biofouling, biocorrosion, and the persistence and transmission of harmful or pathogenic microorganisms and their genetic determinants. The processes and mechanisms of colonization as well as key players among the surface-associated microbiota have been studied for several decades. Accumulating evidence indicates that specific cell-surface, cell-cell, and interpopulation interactions shape the composition, structure, spatiotemporal dynamics, and functions of surface-associated microbial communities. Several key microbial processes and mechanisms, including (i) surface, population, and community sensing and signaling, (ii) intraspecies and interspecies communication and interaction, and (iii) the regulatory balance between cooperation and competition, have been identified as critical for the microbial surface association lifestyle. In this review, recent progress in the study of marine microbial surface colonization and biofilm development is synthesized and discussed. Major gaps in our knowledge remain. We pose questions for targeted investigation of surface-specific community-level microbial features, answers to which would advance our understanding of surface-associated microbial community ecology and the biogeochemical functions of these communities at levels from molecular mechanistic details through systems biological integration.

Optimization of multilocus sequence analysis for identification of species in the genus Vibrio.

Multilocus sequence analysis (MLSA) is an important method for identification of taxa that are not well differentiated by 16S rRNA gene sequences alone. In this procedure, concatenated sequences of selected genes are constructed and then analyzed. The effects that the number and the order of genes used in MLSA have on reconstruction of phylogenetic relationships were examined. The recA, rpoA, gapA, 16S rRNA gene, gyrB, and ftsZ sequences from 56 species of the genus Vibrio were used to construct molecular phylogenies, and these were evaluated individually and using various gene combinations. Phylogenies from two-gene sequences employing recA and rpoA in both possible gene orders were different. The addition of the gapA gene sequence, producing all six possible concatenated sequences, reduced the differences in phylogenies to degrees of statistical (bootstrap) support for some nodes. The overall statistical support for the phylogenetic tree, assayed on the basis of a reliability score (calculated from the number of nodes having bootstrap values of ≥ 80 divided by the total number of nodes) increased with increasing numbers of genes used, up to a maximum of four. No further improvement was observed from addition of the fifth gene sequence (ftsZ), and addition of the sixth gene (gyrB) resulted in lower proportions of strongly supported nodes. Reductions in the numbers of strongly supported nodes were also observed when maximum parsimony was employed for tree construction. Use of a small number of gene sequences in MLSA resulted in accurate identification of Vibrio species.

Genes similar to the Vibrio parahaemolyticus virulence-related genes tdh, tlh, and vscC2 occur in other vibrionaceae species isolated from a pristine estuary.

Detection of the human pathogen Vibrio parahaemolyticus often relies on molecular biological analysis of species-specific virulence factor genes. These genes have been employed in determinations of V. parahaemolyticus population numbers and the prevalence of pathogenic V. parahaemolyticus strains. Strains of the Vibrionaceae species Photobacterium damselae, Vibrio diabolicus, Vibrio harveyi, and Vibrio natriegens, as well as strains similar to Vibrio tubiashii, were isolated from a pristine salt marsh estuary. These strains were examined for the V. parahaemolyticus hemolysin genes tdh, trh, and tlh and for the V. parahaemolyticus type III secretion system 2α gene vscC2 using established PCR primers and protocols. Virulence-related genes occurred at high frequencies in non-V. parahaemolyticus Vibrionaceae species. V. diabolicus was of particular interest, as several strains were recovered, and the large majority (>83%) contained virulence-related genes. It is clear that detection of these genes does not ensure correct identification of virulent V. parahaemolyticus. Further, the occurrence of V. parahaemolyticus-like virulence factors in other vibrios potentially complicates tracking of outbreaks of V. parahaemolyticus infections.

Sublethal effects of the antibiotic tylosin on estuarine benthic microalgal communities.

Pharmaceuticals are common chemical contaminants in estuaries receiving effluent from wastewater and sewage treatment facilities. The purpose of this research was to examine benthic microalgal (BMA) community responses to sublethal exposures to tylosin, a common and environmentally persistent antibiotic. Bioassays, using concentrations of 0.011-218 µmol tylosin l(-1), were performed on intertidal muddy sediments from North Inlet Estuary, SC. Exposure to tylosin resulted in a reduction in total BMA biomass and primary productivity. Furthermore, exposure seemed to retard diatom growth while having a minimal effect on cyanobacteria biomass. Estuarine systems receiving chronic inputs of trace concentrations of tylosin, as well as other antibiotics, may experience significant reductions in BMA biomass and primary productivity. Given the well-documented role of BMA in the trophodynamics of estuaries, these impacts will likely be manifested in higher trophic levels with possible impairments of the structure and function of these sensitive systems.

High frequency of virulence factor genes tdh, trh, and tlh in Vibrio parahaemolyticus strains isolated from a pristine estuary.

Virulence factor genes encoding the thermostable direct hemolysin (tdh) and the thermostable direct hemolysin-related hemolysin (trh) are strongly correlated with virulence of the emergent human pathogen Vibrio parahaemolyticus. The gene encoding the thermolabile hemolysin (tlh) is also considered a signature molecular marker for the species. These genes are typically reported in very low percentages (1 to 2%) of nonclinical strains. V. parahaemolyticus strains were isolated from various niches within a pristine estuary (North Inlet, SC) and were screened for these genes using both newly designed PCR primers and more commonly used primers. DNA sequences of tdh and trh were recovered from 48% and 8.3%, respectively, of these North Inlet strains. The recovery of pathogenic V. parahaemolyticus strains in such high proportions from an estuarine ecosystem that is virtually free of anthropogenic influences indicates the potential for additional, perhaps environmental roles of the tdh and trh genes.

Specificity of Salt Marsh Diazotrophs for Vegetation Zones and Plant Hosts: Results from a North American marsh.

Salt marshes located on the east coast of temperate North America are highly productive, typically nitrogen-limited, and support diverse assemblages of free-living nitrogen fixing (diazotrophic) bacteria. This article reviews and analyzes data from North Inlet estuary (SC, USA), addressing diazotroph assemblage structure and the influence of plant host and environmental conditions on the assemblage. The North Inlet estuary is a salt marsh ecosystem in which anthropogenic influences are minimal and the distributions of diazotrophs are governed by the natural biota and dynamics of the system. Denaturing gradient gel electrophoresis fingerprinting and phylogenetic analyses of recovered sequences demonstrated that the distributions of some diazotrophs reflect plant host specificity and that diazotroph assemblages distributed across marsh gradients are also heavily influenced by edaphic conditions. Broadly distributed diazotrophs that are capable of maintaining populations under all environmental conditions spanning such gradients are also present in these assemblages. Statistical analyses indicate that the structures of diazotroph assemblages in different vegetation zones are significantly (p < 0.01) different. New data presented here demonstrate the heterogeneity of salt marsh rhizosphere microenvironments, and corroborate previous findings from different plant hosts growing at several locations within this estuary. The data from these collected works support the hypothesis that the biogeography of microorganisms is non-random and these biogeographic patterns are predictable.

Infaunal burrows are enrichment zones for Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a species that includes strains known to be pathogenic in humans, and other Vibrionaceae are common, naturally occurring bacteria in coastal environments. Understanding the ecology and transport of these organisms within estuarine systems is fundamental to predicting outbreaks of pathogenic strains. Infaunal burrows serve as conduits for increased transport of tidal waters and V. parahaemolyticus cells by providing large open channels from the sediment to salt marsh tidal creeks. An extensive seasonal study was conducted at the North Inlet Estuary in Georgetown, SC, to quantify Vibrionaceae and specifically V. parahaemolyticus bacteria in tidal water, fiddler crab (Uca pugilator, Uca pugnax) burrow water, and interstitial pore water. Numbers of V. parahaemolyticus bacteria were significantly higher within burrow waters (4,875 CFU ml(-1)) than in creek water (193 CFU ml(-1)) and interstitial pore water (128 CFU ml(-1)), demonstrating that infaunal burrows are sites of V. parahaemolyticus enrichment. A strong seasonal trend of increased abundances of Vibrionaceae and V. parahaemolyticus organisms during the warmer months of May through September was observed. Multilocus sequence typing (MLST) analysis of isolates presumed to be V. parahaemolyticus from creek water, pore water, and burrow water identified substantial strain-level genetic variability among V. parahaemolyticus bacteria. Analysis of carbon substrate utilization capabilities of organisms presumed to be V. parahaemolyticus also indicated physiological diversity within this clade, which helps to explain the broad distribution of these strains within the estuary. These burrows are "hot spots" of Vibrionaceae and V. parahaemolyticus cell numbers and strain diversity and represent an important microhabitat.

Celerinatantimonas diazotrophica gen. nov., sp. nov., a nitrogen-fixing bacterium representing a new family in the Gammaproteobacteria, Celerinatantimonadaceae fam. nov.

Five strains representing a novel family within the Gammaproteobacteria were isolated from the estuarine grasses Spartina alterniflora and Juncus roemerianus. All strains were facultatively anaerobic, Gram-negative, short, motile, polar monotrichous rods that were mesophilic, oxidase-negative, catalase-positive, had DNA G+C contents of 41.5-44.4 mol% and required seawater salts or NaCl. Growth was observed at pH 3.5-8.0. Polar lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, aminophospholipid, phospholipids and unidentified aminolipids were found in the representative strain S-G2-2(T). The major menaquinone and ubiquinone were MK-8 (100 %) and Q-8 (93 %), respectively. Predominant fatty acids present were C(12 : 0) aldehyde and/or unknown fatty acid 10.9525 (MIDI designation) and/or iso-C(16 : 1) I/C(14 : 0) 3-OH, C(16 : 1)ω7c/C(16 : 1)ω6c, C(16 : 0), C(17 : 0) cyclo and C(18 : 1)ω7c and/or C(18 : 1)ω6c. The nearly full-length 16S rRNA gene sequences of the strains were very similar (99-100 % similarity), and the strains were identified as members of the same species by DNA-DNA relatedness measurements. 16S rRNA gene sequence analysis revealed that the strains formed a monophyletic lineage within the order Alteromonadales. All five strains fixed N(2). Analysis of partial nifH gene sequences also revealed a monophyletic lineage within the Gammaproteobacteria, and the sequences were dissimilar to those of any previously described diazotroph. Differences between the novel strains and other members of the Alteromonadales include the inability to produce cytochrome oxidase. The novel strains were metabolically versatile. On the basis of the information described above, the new genus and species Celerinatantimonas diazotrophica gen. nov., sp. nov. are proposed to accommodate the five strains within a new family, Celerinatantimonadaceae fam. nov. The type strain of Celerinatantimonas diazotrophica is S-G2-2(T) ( = ATCC BAA-1368(T)  = DSM 18577(T)).

Responses of salt marsh plant rhizosphere diazotroph assemblages to changes in marsh elevation, edaphic conditions and plant host species.

An important source of new nitrogen in salt marsh ecosystems is microbial diazotrophy (nitrogen fixation). The diazotroph assemblages associated with the rhizospheres (sediment directly affected by the roots) of salt marsh plants are highly diverse, somewhat stable, and consist mainly of novel organisms. In Crab Haul Creek Basin, North Inlet, SC, the distribution of plant types into discrete zones is dictated by relatively minor differences in marsh elevation and it was hypothesized that the biotic and abiotic properties of the plant zones would also dictate the composition of the rhizosphere diazotroph assemblages. Over a period of 1 year, rhizosphere sediments were collected from monotypic stands of the black needlerush, Juncus roemerianus, the common pickleweed, Salicornia virginica, the short and tall growth forms of the smooth cordgrass Spartina alterniflora, and a mixed zone of co-occurring S. virginica and short form, S. alterniflora. DNA was extracted, purified and nifH sequences PCR amplified for denaturing gradient gel electrophoresis (DGGE) analysis to determine the composition of the diazotroph assemblages. The diazotroph assemblages were strongly influenced by season, abiotic environmental parameters and plant host. Sediment chemistry and nitrogen fixation activity were also significantly influenced by seasonal changes. DGGE bands that significantly affected seasonal and zone specific clustering were identified and most of these sequences were from novel diazotrophs, unaffiliated with any previously described organisms. At least one third of the recovered nifH sequences were from a diverse assemblage of Chlorobia, and γ-, α-, ß- and δ-Proteobacteria. Diazotrophs that occurred throughout the growing season and among all zones (frequently detected) were also mostly novel. These significant sequences indicated that diazotrophs driving the structure of the assemblages were diverse, versatile, and some were ubiquitous while others were seasonally responsive. Several ubiquitous sequences were closely related to sequences of actively N(2) fixing diazotrophs previously recovered from this system. These sequences from ubiquitous and versatile organisms likely indicate the diazotrophs in these rhizosphere assemblages that significantly contribute to ecosystem function.

Seasonal variability of diazotroph assemblages associated with the rhizosphere of the salt marsh cordgrass, Spartina alterniflora.

Nitrogen fixation is the primary N source in the highly productive but N-limited North Inlet, SC, USA salt marsh system. The diverse assemblages of nitrogen-fixing (diazotrophic) bacteria associated with the rhizospheres of the short and tall growth forms of Spartina alterniflora were analyzed at two sites, Crab Haul Creek and Goat Island, which are in different tidal creek drainage systems in this marsh. The sites differed in proximity to the main channel for tidal intrusion and in several edaphic parameters. We hypothesized that either the differing abiotic environmental regimes of the two sites or the variation due to seasonal effects result in differences in the diazotroph assemblage. Rhizosphere samples were collected seasonally during 1999 and 2000. DNA was purified and nifH amplified for denaturing gradient gel electrophoresis (DGGE) analysis of diazotroph assemblage composition. Principal components analysis was used to analyze the binary DGGE band position data. Season strongly influenced assemblage composition and biplots were used to identify bands that significantly affected the seasonal and site-specific clustering. The types of organisms that were most responsive to seasonal or site variability were identified on the basis of DGGE band sequences. Seasonally responsive members of the anaerobic diazotrophs were detected during the winter and postsenescence conditions and may have been responsible for elevated pore water sulfide concentrations. Sequences from a diverse assemblage of Gammaproteobacteria were predominant during growth periods of S. alterniflora. Abiotic environmental parameters strongly influenced both the S. alterniflora and the diazotrophic bacterial assemblages associated with this keystone salt marsh plant species.

Production of the phytohormone indole-3-acetic acid by estuarine species of the genus Vibrio.

Strains of Vibrio spp. isolated from roots of the estuarine grasses Spartina alterniflora and Juncus roemerianus produce the phytohormone indole-3-acetic acid (IAA). The colorimetric Salkowski assay was used for initial screening of IAA production. Gas chromatography-mass spectroscopy (GC-MS) was then employed to confirm and quantify IAA production. The accuracy of IAA quantification by the Salkowski assay was examined by comparison to GC-MS assay values. Indole-3-acetamide, an intermediate in IAA biosynthesis by the indole-3-acetamide pathway, was also identified by GC-MS. Multilocus sequence typing of concatenated 16S rRNA, recA, and rpoA genes was used for phylogenetic analysis of environmental isolates within the genus Vibrio. Eight Vibrio type strains and five additional species-level clades containing a total of 16 environmental isolates and representing five presumptive new species were identified as IAA-producing Vibrio species. Six additional environmental isolates similar to four of the Vibrio type strains were also IAA producers. To our knowledge, this is the first report of IAA production by species of the genus Vibrio or by bacteria isolated from an estuarine environment.

Analysis of a diverse assemblage of diazotrophic bacteria from Spartina alterniflora using DGGE and clone library screening.

Methods to assess the diversity of the diazotroph assemblage in the rhizosphere of the salt marsh cordgrass, Spartina alterniflora were examined. The effectiveness of nifH PCR-denaturing gradient gel electrophoresis (DGGE) was compared to that of nifH clone library analysis. Seventeen DGGE gel bands were sequenced and yielded 58 nonidentical nifH sequences from a total of 67 sequences determined. A clone library constructed using the GC-clamp nifH primers that were employed in the PCR-DGGE (designated the GC-Library) yielded 83 nonidentical sequences from a total of 257 nifH sequences. A second library constructed using an alternate set of nifH primers (N-Library) yielded 83 nonidentical sequences from a total of 138 nifH sequences. Rarefaction curves for the libraries did not reach saturation, although the GC-Library curve was substantially dampened and appeared to be closer to saturation than the N-Library curve. Phylogenetic analyses showed that DGGE gel band sequencing recovered nifH sequences that were frequently sampled in the GC-Library, as well as sequences that were infrequently sampled, and provided a species composition assessment that was robust, efficient, and relatively inexpensive to obtain. Further, the DGGE method permits a large number of samples to be examined for differences in banding patterns, after which bands of interest can be sampled for sequence determination.

Trophic interaction of the aerotolerant anaerobe Clostridium intestinale and the acetogen Sporomusa rhizae sp. nov. isolated from roots of the black needlerush Juncus roemerianus.

Acetogens were enumerated from root homogenates of the black needlerush Juncus roemerianus obtained from a nearly pristine salt marsh. An isolated colony, ST1, yielded acetogenic activity and was initially thought to be a pure culture; however, ST1 was subsequently found to be composed of an aerotolerant fermentative anaerobe (RC) and an acetogen (RS(T)) ((T) indicates type strain). The two spore-forming mesophiles were separated by selective cultivation under conditions favouring the growth of either RC or RS(T). The 16S rRNA gene sequence of RC was 99 % similar to that of Clostridium intestinale, indicating that RC was a new isolate of this clostridial species. The rRNA gene sequence most similar to that of RS(T) was only 96 % similar to that of RS(T) and was from a species of the acetogenic genus Sporomusa, indicating that RS(T) was a new sporomusal species; the name Sporomusa rhizae sp. nov. is proposed. RC grew at the expense of saccharides. H(2)-forming butyrate fermentation was the primary catabolism utilized by RC under anoxic conditions, while homolactate fermentation was the primary catabolism under oxic conditions. RC consumed O(2) and tolerated 20 % O(2) in the headspace of shaken broth cultures. In contrast, RS(T) was acetogenic, utilized H(2), lactate and formate, did not utilize saccharides, and could not tolerate high concentrations of O(2). RS(T) grew by trophic interaction with RC on saccharides via the uptake of H(2), and, to a lesser extent, lactate and formate produced by RC. Co-cultures of the two organisms yielded high amounts of acetate. These results indicate that (i) previously uncharacterized species of Sporomusa are associated with Juncus roots and (ii) trophic links to O(2)-consuming aerotolerant anaerobes might contribute to the in situ activities and survival strategies of acetogens in salt marsh rhizospheres, a habitat subject to gradients of plant-derived O(2).

Community-level analysis: key genes of CO2-reductive acetogenesis.

CO2-reductive acetogenic bacteria are ubiquitous in anaerobic habitats and are physiologically and phylogenetically diverse. The latter characteristics have rendered their diversity in natural environments, their distributions, and their ecological function(s) difficult to assess. Recently introduced polymerase chain reaction (PCR) primers for specific amplification of the structural gene encoding formyltetrahydrofolate synthetase (FTHFS, EC 6.3.4.3), a key enzyme in the acetyl-CoA pathway of acetogenesis, have facilitated studies of acetogen diversity and ecology. These primers amplify an approximately 1100-bp segment of the FTHFS gene. FTHFS sequences have been recovered from authentic acetogens, from sulfate reducing bacteria, and from a variety of other nonacetogenic bacteria. Phylogenetic analyses segregated these sequences into distinct clusters, only one of which contained sequences from known acetogens. This chapter describes the PCR primers, defines conditions for successful amplification of FTHFS sequences, and details the phylogenetic analysis of the FTHFS sequences. Information on the types of sequences that have been recovered from natural acetogen habitats and how they have been interpreted is also included.