Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for nitrate respiration and denitrification have not been well characterized. In this study, we performed a clone library analysis of >1,000 clones of the nearly full-length 16S rRNA gene to characterize bacterial community structure in rice paddy soil. We also identified potential key players in nitrate respiration and denitrification by comparing the community structures of soils with strong denitrifying activity to those of soils without denitrifying activity. Clone library analysis showed that bacteria belonging to the phylum Firmicutes, including a unique Symbiobacterium clade, dominated the clones obtained in this study. Using the template match method, several operational taxonomic units (OTUs), most belonging to the orders Burkholderiales and Rhodocyclales, were identified as OTUs that were specifically enriched in the sample with strong denitrifying activity. Almost one-half of these OTUs were classified in the genus Herbaspirillum and appeared >10-fold more frequently in the soils with strong denitrifying activity than in the soils without denitrifying activity. Therefore, OTUs related to Herbaspirillum are potential key players in nitrate respiration and denitrification under the conditions used.
Nineteen soils, three freshwater lake sediments, and oxidized poultry manure were examined to determine the dominant denitrifier populations. The samples, most shown or expected to support active denitrification, were from eight countries and included rice paddy, temperate agricultural, rain forest, organic, and waste-treated soils. Over 1,500 organisms that could grow anaerobically on nitrate agar were isolated. After purification, 146 denitrifiers were obtained, as verified by production of N2 from NO3-. These isolates were characterized by 52 properties appropriate for the Pseudomonas-Alcaligenes group. Numerical taxonomic procedures were used to group the isolates and compare them with nine known denitrifier species. The major group isolated was representative of Pseudonomas fluorescens biotype II. The second most prevalent group was representative of Alcaligenes. Other Pseudomonas species as well as members of the genus Flavobacterium, the latter previously not known to denitrify, also were identified. One-third of the isolates could not utilize glucose or other carbohydrates as sole carbon sources. Significantly, none of the numerically dominant denitrifiers we isolated resembled the most studied species: Pseudomonas denitrificans, Pseudomonas perfectomarinus, and Paracoccus denitrificans. Denitrification appears to be a property of a very diverse group of gram-negative, motile bacteria, as shown by the large number (22.6%) of ungrouped organisms. The diversity of denitrifiers from a given sample was usually high, with at least two groups present. Denitrifiers, nitrite accumulators, and organisms capable of anaerobic growth were present in the ratio of 0.20±0.23:0.81±0.23:1. There were few correlations between their numbers and the sample characteristics measured. However, the temperatures at which isolates could grow were significantly related to the temperatures of the environments from which they were isolated. Regression analysis revealed few relationships between physical parameters and bacterial types, save for the anaerobe numbers, in which 94% of the variance could be accounted for.
Malaria research often requires isolation of individually infected red blood cells (RBCs) or a homogenous parasite population derived from a single parasite (clone). Traditionally, isolation of individual, parasitized RBCs or parasite cloning is achieved by limiting dilution or micromanipulation. This protocol describes a method for more efficient cloning of the malaria parasite, which uses a cell sorter to rapidly isolate singly Plasmodium falciparum-infected RBCs. By gating the parameters of forward angle light scatter (FSC) and side angle light scatter (SSC) in a cell sorter, singly-infected RBCs can be isolated and automatically deposited into a 96-well culture plate within one minute. To include a Percoll purification step, the entire procedure to seed a 96-well plate with singly-infected RBCs takes less than 40 min. This highly efficient single-cell sorting protocol should be useful for cloning of both laboratory parasite populations from genetic manipulation experiments and clinical samples.
malaria parasite; Plasmodium falciparum; parasite cloning; single-cell sorting
We examined the competitive relationship between two isolates of denitrifying bacteria, both of which grow well under aerobic conditions but differ in their ability to grow under denitrifying conditions. The growth and persistence of the two isolates, added to sterile soil or added to soil previously colonized by the other isolate, were monitored under aerobic and denitrifying (anaerobic) conditions. When isolates were added together to sterile soil, the isolate added at the higher density reduced the growth of the isolate added at the lower density. The magnitude of the growth reduction varied depending on the competitive abilities of the individual isolates and the aeration state of the soil. Prior colonization of soil with one of the isolates conferred a competitive advantage on the colonized isolate but did not lead to the disappearance of the challenging isolate. Fluctuations in aeration state caused large changes in the population density of one isolate and altered the competitive relationship between the two isolates. The competitive effectiveness of each isolate varied with cell density, the degree of prior colonization of the soil by the other isolate, and the aeration state of the soil.
The growth and survival of several rifampin-resistant isolates of denitrifying bacteria were examined under anaerobic (denitrifying) and aerobic conditions. Two isolates added to nonsterile Bruno soil at densities of between 10(4) and 10(6) CFU g dry soil-1 exhibited an initial period of growth followed by a gradual decline in numbers. After 28 days, both isolates maintained viable populations of between 10(4) and 10(5) CFU g dry soil-1 under both denitrifying and aerobic conditions. One of the isolates consistently grew better under denitrifying conditions, and the other isolate consistently grew better under aerobic conditions. The relative pattern of denitrifying versus aerobic growth for each organism was not affected by the addition of glucose. The growth yields of the two isolates varied with soil type, but the relative pattern of denitrifying versus aerobic growth was consistent in three soils with greatly different properties. Five of nine isolates introduced into Bruno soil at low population densities (approximately 10(5) CFU g dry soil-1) exhibited better growth after 2 days under denitrifying conditions. It was not possible to predict the prevalence of the denitrifying or aerobic mode of growth in nonsterile soil from the growth characteristics of the isolates in pure cultures or sterile soil.
In terrestrial subsurface environments where nitrate is a critical groundwater contaminant, few cultivated representatives are available to verify the metabolism of organisms that catalyze denitrification. In this study, five species of denitrifying bacteria from three phyla were isolated from subsurface sediments exposed to metal radionuclide and nitrate contamination as part of the U.S. Department of Energy's Oak Ridge Integrated Field Research Challenge (OR-IFRC). Isolates belonged to the genera Afipia and Hyphomicrobium (Alphaproteobacteria), Rhodanobacter (Gammaproteobacteria), Intrasporangium (Actinobacteria), and Bacillus (Firmicutes). Isolates from the phylum Proteobacteria were complete denitrifiers, whereas the Gram-positive isolates reduced nitrate to nitrous oxide. rRNA gene analyses coupled with physiological and genomic analyses suggest that bacteria from the genus Rhodanobacter are a diverse population of denitrifiers that are circumneutral to moderately acidophilic, with a high relative abundance in areas of the acidic source zone at the OR-IFRC site. Based on genome analysis, Rhodanobacter species contain two nitrite reductase genes and have not been detected in functional-gene surveys of denitrifying bacteria at the OR-IFRC site. Nitrite and nitrous oxide reductase gene sequences were recovered from the isolates and from the terrestrial subsurface by designing primer sets mined from genomic and metagenomic data and from draft genomes of two of the isolates. We demonstrate that a combination of cultivation and genomic and metagenomic data is essential to the in situ characterization of denitrifiers and that current PCR-based approaches are not suitable for deep coverage of denitrifiers. Our results indicate that the diversity of denitrifiers is significantly underestimated in the terrestrial subsurface.
We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042–5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 × 107 to 2.5 × 108 cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.
Despite the fact that rice paddy fields (RPFs) are contributing 10 to 25% of global methane emissions, the organisms responsible for methane production in RPFs have remained uncultivated and thus uncharacterized. Here we report the isolation of a methanogen (strain SANAE) belonging to an abundant and ubiquitous group of methanogens called rice cluster I (RC-I) previously identified as an ecologically important microbial component via culture-independent analyses. To enrich the RC-I methanogens from rice paddy samples, we attempted to mimic the in situ conditions of RC-I on the basis of the idea that methanogens in such ecosystems should thrive by receiving low concentrations of substrate (H2) continuously provided by heterotrophic H2-producing bacteria. For this purpose, we developed a coculture method using an indirect substrate (propionate) in defined medium and a propionate-oxidizing, H2-producing syntroph, Syntrophobacter fumaroxidans, as the H2 supplier. By doing so, we significantly enriched the RC-I methanogens and eventually obtained a methanogen within the RC-I group in pure culture. This is the first report on the isolation of a methanogen within RC-I.
Earthworms emit nitrous oxide (N2O) and dinitrogen (N2). It has been hypothesized that the in situ conditions of the earthworm gut activates ingested soil denitrifiers during gut passage and leads to these in vivo emissions (M. A. Horn, A. Schramm, and H. L. Drake, Appl. Environ. Microbiol. 69:1662-1669, 2003). This hypothesis implies that the denitrifiers in the earthworm gut are not endemic to the gut but rather are regular members of the soil denitrifier population. To test this hypothesis, the denitrifier populations of gut and soil from three different sites were comparatively assessed by sequence analysis of nosZ, the gene for the terminal enzyme in denitrification, N2O reductase. A total of 182 and 180 nosZ sequences were retrieved from gut and soil, respectively; coverage of gene libraries was 79 to 100%. Many of the nosZ sequences were heretofore unknown, clustered with known soil-derived sequences, or were related to N2O reductases of the genera Bradyrhizobium, Brucella, Dechloromonas, Flavobacterium, Pseudomonas, Ralstonia, and Sinorhizobium. Although the numbers of estimators for genotype richness of sequence data from the gut were higher than those of soil, only one gut-derived nosZ sequence did not group phylogenetically with any of the soil-derived nosZ sequences. Thus, the phylogenies of nosZ from gut and soil were not dissimilar, indicating that gut denitrifiers are soil derived.
A mechanistic approach is presented to describe oxidation of the greenhouse gas methane in the rice rhizosphere of flooded paddies by obligate methanotrophic bacteria. In flooded rice paddies these methanotrophs compete for available O2 with other types of bacteria. Soil incubation studies and most-probable-number (MPN) counts of oxygen consumers show that microbial oxygen consumption rates were dominated by heterotrophic and methanotrophic respiration. MPN counts of methanotrophs showed large spatial and temporal variability. The most abundant methanotrophs (a Methylocystis sp.) and heterotrophs (a Pseudomonas sp. and a Rhodococcus sp.) were isolated and characterized. Growth dynamics of these bacteria under carbon and oxygen limitations are presented. Theoretical calculations based on measured growth dynamics show that methanotrophs were only able to outcompete heterotrophs at low oxygen concentrations (frequently <5 μM). The oxygen concentration at which methanotrophs won the competition from heterotrophs did not depend on methane concentration, but it was highly affected by organic carbon concentrations in the paddy soil. Methane oxidation was severely inhibited at high acetate concentrations. This is in accordance with competition experiments between Pseudomonas spp. and Methylocystis spp. carried out at different oxygen and carbon concentrations. Likely, methane oxidation mainly occurs at microaerophilic and low-acetate conditions and thus not directly at the root surface. Acetate and oxygen concentrations in the rice rhizosphere are in the critical range for methane oxidation, and a high variability in methane oxidation rates is thus expected.
An evolutionary algorithm was applied to study the complex interactions between medium parameters and their effects on the isolation of denitrifying bacteria, both in number and in diversity. Growth media with a pH of 7 and a nitrogen concentration of 3 mM, supplemented with 1 ml of vitamin solution but not with sodium chloride or riboflavin, were the most successful for the isolation of denitrifiers from activated sludge. The use of ethanol or succinate as a carbon source and a molar C/N ratio of 2.5, 20, or 25 were also favorable. After testing of 60 different medium parameter combinations and comparison with each other as well as with the standard medium Trypticase soy agar supplemented with nitrate, three growth media were highly suitable for the cultivation of denitrifying bacteria. All evaluated isolation conditions were used to study the cultivable denitrifier diversity of activated sludge from a municipal wastewater treatment plant. One hundred ninety-nine denitrifiers were isolated, the majority of which belonged to the Betaproteobacteria (50.4%) and the Alphaproteobacteria (36.8%). Representatives of Gammaproteobacteria (5.6%), Epsilonproteobacteria (2%), and Firmicutes (4%) and one isolate of the Bacteroidetes were also found. This study revealed a much more diverse denitrifying community than that previously described in cultivation-dependent research on activated sludge.
A 10-year-long field trial (between 2001 and 2010) was conducted to investigate the effect of paddy-upland rotation on rice yield, soil properties, and bacteria community diversity. Six types of paddy-upland crop rotations were evaluated: rice-fallow (control; CK), rice-rye grass (RR), rice-potato with rice straw mulches (RP), rice-rapeseed with straw incorporated into soil at flowering (ROF), rice-rapeseed incorporated in soil after harvest (ROM), and rice-Chinese milk vetch (RC). Analysis of terminal restriction fragment length polymorphism (T-RFLP) was used to determine microbial diversity among rotations. Rice yield increased for upland crops planted during the winter. RC had the highest average yield of 7.74 t/ha, followed by RR, RP, ROM, and ROF. Soil quality differences among rotations were found. RC and RP improved the soil mean weight diameter (MWD), which suggested that rice rotated with milk vetch and potato might improve the paddy soil structure. Improved total nitrogen (TN) and soil organic matter (SOM) were also found in RC and RP. The positive relationship between yield and TN/SOM might provide evidence for the effect of RC rotation on rice yield. A strong time dependency of soil bacterial community diversity was also found.
In continuously cropped, irrigated rice fields, soil microbial biomass as measured by total phospholipid fatty acid concentrations declined during the second half of the crop cycle. This decline was also observed in other components of the microbial community assessed by viable counts, including denitrifiers and sporeformers. Simultaneous with total biomass decline was the increase in potential indicators of nutrient stress--such as ratios of cyclopropanol ((Sigma)[cy/(omega)7c]) and trans ((Sigma)[(omega)7t/(omega)7c]) phospholipid fatty acids--in plain crop soil but not in the rhizosphere. Polyhydroxyalkanoate levels were enhanced in the root environment of mature rice. Polyunsaturated eukaryotic biomarkers accounted for only 13 to 16 mol% of the total phospholipids, including 2 mol% of 18:2(omega)6, which is considered a fungal biomarker. Single biomarkers for defined physiological groups of bacteria did not follow the declining trend of total microbial biomass. Signature compounds for gram-positive and gram-negative fermenters (plasmalogen phospholipids), methanogenic bacteria (diether lipids), and methanotrophs (18:1(omega)8c) increased as the crop approached maturity. Methanotrophs were not particularly enriched in the rhizosphere. Methanogenic biomarkers were, however, most abundant in root extracts from mature rice plants. Assuming that soil microbial biomass plays a significant role as a passive nutrient pool, its reduction during the second half of the cropping season suggests a mechanism that may ultimately contribute to declining productivity in irrigated, continuous rice cropping systems.
Oligotrophic denitrifying bacteria, including those belonging to the genera Herbaspirillum, Azospirillum, and Bradyrhizobium, were obtained using a single-cell isolation technique. The taxonomic composition of the denitrifier population was similar to those assessed by previous culture-independent studies. The sequencing of nitrite reductase and N2O reductase genes of these strains revealed previously unknown links between 16S rRNA and the denitrification-functional gene phylogenies. In particular, we identified Bradyrhizobium strains that harbor nirS sequences previously detected only in culture-independent studies.
The effects of air drying soil on denitrifying enzyme activity, denitrifier numbers, and rates of N gas loss from soil cores were measured. Only 29 and 16% of the initial denitrifying enzyme activity in fresh, near field capacity samples of Maury and Donerail soils, respectively, were lost after 7 days of air drying. The denitrifying activity of bacteria added to soil and activity recently formed in situ were not stable during drying. When dried and moist soil cores were irrigated, evolution of N gas began, and it maximized sooner in the dried cores. This suggests that the persistence of denitrifying enzymes permits accelerated denitrification when dried soils are remoistened. Enzyme activity increased significantly in these waterlogged cores, but fluctuations in enzyme activity were small compared with fluctuations in actual denitrification rate, and enzyme activities were always greater than denitrification rates. Apparent numbers of isolatable denitrifiers (most-probable-number counts) decreased more than enzyme activity as the soils were dried, but after the soils were rewetted, the extent of apparent growth was not consistently related to the magnitude of N loss. We hypothesize that activation-inactivation of existing enzymes by soil O2 is of greater significance in transient denitrification events than is growth of denitrifiers or synthesis of new enzymes.
Two kinds of phenol-degrading denitrifying bacteria, Azoarcus sp. strain CC-11 and spiral bacterial strain CC-26, were isolated from the same enrichment culture after 1 and 3 years of incubation, respectively. Both strains required ferrous ions for growth, but strain CC-26 grew better than strain CC-11 grew under iron-limited conditions, which may have resulted in the observed change in the phenol-degrading bacteria during the enrichment process. Strain CC-26 grew on phenol, benzoate, and other aromatic compounds under denitrifying conditions. Phylogenetic analysis of 16S ribosomal DNA sequences revealed that this strain is most closely related to a Magnetospirillum sp., a member of the α subclass of the class Proteobacteria, and is the first strain of a denitrifying aromatic compound-degrading bacterium belonging to this group. Unlike previously described Magnetospirillum strains, however, this strain did not exhibit magnetotaxis. It grew on phenol only under denitrifying conditions. Other substrates, such as acetate, supported aerobic growth, and the strain exhibited microaerophilic features.
Real-time quantitative PCR assays, targeting part of the ammonia-monooxygenase (amoA), nitrous oxide reductase (nosZ), and 16S rRNA genes were coupled with 15N pool dilution techniques to investigate the effects of long-term agricultural management practices on potential gross N mineralization and nitrification rates, as well as ammonia-oxidizing bacteria (AOB), denitrifier, and total bacterial community sizes within different soil microenvironments. Three soil microenvironments [coarse particulate organic matter (cPOM; >250 μm), microaggregate (53–250 μm), and silt-and-clay fraction (<53 μm)] were physically isolated from soil samples collected across the cropping season from conventional, low-input, and organic maize-tomato systems (Zea mays L.- Lycopersicum esculentum L.). We hypothesized that (i) the higher N inputs and soil N content of the organic system foster larger AOB and denitrifier communities than in the conventional and low-input systems, (ii) differences in potential gross N mineralization and nitrification rates across the systems correspond with AOB and denitrifier abundances, and (iii) amoA, nosZ, and 16S rRNA gene abundances are higher in the microaggregates than in the cPOM and silt-and-clay microenvironments. Despite 13 years of different soil management and greater soil C and N content in the organic compared to the conventional and low-input systems, total bacterial communities within the whole soil were similar in size across the three systems (~5.15×108 copies g−1 soil). However, amoA gene densities were ~2 times higher in the organic (1.75×108 copies g−1 soil) than the other systems at the start of the season and nosZ gene abundances were ~2 times greater in the conventional (7.65×107 copies g−1 soil) than in the other systems by the end of the season. Because organic management did not consistently lead to larger AOB and denitrifier communities than the other two systems, our first hypothesis was not corroborated. Our second hypothesis was also not corroborated because canonical correspondence analyses revealed that AOB and denitrifier abundances were decoupled from potential gross N mineralization and nitrification rates and from inorganic N concentrations. Our third hypothesis was supported by the overall larger nitrifier, denitrifier, and total bacterial communities measured in the soil microaggregates compared to the cPOM and silt-and-clay. These results suggest that the microaggregates are microenvironments that preferentially stabilize C, and concomitantly promote the growth of nitrifier and denitrifier communities, thereby serving as potential hotspots for N2O losses.
amoA; nosZ; soil microenvironments; N transformation rates; cropping system
We investigated communities of denitrifying bacteria from adjacent meadow and forest soils. Our objectives were to explore spatial gradients in denitrifier communities from meadow to forest, examine whether community composition was related to ecological properties (such as vegetation type and process rates), and determine phylogenetic relationships among denitrifiers. nosZ, a key gene in the denitrification pathway for nitrous oxide reductase, served as a marker for denitrifying bacteria. Denitrifying enzyme activity (DEA) was measured as a proxy for function. Other variables, such as nitrification potential and soil C/N ratio, were also measured. Soil samples were taken along transects that spanned meadow-forest boundaries at two sites in the H. J. Andrews Experimental Forest in the Western Cascade Mountains of Oregon. Results indicated strong functional and structural community differences between the meadow and forest soils. Levels of DEA were an order of magnitude higher in the meadow soils. Denitrifying community composition was related to process rates and vegetation type as determined on the basis of multivariate analyses of nosZ terminal restriction fragment length polymorphism profiles. Denitrifier communities formed distinct groups according to vegetation type and site. Screening 225 nosZ clones yielded 47 unique denitrifying genotypes; the most dominant genotype occurred 31 times, and half the genotypes occurred once. Several dominant and less-dominant denitrifying genotypes were more characteristic of either meadow or forest soils. The majority of nosZ fragments sequenced from meadow or forest soils were most similar to nosZ from the Rhizobiaceae group in α-Proteobacteria species. Denitrifying community composition, as well as environmental factors, may contribute to the variability of denitrification rates in these systems.
A field experiment was conducted to (i) examine the diurnal and seasonal soil carbon dioxide (CO2) fluxes pattern in rice paddy fields in central China and (ii) assess the role of floodwater in controlling the emissions of CO2 from soil and floodwater in intermittently draining rice paddy soil. The soil CO2 flux rates ranged from −0.45 to 8.62 µmol.m−2.s−1 during the rice-growing season. The net effluxes of CO2 from the paddy soil were lower when the paddy was flooded than when it was drained. The CO2 emissions for the drained conditions showed distinct diurnal variation with a maximum efflux observed in the afternoon. When the paddy was flooded, daytime soil CO2 fluxes reversed with a peak negative efflux just after midday. In draining/flooding alternating periods, a sudden pulse-like event of rapidly increasing CO2 efflux occured in response to re-flooding after draining. Correlation analysis showed a negative relation between soil CO2 flux and temperature under flooded conditions, but a positive relation was found under drained conditions. The results showed that draining and flooding cycles play a vital role in controlling CO2 emissions from paddy soils.
The distribution and phylogenetic affiliations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria in activated sludge were studied by a polyphasic approach including culture-independent biomarker and molecular analyses as well as cultivation methods. A total of 23 strains of PHBV-degrading denitrifiers were isolated from activated sludges from different sewage treatment plants. 16S ribosomal DNA (rDNA) sequence comparisons showed that 20 of the isolates were identified as members of the family Comamonadaceae, a major group of β-Proteobacteria. When the sludges from different plants were acclimated with PHBV under denitrifying conditions in laboratory scale reactors, the nitrate removal rate increased linearly during the first 4 weeks and reached 20 mg NO3−-N h−1 g of dry sludge−1 at the steady state. The bacterial-community change in the laboratory scale sludges during the acclimation was monitored by rRNA-targeted fluorescence in situ hybridization and quinone profiling. Both approaches showed that the population of β-Proteobacteria in the laboratory sludges increased sharply during acclimation regardless of their origins. 16S rDNA clone libraries were constructed from two different acclimated sludges, and a total of 37 clones from the libraries were phylogenetically analyzed. Most of the 16S rDNA clones were grouped with members of the family Comamonadaceae. The results of our polyphasic approach indicate that β-Proteobacteria, especially members of the family Comamonadaceae, are primary PHBV-degrading denitrifiers in activated sludge. Our data provide useful information for the development of a new nitrogen removal system with solid biopolymer as an electron donor.
Tularemia is a geographically widespread, severely debilitating, and occasionally lethal disease in humans. It is caused by infection by a gram-negative bacterium, Francisella tularensis. In order to better understand its potency as an etiological agent as well as its potential as a biological weapon, we have completed draft assemblies and report the first complete genomic characterization of five strains belonging to the following different Francisella subspecies (subsp.): the F. tularensis subsp. tularensis FSC033, F. tularensis subsp. holarctica FSC257 and FSC022, and F. tularensis subsp. novicida GA99-3548 and GA99-3549 strains. Here, we report the sequencing of these strains and comparative genomic analysis with recently available public Francisella sequences, including the rare F. tularensis subsp. mediasiatica FSC147 strain isolate from the Central Asian Region. We report evidence for the occurrence of large-scale rearrangement events in strains of the holarctica subspecies, supporting previous proposals that further phylogenetic subdivisions of the Type B clade are likely. We also find a significant enrichment of disrupted or absent ORFs proximal to predicted breakpoints in the FSC022 strain, including a genetic component of the Type I restriction-modification defense system. Many of the pseudogenes identified are also disrupted in the closely related rarely human pathogenic F. tularensis subsp. mediasiatica FSC147 strain, including modulator of drug activity B (mdaB) (FTT0961), which encodes a known NADPH quinone reductase involved in oxidative stress resistance. We have also identified genes exhibiting sequence similarity to effectors of the Type III (T3SS) and components of the Type IV secretion systems (T4SS). One of the genes, msrA2 (FTT1797c), is disrupted in F. tularensis subsp. mediasiatica and has recently been shown to mediate bacterial pathogen survival in host organisms. Our findings suggest that in addition to the duplication of the Francisella Pathogenicity Island, and acquisition of individual loci, adaptation by gene loss in the more recently emerged tularensis, holarctica, and mediasiatica subspecies occurred and was distinct from evolutionary events that differentiated these subspecies, and the novicida subspecies, from a common ancestor. Our findings are applicable to future studies focused on variations in Francisella subspecies pathogenesis, and of broader interest to studies of genomic pathoadaptation in bacteria.
Tularemia is a zoonotic disease that is widely disseminated throughout the Northern Hemisphere and is caused by different strain types of bacteria belonging to the genus Francisella. In general, Francisella tularensis subspecies are able to infect a wide range of mammals including humans and are often transmitted via insect vectors such as ticks. Depending on the strain and route of infection the disease may be fatal in humans. In order to better understand F. tularensis as an etiological agent as well as its potential as a biological weapon, we have completed draft sequence assemblies of five globally diverse strains. We have performed a comparative analysis of these sequences with other available public Francisella sequences of strains of differing virulence. Our analysis suggests that genome rearrangements and gene loss in specific Francisella subspecies may underlie the evolution of niche adaptation and virulence of this pathogen.
A system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128); the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained with nondenitrifying bacteria or with strains of the other nir type. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying one generally amplifying primer combination for each nir gene developed in this study to total DNA preparations from aquatic habitats.
Propionate-forming bacteria seem to be abundant in anoxic rice paddy soil, but biogeochemical investigations show that propionate is not a correspondingly important intermediate in carbon flux in this system. Mixed cultures of Opitutus terrae strain PB90-1, a representative propionate-producing bacterium from rice paddy soil, and the hydrogenotrophic Methanospirillum hungatei strain SK maintained hydrogen partial pressures similar to those in the soil. The associated shift away from propionate formation observed in these cultures helps to reconcile the disparity between microbiological and biogeochemical studies.
The soil-dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis. Extreme structuring of genotype and genotypic frequency has been demonstrated for B. pseudomallei in uncultivated land, but its distribution and genetic diversity in agricultural land where most human infections are probably acquired is not well defined.
Fixed-interval soil sampling was performed in a rice paddy in northeast Thailand in which 100 grams of soil was sampled at a depth of 30 cm from 10×10 sampling points each measuring 2.5 m by 2.5 m. Soil was cultured for the presence of B. pseudomallei and genotyping of colonies present on primary culture plates was performed using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).
B. pseudomallei was cultured from 28/100 samples. Genotyping of 630 primary colonies drawn from 11 sampling points demonstrated 10 PFGE banding pattern types, which on MLST were resolved into 7 sequence types (ST). Overlap of genotypes was observed more often between sampling points that were closely positioned. Two sampling points contained mixed B. pseudomallei genotypes, each with a numerically dominant genotype and one or more additional genotypes present as minority populations.
Genetic diversity and structuring of B. pseudomallei exists despite the effects of flooding and the physical and chemical processes associated with farming. These findings form an important baseline for future studies of environmental B. pseudomallei.
Burkholderia pseudomallei is the cause of melioidosis, a serious human infection most commonly diagnosed in southeast Asia and northern Australia. The organism lives in the soil in a specific geographical distribution and infection results from bacterial inoculation, inhalation or ingestion. The purpose of this study was to define the distribution and genetic diversity of B. pseudomallei in agricultural land where most human infections probably occur. We performed soil sampling and culture for the presence of B. pseudomallei in 100 equally spaced points within a rice paddy in northeast Thailand, and undertook genotyping of primary culture plate colonies from 11 sampling points. We identified 7 different genotypes, with relatively limited overlap between different sampling points. Two samples contained more than one B. pseudomallei genotype, in which a numerically dominant genotype coexisted with one or more additional genotypes present as a minority population. We conclude that genetic diversity and structuring of B. pseudomallei exists despite the effects of flooding and the physical and chemical processes associated with farming. These findings inform future efforts to define B. pseudomallei in the environment, and should be considered during the design stage of studies comparing B. pseudomallei isolated from the environment and from patients with invasive disease.
An autoradiographic method was developed to screen for and isolate soil microorganisms which accumulate zinc (Zn). Diluted soil samples (Rubicon fine sand, Entic Haplorthods [pH 5.9]) were plated on soil extract-glucose agar containing radioactive 65Zn. After 7 days of incubation, individual colonies which accumulated sufficient 65Zn could be detected by autoradiography. These colonies were isolated and confirmed as Zn accumulators in pure culture by using the autoradiographic plate technique. Most Zn accumulators were filamentous fungi, identified as Penicillium janthinellum, Aspergillus fumigatus, and Paecilomyces sp. Isolates of Penicillium janthinellum were the most common Zn accumulators. The most abundant Zn-accumulating bacteria were Bacillus spp. The validity of the autoradiographic plate technique to differentiate soil microbes which accumulate Zn was examined independently by energy dispersive X-ray analysis in a scanning electron microscope. This method confirmed that fungal isolates which gave positive autoradiographic responses in the plate assay bioaccumulated more Zn in their biomass than fungal isolates from the same soil sample which gave negative autoradiographic responses. Thus, this technique can be applied to specifically screen for and isolate microbes from the environment which bioaccumulate Zn.