Impacts of Hurricanes Katrina and Rita on the microbial landscape of the New Orleans area.

Floodwaters in New Orleans from Hurricanes Katrina and Rita were observed to contain high levels of fecal indicator bacteria and microbial pathogens, generating concern about long-term impacts of these floodwaters on the sediment and water quality of the New Orleans area and Lake Pontchartrain. We show here that fecal indicator microbe concentrations in offshore waters from Lake Pontchartrain returned to prehurricane concentrations within 2 months of the flooding induced by these hurricanes. Vibrio and Legionella species within the lake were more abundant in samples collected shortly after the floodwaters had receded compared with samples taken within the subsequent 3 months; no evidence of a long-term hurricane-induced algal bloom was observed. Giardia and Cryptosporidium were detected in canal waters. Elevated levels of fecal indicator bacteria observed in sediment could not be solely attributed to impacts from floodwaters, as both flooded and nonflooded areas exhibited elevated levels of fecal indicator bacteria. Evidence from measurements of Bifidobacterium and bacterial diversity analysis suggest that the fecal indicator bacteria observed in the sediment were from human fecal sources. Epidemiologic studies are highly recommended to evaluate the human health effects of the sediments deposited by the floodwaters.

Vertical distribution of nitrogen-fixing phylotypes in a meromictic, hypersaline lake.

We investigated the diversity of nitrogenase genes in the alkaline, moderately hypersaline Mono Lake, California to determine (1) whether nitrogen-fixing (diazotrophic) populations were similar to those in other aquatic environments and (2) if there was a pattern of distribution of phylotypes that reflected redox conditions, as well as (3) to identify populations that could be important in N dynamics in this nitrogen-limited lake. Mono Lake has been meromictic for almost a decade and has steep gradients in oxygen and reduced compounds that provide a wide range of aerobic and anaerobic habitats. We amplified a fragment of the nitrogenase gene (nifH) from planktonic DNA samples collected at three depths representing oxygenated surface waters, the oxycline, and anoxic, ammonium-rich deep waters. Forty-three percent of the 90 sequences grouped in nifH Cluster I. The majority of clones (57%) grouped in Cluster III, which contains many known anaerobic bacteria. Cluster I and Cluster III sequences were retrieved at every depth indicating little vertical zonation in sequence types related to the prominent gradients in oxygen and ammonia. One group in Cluster I was found most often at every depth and accounted for 29% of all the clones. These sequences formed a subcluster that contained other environmental clones, but no cultivated representatives. No significant nitrogen fixation was detected by the 15N2 method after 48 h of incubation of surface, oxycline, or deep waters, suggesting that pelagic diazotrophs were contributing little to nitrogen fluxes in the lake. The failure to measure any significant nitrogen fixation, despite the detection of diverse and novel nitrogenase genes throughout the water column, raises interesting questions about the ecological controls on diazotrophy in Mono Lake and the distribution of functional genes in the environment.

Phylogenetic screening of ribosomal RNA gene-containing clones in Bacterial Artificial Chromosome (BAC) libraries from different depths in Monterey Bay.

Marine picoplankton are central mediators of many oceanic biogeochemical processes, but much of their biology and ecology remains ill defined. One approach to better defining these environmentally significant microbes involves the acquisition of genomic data that can provide information about genome content, metabolic capabilities, and population variability in picoplankton assemblages. Previously, we constructed and phylogenetically screened a Bacterial Artificial Chromosome (BAC) library from surface water picoplankton of Monterey Bay. To further describe niche partitioning, metabolic variability, and population structure in coastal picoplankton populations, we constructed and compared several picoplankton BAC libraries recovered from different depths in Monterey Bay. To facilitate library screening, a rapid technique was developed (ITS-LH-PCR) to identify and quantify ribosomal RNA (rRNA) gene-containing BAC clones in BAC libraries. The approach exploited natural length variations in the internal transcribed spacer (ITS) located between SSU and LSU rRNA genes, as well as the presence and location of tRNA-alanine coding genes within the ITS. The correspondence between ITS-LH-PCR fragment sizes and 16S rRNA gene phylogenies facilitated rapid identification of rRNA genes in BAC clones without requiring direct DNA sequencing. Using this approach, 35 phylogenetic groups (previously identified by cultivation or PCR-based rRNA gene surveys) were detected and quantified among the BAC clones. Since the probability of recovering chimeric rRNA gene sequences in large insert BAC clones was low, we used these sequences to identify potentially chimeric sequences from previous PCR amplified clones deposited in public databases. Full-length SSU rRNA gene sequences from picoplankton BAC libraries, cultivated bacterioplankton, and nonchimeric RNA genes were then used to refine phylogenetic analyses of planktonic marine gamma Proteobacteria, Roseobacter, and Rhodospirillales species.

Unicellular cyanobacteria fix N2 in the subtropical North Pacific Ocean.

Fixed nitrogen (N) often limits the growth of organisms in terrestrial and aquatic biomes, and N availability has been important in controlling the CO2 balance of modern and ancient oceans. The fixation of atmospheric dinitrogen gas (N2) to ammonia is catalysed by nitrogenase and provides a fixed N for N-limited environments. The filamentous cyanobacterium Trichodesmium has been assumed to be the predominant oceanic N2-fixing microorganism since the discovery of N2 fixation in Trichodesmium in 1961 (ref. 6). Attention has recently focused on oceanic N2 fixation because nitrogen availability is generally limiting in many oceans, and attempts to constrain the global atmosphere-ocean fluxes of CO2 are based on basin-scale N balances. Biogeochemical studies and models have suggested that total N2-fixation rates may be substantially greater than previously believed but cannot be reconciled with observed Trichodesmium abundances. It is curious that there are so few known N2-fixing microorganisms in oligotrophic oceans when it is clearly ecologically advantageous. Here we show that there are unicellular cyanobacteria in the open ocean that are expressing nitrogenase, and are abundant enough to potentially have a significant role in N dynamics.

A simple, rapid method for demonstrating bacterial flagella.

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.

Dynamics of bacterial community composition and activity during a mesocosm diatom bloom.

Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by a Thalassiosira sp., which peaked 9 days after enrichment ( approximately 24 microg of chlorophyll a liter(-1)). At this time bacterial abundance abruptly decreased from 2.8 x 10(6) to 0.75 x 10(6) ml(-1), and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-microm size fraction towards the >1.0-microm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized alpha-Proteobacteria- and Cytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, beta-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.

Bacteria-organic matter coupling and its significance for oceanic carbon cycling.

This paper synthesizes current ideas on the role of the microbial loop in carbon fluxes in the ocean and proposes some directions for future research. Organic matter flux into bacteria is highly variable, which can significantly influence the pathways of carbon flow in the ocean. A goal for future research is to elucidate the mechanistic bases of bacteria-organic matter coupling. This research should take into consideration the micrometer-scale distribution of bacteria and the composition, structure, and dynamics of the organic matter field in the bacterium's microhabitat. The ideas on the interactions of bacteria with the particulate organic phase need to be revised in view of recent findings of highly abundant, previously unknown particles ranging in size from nanometers to hundreds of micrometers. The "hot-spots" in the distribution of organic matter and remineralized nutrients can influence the rates as well as the direction of biogeochemical fluxes. Slow-to-degrade dissolved organic matter (DOM) may be produced because of loose bacteria-organic matter coupling resulting in DOM storage. Its use at a later time and place has profound implications for carbon fluxes and food web dynamics. A fundamental research need for the future is to understand the ecological interactions among the members of the microbial loop in an appropriate microhabitat context. While this goal was previously intractable, new molecular and optical techniques should make it possible to understand the biogeochemical activities of the microbial loop in terms of the ecology and evolution of pelagic microbial communities.

Sufentanil, morphine, met-enkephalin, and kappa-agonist (U-50,488H) inhibit substance P release from primary sensory neurons: a model for presynaptic spinal opioid actions.

An in vitro model system for analysis of presynaptic inhibitory actions of spinal opioids has been applied. Embryonic sensory neurons derived from chick dorsal root ganglia were grown in primary cell culture, and the release of substance P was evoked by electrical field stimulation during exposure to drugs with well-demonstrated affinity for opioid receptors. This allowed a pharmacologic characterization of the inhibitory actions of specific opioid agonists on the release of substance P as measured by radioimmunoassay (RIA). Sufentanil (0.5 microM), a high affinity mu receptor agonist, U-50,488H (25 microM), a selective kappa receptor agonist, and morphine (10 microM), an agonist with high affinity for mu and delta receptors, inhibited the evoked release of substance P by approximately 60%, 40%, and 50%, respectively. For sufentanil the response was demonstrated to be dose-dependent. As is the case for its analgesic action in vivo, morphine was approximately 50-fold less potent than sufentanil on a molar basis in this assay. The actions of sufentanil, U-50-488H and morphine were mimicked by the endogenous opioid peptide met-enkephalin, and its stable synthetic analog D-ala2-met5-enkephalinamide (DAME). Naloxone (25 microM), an opioid receptor antagonist, blocked the inhibitory action of sufentanil (0.5 microM), morphine (5 microM), and DAME (5 microM), but not U-50,488H (10 microM). The action of U-50,488H was partially blocked by the antagonist naltrexone (25 microM). Stereo-selectivity of agonist action was confirmed by the failure of dextrorphan (50 microM), an inactive opioid isomer, to inhibit the release of substance P.(ABSTRACT TRUNCATED AT 250 WORDS)