Ashvini Chauhan

Syntrophic-Methanogenic Associations along a Nutrient Gradient in the Florida Everglades

ABSTRACT Nutrient runoff from the Everglades Agricultural Area resulted in a well-documented gradient of phosphorus concentrations in soil and water, with concomitant ecosystem-level changes, in the northern Florida Everglades. It was recently reported that sulfate-reducing prokaryote assemblage composition, numbers, and activities are dependent on position along the gradient (H. Castro, K. R. Reddy, and A. Ogram, Appl. Environ. Microbiol. 68:6129-6137, 2002). The present study utilized a combination of culture- and non-culture-based approaches to study differences in composition of assemblages of syntrophic and methanogenic microbial communities in eutrophic, transition, and oligotrophic areas along the phosphorus gradient. Methanogenesis rates were much higher in eutrophic and transition regions, and sequence analysis of 16S rRNA gene clone libraries constructed from samples taken from these regions revealed differences in composition and activities of syntroph-methanogen consortia. Methanogens from eutrophic and transition regions were almost exclusively composed of hydrogenotrophic methanogens, with approximately 10,000-fold-greater most probable numbers of hydrogenotrophs than of acetotrophs. Most cultivable strains from eutrophic and transition regions clustered within novel lineages. In non-culture-based studies to enrich syntrophs, most bacterial and archaeal clones were either members of novel lineages or closely related to uncultivated environmental clones. Novel cultivable Methanosaeta sp. and fatty acid-oxidizing bacteria related to the genera Syntrophomonas and Syntrophobacter were observed in microcosms containing soil from eutrophic regions, and different lines of evidence indicated the existence of novel syntrophic association in eutrophic regions.

Impact of sideways and bottom-up control factors on bacterial community succession over a tidal cycle

In aquatic systems, bacterial community succession is a function of top-down and bottom-up factors, but little information exists on “sideways” controls, such as bacterial predation by Bdellovibrio-like organisms (BLOs), which likely impacts nutrient cycling within the microbial loop and eventual export to higher trophic groups. Here we report transient response of estuarine microbiota and BLO spp. to tidal-associated dissolved organic matter supply in a river-dominated estuary, Apalachicola Bay, Florida. Both dissolved organic carbon and dissolved organic nitrogen concentrations oscillated over the course of the tidal cycle with relatively higher concentrations observed at low tide. Concurrent with the shift in dissolved organic matter (DOM) supply at low tide, a synchronous increase in numbers of bacteria and predatorial BLOs were observed. PCR-restriction fragment length polymorphism of small subunit rDNA, cloning, and sequence analyses revealed distinct shifts such that, at low tide, significantly higher phylotype abundances were observed from γ-Proteobacteria, δ-Proteobacteria, Bacteroidetes, and high G+C Gram-positive bacteria. Conversely, diversity of α-Proteobacteria, β-Proteobacteria, and Chlamydiales-Verrucomicrobia group increased at high tides. To identify metabolically active BLO guilds, tidal microcosms were spiked with six 13C-labeled bacteria as potential prey and studied using an adaptation of stable isotope probing. At low tide, representative of higher DOM and increased prey but lower salinity, BLO community also shifted such that mesohaline clusters I and VI were more active; with an increased salinity at high tide, halotolerant clusters III, V, and X were predominant. Eventually, 13C label was identified from higher micropredators, indicating that trophic interactions within the estuarine microbial food web are potentially far more complex than previously thought.

Fatty Acid-Oxidizing Consortia along a Nutrient Gradient in the Florida Everglades†

ABSTRACT The Florida Everglades is one of the largest freshwater marshes in North America and has been subject to eutrophication for decades. A gradient in P concentrations extends for several kilometers into the interior of the northern regions of the marsh, and the structure and function of soil microbial communities vary along the gradient. In this study, stable isotope probing was employed to investigate the fate of carbon from the fermentation products propionate and butyrate in soils from three sites along the nutrient gradient. For propionate microcosms, 16S rRNA gene clone libraries from eutrophic and transition sites were dominated by sequences related to previously described propionate oxidizers, such as Pelotomaculum spp. and Syntrophobacter spp. Significant representation was also observed for sequences related to Smithella propionica, which dismutates propionate to butyrate. Sequences of dominant phylotypes from oligotrophic samples did not cluster with known syntrophs but with sulfate-reducing prokaryotes (SRP) and Pelobacter spp. In butyrate microcosms, sequences clustering with Syntrophospora spp. and Syntrophomonas spp. dominated eutrophic microcosms, and sequences related to Pelospora dominated the transition microcosm. Sequences related to Pelospora spp. and SRP dominated clone libraries from oligotrophic microcosms. Sequences from diverse bacterial phyla and primary fermenters were also present in most libraries. Archaeal sequences from eutrophic microcosms included sequences characteristic of Methanomicrobiaceae, Methanospirillaceae, and Methanosaetaceae. Oligotrophic microcosms were dominated by acetotrophs, including sequences related to Methanosarcina, suggesting accumulation of acetate.

Degradation of 4-nitrocatechol by Burkholderia cepacia: a plasmid-encoded novel pathway.

Pseudomonas cepacia RKJ200 (now described as Burkholderia cepacia) has been shown to utilize p‐nitrophenol (PNP) as sole carbon and energy source. The present work demonstrates that RKJ200 utilizes 4‐nitrocatechol (NC) as the sole source of carbon, nitrogen and energy, and is degraded with concomitant release of nitrite ions. Several lines of evidence, including thin layer chromatography, gas chromatography, 1H‐nuclear magnetic resonance, gas chromatography–mass spectrometry, spectral analyses and quantification of intermediates by high performance liquid chromatography, have shown that NC is degraded via 1,2,4‐benzenetriol (BT) and hydroquinone (HQ) formation. Studies carried out on a PNP– derivative and a PNP+ transconjugant also demonstrate that the genes for the NC degradative pathway reside on the plasmid present in RKJ200; the same plasmid had earlier been shown to encode genes for PNP degradation, which is also degraded via HQ formation. It is likely, therefore, that the same sets of genes encode the further metabolism of HQ in NC and PNP degradation.

Phylogeny of Acetate-Utilizing Microorganisms in Soils along a Nutrient Gradient in the Florida Everglades

ABSTRACT The consumption of acetate in soils taken from a nutrient gradient in the northern Florida Everglades was studied by stable isotope probing. Bacterial and archaeal 16S rRNA gene clone libraries from eutrophic and oligotrophic soil microcosms strongly suggest that a significant amount of acetate is consumed by syntrophic acetate oxidation in nutrient-enriched soil.

Bacterial quorum sensing: functional features and potential applications in biotechnology.

Quorum sensing (QS) represents an exceptional pattern of cell-to-cell communication in bacteria using self-synthesized signalling molecules known as autoinducers. Various features regulated by QS in bacteria include virulence, biofilm formation, sporulation, genetic competence and bioluminescence, among others. Other than the diverse signalling properties of autoinducers, there are non-signalling properties also associated with these signalling molecules which make them potential antimicrobial agents and metal chelators. Additionally, QS signal antagonism has also been shown to be a promising alternative for blocking pathogenic diseases. Besides, QS has impressive design features useful in tissue engineering and biosensor technology. Although many aspects of QS are well understood, several other features remain largely unknown, especially in biotechnology applications. This review focuses on the functional features and potential applications of QS signalling molecules in biotechnology.

Chemotaxis of a Ralstonia sp. SJ98 toward co-metabolizable nitroaromatic compounds

We have earlier reported chemotaxis of a Gram-negative, motile Ralstonia sp. SJ98 towards p-nitrophenol (PNP), 4-nitrocatechol (NC), o-nitrobenzoate (ONB), p-nitrobenzoate (PNB), and 3-methyl-4-nitrophenol (MNP) that also served as sole source of carbon and energy to the strain [S.K. Samanta, B. Bhushan, A. Chauhan, R.K. Jain, Biochem. Biophy. Res. Commun. 269 (2000) 117; B. Bhushan, S.K. Samanta, A. Chauhan, A.K. Chakraborti, R.K. Jain, Biochem. Biophy. Res. Commun. 275 (2000) 129]. In this paper, we report chemotaxis of a Ralstonia sp. SJ98 toward seven different nitroaromatic compounds (NACs) by drop assay, swarm plate assay, and capillary assay. These NACs do not serve as sole carbon and energy source to strain SJ98 but are partially transformed in the presence of an alternate carbon source such as succinate. This is the first report showing chemotaxis of a bacterial strain toward co-metabolizable NACs.

Halobacteriovorax, an underestimated predator on bacteria: potential impact relative to viruses on bacterial mortality

Predation on bacteria and accompanying mortality are important mechanisms in controlling bacterial populations and recycling of nutrients through the microbial loop. The agents most investigated and seen as responsible for bacterial mortality are viruses and protists. However, a body of evidence suggests that predatory bacteria such as the Halobacteriovorax (formerly Bacteriovorax), a Bdellovibrio-like organism, contribute substantially to bacterial death. Until now, conclusive evidence has been lacking. The goal of this study was to better understand the contributors to bacterial mortality by addressing the poorly understood role of Halobacteriovorax and how their role compares with that of viruses. The results revealed that when a concentrated suspension of Vibrio parahaemolyticus was added into microcosms of estuarine waters, the native Halobacteriovorax were the predators that responded first and most rapidly. Their numbers increased by four orders of magnitude, whereas V. parahaemolyticus prey numbers decreased by three orders of magnitude. In contrast, the extant virus population showed little increase and produced little change in the prey density. An independent experiment with stable isotope probing confirmed that Halobacteriovorax were the predators primarily responsible for the mortality of the V. parahaemolyticus. The results show that Halobacteriovorax have the potential to be significant contributors to bacterial mortality, and in such cases, predation by Halobacteriovorax may be an important mechanism of nutrient recycling. These conclusions add another dimension to bacterial mortality and the recycling of nutrients.

Response of Bdellovibrio and like organisms (BALOs) to the migration of naturally occurring bacteria to chemoattractants.

A dual culture-based and non–culture-based approach was applied to characterize predator bacterial groups in surface water samples collected from Apalachicola Bay, Florida. Chemotaxis drop assays were performed on concentrated samples in an effort to isolate predator bacteria by their chemotactic ability. Yeast extract (YE) and casamino acids (CA) proved to be strong chemoattractants and resulted in three visibly distinct bands; however, dextrose, succinate, pyruvate, and concentrated cells of Vibrio parahaemolyticus P5 as prey did not elicit any response. The three distinct bands from YE and CA were separately collected to identify the chemotactic microbial assemblages. Plaque-forming unit assays from different chemotaxis bands with P5 as prey indicated 5- (CA) to 10-fold (YE) higher numbers of predator bacteria in the outermost chemotactic bands. Polymerase chain reaction–restriction fragment length polymorphism and 16S rDNA sequencing of clones from different chemotaxis bands resulted in identification of Pseudoalteromonas spp., Marinomonas spp., and Vibrio spp., with their numbers inversely proportional to the numbers of predators—i.e., Bdellovibrio spp. and Bacteriovorax spp—in the chemotaxis bands. This study indicates that predatorial bacteria potentially respond to high densities of microbial biomass in aquatic ecosystems and that chemotaxis drop assay may be an alternate culture-independent method to characterize predatorial bacterial guilds from the environment.

Whole-Genome Sequences of Five Oyster-Associated Bacteria Show Potential for Crude Oil Hydrocarbon Degradation

ABSTRACT Draft genome sequences of oyster-associated Pseudomonas stutzeri strain MF28, P. alcaligenes strain OT69, P. aeruginosa strain WC55, Stenotrophomonas maltophilia strain MF89, and Microbacterium maritypicum strain MF109 are reported. Genome-wide surveys of these isolates suggest that the oyster microbiome, which remains largely understudied, has a strong potential to degrade crude oil.