Spatial and Temporal Distribution of Two Diazotrophic Bacteria in the Chesapeake Bay

The aim of this study was to initiate autecological studies on uncultivated natural populations of diazotrophic bacteria by examining the distribution of specific diazotrophs in the Chesapeake Bay. By use of quantitative PCR, the abundance of two nifH sequences (907h22 and 912h4) was quantified in water samples collected along a transect from the head to the mouth of the Chesapeake Bay during cruises in April and October 2001 and 2002. Standard curves for the quantitative PCR assays demonstrated that the relationship between gene copies and cycle threshold was linear and highly reproducible from 1 to 10⁷ gene copies. The maximum number of 907h22 gene copies detected was approximately 140 ml⁻¹ and the maximum number of 912h4 gene copies detected was approximately 340 ml⁻¹. Sequence 912h4 was most abundant at the mouth of the Chesapeake Bay, and in general, its abundance increased with increasing salinity, with the highest abundances observed in April 2002. Overall, the 907h22 phylotype was most abundant at the mid-bay station. Additionally, 907h22 was most abundant in the April samples from the mid-bay and mouth of the Chesapeake Bay. Despite the fact that the Chesapeake Bay is rarely nitrogen limited, our results show that individual nitrogen-fixing bacteria have distinct nonrandom spatial and seasonal distributions in the Chesapeake Bay and are either distributed by specific physical processes or adapted to different environmental niches.     

Genetic Diversity of Vibrio cholerae in Chesapeake Bay Determined by Amplified Fragment Length Polymorphism Fingerprinting

Vibrio cholerae is indigenous to the aquatic environment, and serotype non-O1 strains are readily isolated from coastal waters. However, in comparison with intensive studies of the O1 group, relatively little effort has been made to analyze the population structure and molecular evolution of non-O1 V. cholerae. In this study, high-resolution genomic DNA fingerprinting, amplified fragment length polymorphism (AFLP), was used to characterize the temporal and spatial genetic diversity of 67 V. cholerae strains isolated from Chesapeake Bay during April through July 1998, at four different sampling sites. Isolation of V. cholerae during the winter months (January through March) was unsuccessful, as observed in earlier studies (J. H. L. Kaper, R. R. Colwell, and S. W. Joseph, Appl. Environ. Microbiol. 37:91–103, 1979). AFLP fingerprints subjected to similarity analysis yielded a grouping of isolates into three large clusters, reflecting time of the year when the strains were isolated. April and May isolates were closely related, while July isolates were genetically diverse and did not cluster with the isolates obtained earlier in the year. The results suggest that the population structure of V. cholerae undergoes a shift in genotype that is linked to changes in environmental conditions. From January to July, the water temperature increased from 3°C to 27.5°C, bacterial direct counts increased nearly an order of magnitude, and the chlorophyll a concentration tripled (or even quadrupled at some sites). No correlation was observed between genetic similarity among isolates and geographical source of isolation, since isolates found at a single sampling site were genetically diverse and genetically identical isolates were found at several of the sampling sites. Thus, V. cholerae populations may be transported by surface currents throughout the entire Bay, or, more likely, similar environmental conditions may be selected for a specific genotype. The dynamic nature of the population structure of this bacterial species in Chesapeake Bay provides new insight into the ecology and molecular evolution of V. cholerae in the natural environment.     

Axial Dynamics, Stability, and Interspecies Similarity of Bacterial Community Structure in the Highly Compartmentalized Gut of Soil-Feeding Termites (Cubitermes spp.)

The highly compartmentalized gut of soil-feeding termites is characterized by pronounced axial dynamics in physicochemical conditions and microbial processes. In a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6007-6017, 2003), we demonstrated that the variety of physicochemical conditions in the different gut compartments of Cubitermes spp. is reflected in the diversity of the respective intestinal microbial communities. Here, we used molecular fingerprints of 16S rRNA genes of the bacterial community, obtained by terminal restriction fragment length polymorphism (T-RFLP) analysis, to describe the axial dynamics of the bacterial community structure in the different gut sections. Comparison of the T-RFLP profiles with the predicted terminal restriction fragments of the clones in clone libraries of the gut segments in Cubitermes orthognathus confirmed that all hindgut sections harbored distinct bacterial communities. Morisita indices of community similarity, calculated by comparing the different patterns, revealed large differences between the bacterial communities of soil, gut, and nest material and also among the individual gut sections. By contrast, comparison of the homologous gut segments of different Cubitermes species indicated that the three termite species investigated possessed a similar, gut-specific microbiota that remained comparatively stable even during several months of maintenance in the laboratory.     

Nitrogen-fixing phylotypes of Chesapeake Bay and Neuse River estuary sediments

Sediments often exhibit low rates of nitrogen fixation, despite the presence of elevated concentrations of inorganic nitrogen. The organisms that potentially fix nitrogen in sediments have not previously been identified. Amplification of nifH genes with degenerate primers was used to assess the diversity of diazotrophs in two distinct sediment systems, anoxic muds of Chesapeake Bay and shallow surficial sediments of the Neuse River. Phylogenetic analysis revealed that sequences obtained from mid-Chesapeake Bay, which receive high organic loading and are highly reducing, clustered closely with each other and with known anaerobic microorganisms, suggesting a low abundance of aerobic or facultative diazotrophs in these sediments. Sulfate reduction dominates in the surface, but methanogenesis becomes more important with depth. A thin (<1 cm) oxidized layer is present only in the spring. No archaeal nifH sequences were obtained from Chesapeake Bay. Sequences of nifH amplified from surficial sediments of the Neuse River were distant from Chesapeake Bay sequences and included nif phylotypes related to sequences previously reported from marine mats and the Spartina rhizosphere. Differences in environmental site characteristics appear to select for different types of sediment diazotrophs, which is reflected in the phylogenetic composition of amplified nifH sequences.     

Genetic diversity of Vibrio cholerae in Chesapeake Bay determined by amplified fragment length polymorphism fingerprinting

Vibrio cholerae is indigenous to the aquatic environment, and serotype non-O1 strains are readily isolated from coastal waters. However, in comparison with intensive studies of the O1 group, relatively little effort has been made to analyze the population structure and molecular evolution of non-O1 V. cholerae. In this study, high-resolution genomic DNA fingerprinting, amplified fragment length polymorphism (AFLP), was used to characterize the temporal and spatial genetic diversity of 67 V. cholerae strains isolated from Chesapeake Bay during April through July 1998, at four different sampling sites. Isolation of V. cholerae during the winter months (January through March) was unsuccessful, as observed in earlier studies (J. H. L. Kaper, R. R. Colwell, and S. W. Joseph, Appl. Environ. Microbiol. 37:91-103, 1979). AFLP fingerprints subjected to similarity analysis yielded a grouping of isolates into three large clusters, reflecting time of the year when the strains were isolated. April and May isolates were closely related, while July isolates were genetically diverse and did not cluster with the isolates obtained earlier in the year. The results suggest that the population structure of V. cholerae undergoes a shift in genotype that is linked to changes in environmental conditions. From January to July, the water temperature increased from 3 degrees C to 27.5 degrees C, bacterial direct counts increased nearly an order of magnitude, and the chlorophyll a concentration tripled (or even quadrupled at some sites). No correlation was observed between genetic similarity among isolates and geographical source of isolation, since isolates found at a single sampling site were genetically diverse and genetically identical isolates were found at several of the sampling sites. Thus, V. cholerae populations may be transported by surface currents throughout the entire Bay, or, more likely, similar environmental conditions may be selected for a specific genotype. The dynamic nature of the population structure of this bacterial species in Chesapeake Bay provides new insight into the ecology and molecular evolution of V. cholerae in the natural environment.     

The effects of pesticides on bacterial nitrogen fixers in peanut-growing area

In the peanut production, the applications of herbicides and fungicides are a common practice. In this work, studies done under field conditions demonstrated that pesticides affected negatively the number and nitrogenase activity of diazotrophic populations of soil. Agrochemical effects were not transient, since these parameters were not recovered to pre-treatment levels even 1 year after pesticides application. Results obtained from greenhouse experiments revealed that the addition of herbicide or fungicides diminished the free-living diazotrophs number reaching levels found in soil amended with the pesticides and that the number of symbiotic diazotrophs was not affected by the insecticide assayed. The soil nitrogenase activity was not affected by fungicides and glyphosate. The effect of pesticides on the nitrogen-fixing bacteria diversity was evaluated both in field and greenhouse experiments. Analysis of clone libraries generated from the amplification of soil nifH gene showed a diminution in the genetic diversity of this bacterial community.     

Molecular Analysis of Diazotroph Diversity in the Rhizosphere of the Smooth Cordgrass, Spartina alterniflora

N₂ fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass, Spartina alterniflora, is an important source of new nitrogen in many salt marsh ecosystems. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified nifH sequence segments was used in previous studies to examine the stability and dynamics of the Spartina rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C. In this study, plugs were taken from gel bands from representative DGGE gels, the nifH amplimers were recovered and cloned, and their sequences were determined. A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the Spartina rhizosphere. We recovered numerous sequences from diazotrophs in the γ subdivision of the division Proteobacteria (γ-Proteobacteria) and from various anaerobic diazotrophs. Diazotrophs in the α-Proteobacteria were poorly represented. None of the Spartina rhizosphere DGGE band sequences were identical to any known or previously recovered environmental nifH sequences. The Spartina rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.     

Development and Testing of a DNA Macroarray To Assess Nitrogenase (nifH) Gene Diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml⁻¹, a half-saturation of signal at 0.26 ng ml⁻¹, and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.     

Prevalence of Betaproteobacterial Sequences in <Emphasis Type="Italic">nifH</Emphasis> Gene Pools Associated with Roots of Modern Rice Cultivars

The diversity and function of nitrogen-fixing bacteria colonizing rice roots are not well understood. A field experiment was conducted to determine the diversity of diazotrophic communities associated with roots of modern rice cultivars using culture-independent molecular analyses of nitrogenase gene (nifH) fragments. Experimental treatments included four modern rice cultivars (Oryza sativa, one Indica, one Japonica and two hybrid rice varieties) and three levels (0, 50, and 100 kg N ha⁻¹) of N (urea) fertilizer application. Cloning and sequencing of 103 partial nifH genes showed that a diverse community of diazotrophs was associated with rice roots. However, the nifH gene fragments belonging to betaproteobacteria were dominant, accounting for nearly half of nifH sequences analyzed across the clone libraries. Most of them were similar to nifH fragments retrieved from wild rice and Kallar grass, with Azoarcus spp. being the closest cultured relatives. Alphaproteobacteria were also detected, but their relative abundance in the nifH gene pools was dramatically decreased with N fertilizer application. In addition, a high fraction of nifH gene pools was affiliated with methylotrophs and methane oxidizers. The sequence analysis was consistent with the terminal restriction fragment-length polymorphism (T-RFLP) fingerprinting of the nifH gene fragments, which showed three of four dominant terminal restriction fragments were mainly related to betaproteobacteria based on in silico digestion of nifH sequences. T-RFLP analyses also revealed that the effects of N fertilizer on the nifH gene diversity retrieved from roots varied according to rice cultivars. In summary, the present study revealed the prevalence of betaproteobacterial sequences among the proteobacteria associated with roots of modern rice cultivars. This group of diazotrophs appeared less sensitive to N fertilizer application than diazotrophic alphaproteobacteria. Furthermore, methylotrophs may also play a role in nitrogen fixation on rice roots. However, it must be noted that due to the potential bias of polymerase chain reaction protocol, the significance of non-proteobacterial diazotrophs such as Firmicutes and anaerobic bacteria is possibly underestimated.     

The complete genome sequence of Bacillus thuringiensis Al Hakam

Bacillus thuringiensis is an insect pathogen that is widely used as a biopesticide (E. Schnepf, N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D. R. Zeigler, and D. H. Dean, Microbiol. Mol. Biol. Rev. 62:775-806, 1998). Here we report the finished, annotated genome sequence of B. thuringiensis Al Hakam, which was collected in Iraq by the United Nations Special Commission (L. Radnedge, P. Agron, K. Hill, P. Jackson, L. Ticknor, P. Keim, and G. Andersen, Appl. Environ. Microbiol. 69:2755-2764, 2003).     

Vertical distribution of diazotrophic bacterial community associated with temperature and oxygen gradients in a subtropical reservoir

Nitrogen-fixing microorganisms play important roles in the structure and function of aquatic ecosystems. However, the diversity and distribution of diazotrophic bacteria along the lake depth continuum are so far poorly understood. In this study, we investigated the dynamic variations of diazotrophs in a subtropical deep reservoir during the stratified period. We applied an in-depth biomolecular approach (DGGE, clone libraries, and quantitative real-time PCR) to explore the nitrogenase (nifH) gene diversity and abundance. The diazotrophic community shifted between the oxic/anoxic interface and the nifH diversity increased with depth. The Cyanobacteria, affiliated to the toxic bloom-forming Cylindrospermopsis raciborskii, were the dominant diazotrophic cluster in the surface waters, whereas diazotrophic Alphaproteobacteria were dominant in the bottom waters. The relationships between microbial and environmental factors clearly demonstrated that the temperature gradient and the oxygen concentration affect the heterogeneity of the diazotrophic community, thereby influencing the entire aquatic nitrogen cycle.     

The complete genome sequence of Campylobacter jejuni strain 81116 (NCTC11828)

Campylobacter jejuni is a major human enteric pathogen that displays genetic variability via genomic reorganization and phase variation. This variability can adversely affect the outcomes and reproducibility of experiments. C. jejuni strain 81116 (NCTC11828) has been suggested to be a genetically stable strain (G. Manning, B. Duim, T. Wassenaar, J. A. Wagenaar, A. Ridley, and D. G. Newell, Appl. Environ. Microbiol. 67:1185-1189, 2001), is amenable to genetic manipulation, and is infective for chickens. Here we report the finished annotated genome sequence of C. jejuni strain 81116.     

Metagenomic Characterization of Chesapeake Bay Virioplankton

Viruses are ubiquitous and abundant throughout the biosphere. In marine systems, virus-mediated processes can have significant impacts on microbial diversity and on global biogeocehmical cycling. However, viral genetic diversity remains poorly characterized. To address this shortcoming, a metagenomic library was constructed from Chesapeake Bay virioplankton. The resulting sequences constitute the largest collection of long-read double-stranded DNA (dsDNA) viral metagenome data reported to date. BLAST homology comparisons showed that Chesapeake Bay virioplankton contained a high proportion of unknown (homologous only to environmental sequences) and novel (no significant homolog) sequences. This analysis suggests that dsDNA viruses are likely one of the largest reservoirs of unknown genetic diversity in the biosphere. The taxonomic origin of BLAST homologs to viral library sequences agreed well with reported abundances of cooccurring bacterial subphyla within the estuary and indicated that cyanophages were abundant. However, the low proportion of Siphophage homologs contradicts a previous assertion that this family comprises most bacteriophage diversity. Identification and analyses of cyanobacterial homologs of the psbA gene illustrated the value of metagenomic studies of virioplankton. The phylogeny of inferred PsbA protein sequences suggested that Chesapeake Bay cyanophage strains are endemic in that environment. The ratio of psbA homologous sequences to total cyanophage sequences in the metagenome indicated that the psbA gene may be nearly universal in Chesapeake Bay cyanophage genomes. Furthermore, the low frequency of psbD homologs in the library supports the prediction that Chesapeake Bay cyanophage populations are dominated by Podoviridae.     

Novel Major Bacterial Candidate Division within a Municipal Anaerobic Sludge Digester

In a previous study, we analyzed the molecular diversity of Planctomycetales by PCR amplification and sequencing of 16S rRNA clone libraries generated from a municipal wastewater plant, using planctomycete-specific and universal primer sets (R. Chouari, D. Le Paslier, P. Daegelen, P. Ginestet, J. Weissenbach, and A. Sghir, Appl. Environ. Microbiol. 69:7354-7363, 2003). Only a small fraction (4%) of the 16S rRNA gene sequences of the digester clone library corresponded to the Planctomycetales division. Importantly, 85.9% of the digester clone sequences are grouped into two different clusters named WWE1 (81.4% of the sequences) and WWE2 (4.5%) and are distantly affiliated with unidentified bacterial sequences retrieved from a methanogenic reactor community and from a termite gut, respectively. In phylogenetic analysis using 16S rRNA gene sequence representatives of the main phylogenetic bacterial divisions, the two clusters are monophyletic, branch apart from each other, and are distantly related to Planctomycetales and other bacterial divisions. A novel candidate division is proposed for WWE1, while the WWE2 cluster strongly affiliates with the recently proposed Lentisphearae phylum. We designed and validated a 16S rRNA probe targeting WWE1 16S rRNA sequences by both fluorescent in situ hybridization (FISH) and dot blot hybridization (DBH). Results of FISH analysis show that WWE1 representative microorganisms are rods or filamentous shaped, while DBH shows that WWE1 accounts for 12% of the total bacterial rRNA within the anaerobic digester. The remaining 16S rRNA gene sequences are affiliated with Verrucomicrobia or recently described candidate divisions with no known pure culture representatives, such as OD1, BRC1, or NBL-UPA2, making up less than 3.5% of the clone library, respectively. This inventory expands the known diversity of the latter bacterial division-level lineages.     

Stable Isotope Probing with 15N2 Reveals Novel Noncultivated Diazotrophs in Soil

Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of nifH gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel ¹⁵N₂-DNA stable isotope probing (⁵N₂-DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from ¹⁵N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the Rhizobiales while the other two represent deeply divergent lineages of noncultivated bacteria within the Betaproteobacteria and Actinobacteria, respectively. Analysis of nifH genes from ¹⁵N-labeled DNA also revealed three microbial groups, one of which was associated with Alphaproteobacteria while the others were associated with two noncultivated groups that are deeply divergent within nifH cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that ¹⁵N₂-DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by Actinobacteria outside of the order Actinomycetales.     

Small-Subunit rRNA Genotyping of Rhizobia Nodulating Australian Acacia spp.

The structure of rhizobial communities nodulating Acacia in southeastern Australia from south Queensland to Tasmania was investigated by a molecular approach. A total of 118 isolates from nodule samples from 13 different Acacia species collected at 44 sites were characterized by small-subunit (SSU) ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. Nine rhizobial genomospecies were identified, and these taxa corresponded to previously described genomospecies (B. Lafay and J. J. Burdon, Appl. Environ. Microbiol. 64:3989–3997, 1998). Eight of these genomospecies belonged to the Bradyrhizobium lineage and accounted for 96.6% of the isolates. The remaining genomospecies corresponded to Rhizobium tropici. For analysis of geographic patterns, results were grouped into five latitudinal regions regardless of host origin. In each region, as observed previously for rhizobial isolates taken from non-Acacia legumes (Lafay and Burdon, Appl. Environ. Microbiol. 64:3989–3997, 1998), rhizobial communities were dominated by one or two genomospecies, the identities of which varied from place to place. Despite this similarity in patterns, the most abundant genomospecies for Acacia isolates differed from the genomospecies found in the non-Acacia-derived rhizobial collection, suggesting that there is a difference in nodulation patterns of the Mimosoideae and the Papilionoideae. Only two genomospecies were both widespread and relatively abundant across the range of sites sampled. Genomospecies A was found in all regions except the most northern sites located in Queensland, whereas genomospecies B was not detected in Tasmania. This suggests that genomospecies A might be restricted to the more temperate regions of Australia, whereas in contrast, genomospecies B occurs in different climatic and edaphic conditions across the whole continent. The latter hypothesis is supported by the presence of genomospecies B in southwestern Australia, based on partial SSU rDNA sequence data (N. D. S. Marsudi, A. R. Glenn, and M. J. Dilworth, Soil Biol. Biochem. 31:1229–1238, 1998).     

New Molecular Screening Tools for Analysis of Free-Living Diazotrophs in Soil

Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.     

Comparison of methods for isolating Campylobacter jejuni from raw milk.

The method of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) was compared with that of Lovett et al. (Appl. Environ. Microbiol. 46:459-462, 1983) for the ability to recover Campylobacter jejuni strains inoculated into raw milk at a concentration of less than 1 cell per g. The method of Lovett et al. gave significantly greater recovery proportions.