Length Heterogeneity Polymerase Chain Reaction Research Articles

Groundwater Depth Overrides Tree-Species Effects on the Structure of Soil Microbial Communities Involved in Nitrogen Cycling in Plantation Forests

Microbial communities found in soil ecosystems play important roles in decomposing organic materials and recycling nutrients. A clear understanding on how biotic and abiotic factors influence the microbial community and its functional role in ecosystems is fundamental to terrestrial biogeochemistry and plant production. The purpose of this study was to investigate microbial communities and functional genes involved in nitrogen cycling as a function of groundwater depth (deep and shallow), tree species (pine and eucalypt), and season (spring and fall). Soil fungal, bacterial, and archaeal communities were determined by length heterogeneity polymerase chain reaction (LH-PCR). Soil ammonia oxidation archaeal (AOA) amoA gene, ammonia oxidation bacterial (AOB) amoA gene, nitrite oxidoreductase nrxA gene, and denitrifying bacterial narG, nirK, nirS, and nosZ genes were further studied using PCR and denaturing gradient gel electrophoresis (DGGE). Soil fungal and bacterial communities remained similar between tree species and groundwater depths, respectively, regardless of season. Soil archaeal communities remained similar between tree species but differed between groundwater depths in the spring only. Archaeal amoA for nitrification and bacterial nirK and nosZ genes for denitrification were detected in DGGE, whereas bacterial amoA and nrxA for nitrification and bacterial narG and nirS genes for denitrification were undetectable. The detected nitrification and denitrification communities varied significantly with groundwater depth. There was no significant difference of nitrifying archaeal amoA or denitrifying nirK communities between different tree species regardless of season. The seasonal difference in microbial communities and functional genes involved in nitrogen cycling suggests microorganisms exhibit seasonal dynamics that likely impact relative rates of nitrification and denitrification.

Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR

The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media.For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loads<5.0 Logcfug−1. Furthermore, LH-PCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products.

Effects of soil management practices on soil microbial communities and development of southern blight in vegetable production

Soil microbial communities and their relationship with major soil factors and disease development under tillage, no-tillage and organic farming practices were studied. Real-time quantitative-PCR (Q-PCR) was performed to quantify soil fungal and bacterial abundance. Length heterogeneity polymerase chain reaction (LH-PCR) combined with cloning and sequencing was used to assess microbial communities. Soil chemical characteristics were significantly different under different management practices. Organic agricultural practice significantly increased soil bacterial and fungal abundances, but not the diversity when compared to tillage and no-tillage management practices. Tillage farming agricultural practice yielded a significant higher bacterial abundance than no-tillage agricultural practice. No difference in fungal abundance was observed between tillage and no-tillage. Bacterial communities in no-tillage and organic cultivation were relatively similar, but significantly different from those in tillage cultivation. Differences of bacterial communities were correlated to soil organic matter (R2=0.558), cation exchange capacity (R2=0.583) and percentage of Mg (R2=0.590). Fungal communities in soil under organic cultivation were significantly different from relatively similar fungal community structure in no-tillage and tillage cultivation treatments. Differences of fungal communities were correlated to soil pH (R2=0.421) and soil organic matter (R2=0.401). In greenhouse assays, severity of southern blight on tomato plants grown in soils collected from the no-tillage was significantly lower than in soils from the tillage field. Different soil management practices have played an important role in altering soil microbial communities and potentially reducing disease incidence of southern blight on tomato.

Use of Length Heterogeneity Polymerase Chain Reaction (LH-PCR) as Non-Invasive Approach for Dietary Analysis of Svalbard Reindeer, Rangifer tarandus platyrhynchus

To efficiently investigate the forage preference of Svalbard reindeer (Rangifer tarandus platyrhynchus), we applied length-heterogeneity polymerase chain reaction (LH-PCR) based on length differences of internal transcribed spacer (ITS) regions of ribosomal RNA (rRNA) to fecal samples from R. tarandus platyrhynchus. A length-heterogeneity (LH) database was constructed using both collected potential food sources of Svalbard reindeer and fecal samples, followed by PCR, cloning and sequencing. In total, eighteen fecal samples were collected between 2011 and 2012 from 2 geographic regions and 15 samples were successfully amplified by PCR. The LH-PCR analysis detected abundant peaks, 18.6 peaks on an average per sample, ranging from 100 to 500 bp in size and showing distinct patterns associated with both regions and years of sample collection. Principal component analysis (PCA) resulted in clustering of 15 fecal samples into 3 groups by the year of collection and region with a statistically significant difference at 99.9% level. The first 2 principal components (PCs) explained 71.1% of the total variation among the samples. Through comparison with LH database and identification by cloning and sequencing, lichens (Stereocaulon sp. and Ochrolechia sp.) and plant species (Salix polaris and Saxifraga oppositifolia) were detected as the food sources that contributed most to the Svalbard reindeer diet. Our results suggest that the use of LH-PCR analysis would be a non-invasive and efficient monitoring tool for characterizing the foraging strategy of Svalbard reindeer. Additionally, combining sequence information would increase its resolving power in identification of foraged diet components.

Influence of nitrogen fertilization on diazotrophic communities in the rhizosphere of the Jerusalem artichoke (Helianthus tuberosus L.)

Diazotrophs in the soil may be influenced by plant factors as well as nitrogen (N) fertilization. In this study, we investigated potential diazotrophic communities in the rhizosphere of the Jerusalem artichoke (Helianthus tuberosus L.) supplied with differing amounts of N. The community structure of N2-fixing bacteria was profiled using the length heterogeneity polymerase chain reaction (LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP) based on a variation in the nifH gene. Higher numbers of diazotrophs were detected by T-RFLP compared to LH-PCR. The lowest number of N2-fixing bacteria was observed in the rhizosphere soil with high N fertilization. T-RFLP was a better method than LH-PCR for profiling microbial diversity of diazotrophs using multidimensional scaling (MDS) and analysis of similarity (ANOSIM) of fingerprints as well as diversity measures. The supply of N fertilizer appeared to negatively influence the abundance of diazotrophs in the rhizophere of the Jerusalem artichoke.

Carbon quantity defines productivity while its quality defines community composition of bacterioplankton in subarctic ponds

Bacterial communities in 16 oligotrophic ponds in Kilpisjarvi, subarctic Finland, were studied to test the hypothesis that dissolved organic carbon (DOC) quantity and quality differently influence bacterioplankton. The ponds were located below and above treeline at 600 m a.s.l., with 2–4 fold higher concentration of DOC below treeline. The concentration of DOC changed during the open-water season with highest values measured in mid-summer. Bacterial production, abundance, biomass were highest in mid-summer and correlated positively with the concentration of DOC. Quality indices of DOC showed that spring differed from the rest of the season. Highest specific UV-absorbance (SUVA) and humification index (HI), ratio a250/a265 and lowest fluorescence index (FI) were found during spring compared to summer and autumn, possibly indicating higher relative importance of allochthonous carbon during spring and a seasonal effect of photo-oxidation. According to Length Heterogeneity Polymerase Chain Reaction (LH-PCR) analyses, bacterial communities in spring were significantly different from those later in the season, possible due to the introduction of terrestrial bacteria associated with higher molecular weight material in spring DOC. Comparison between ponds situated above and below treeline revealed that bacteria were more abundant and productive at lower altitudes, which is probably connected to higher concentrations of DOC. The results also suggest that increased temperature and precipitation induced by global change and consequent higher allochthonous DOC runoff from the catchment could have a strong impact on biomass, productivity and community composition of micro-organisms in subarctic ponds and lakes.

Response of a summertime Antarctic marine ­bacterial community to glucose and ammonium enrichment

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 64:205-220 (2011) - DOI: https://doi.org/10.3354/ame01519 Response of a summertime Antarctic marine ­bacterial community to glucose and ammonium enrichment Hugh W. Ducklow1,*, Kristen M. S. Myers1, Matthew Erickson1, Jean-François Ghiglione2,3, Alison E. Murray4 1Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA 2CNRS, UMR 7621, Laboratoire d’Océanographie Biologique de Banyuls, BP44, 66650 Banyuls-sur-Mer cedex, France 3UPMC Univ Paris 06, UMR 7621, Laboratoire ARAGO, Avenue Fontaulé, BP44, 66650 Banyuls-sur-Mer, France 4Desert Research Institute, Reno, Nevada 89512, USA *Email: hducklow@mbl.edu ABSTRACT: Along the western Antarctic Peninsula, marine bacterioplankton respond to the spring phytoplankton bloom with increases in abundance, production and growth rates, and a seasonal succession in bacterial community composition (BCC). We investigated the response of the bacterial community to experimental additions of glucose and ammonium, alone or in combination, incubated in replicate carboys (each: 50 l) over 10 d in November 2006. Changes in bulk properties (abundance, production rates) in the incubations resembled observations in the nearshore environment over 8 seasons (2001 to 2002 through 2008 to 2009) at Palmer Stn (64.8°S, 64.1°W). Changes in bulk properties and BCC in ammonium-amended carboys were small relative to controls, compared to the glucose-amended treatments. The BCC in Day 0 and Day 10 controls and ammonium treatments were >72% similar when assessed by denaturing-gradient gel electrophoresis (DGGE), length heterogeneity polymerase chain reaction (LH-PCR) and capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) fingerprinting techniques. Bacterial abundance increased 2- to 10-fold and leucine incorporation rates increased 2- to 30-fold in the glucose treatments over 6 d. The BCC in carboys receiving glucose (with or without ammonium) remained >60% similar to that in Day 0 controls at 6 d and evolved to <20% similar to that in Day 0 controls after 10 d incubation. The increases in bacterial production rates, and the changes in BCC, suggest that selection for glucose-utilizing bacteria was slow under the ambient environmental conditions. The results suggest that organic carbon enrichment is a major factor influencing the observed winter-to-summer increase in bacterial abundance and activity. In contrast, the BCC was relatively robust, changing little until after repeated additions of glucose and prolonged (~10 d) incubation. KEY WORDS: Antarctica · Bacterial community composition · Bioassay · Marine bacterioplankton Full text in pdf format NextCite this article as: Ducklow HW, Myers KMS, Erickson M, Ghiglione JF, Murray AE (2011) Response of a summertime Antarctic marine ­bacterial community to glucose and ammonium enrichment. Aquat Microb Ecol 64:205-220. https://doi.org/10.3354/ame01519 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 64, No. 3. Online publication date: September 20, 2011 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2011 Inter-Research.

Bacterial diversity in the polluted water of the Dianchi Lakeshore in China

Dianchi Lake is a typical Chinese eutrophic lake. The bacterial community in the polluted water of the Dianchi lakeshore was investigated by cultivation-independent approaches. The amplicon length heterogeneity polymerase-chain reaction (LH-PCR) was used to detect the major differences in bacterial structure among the nine different sampling sites. Cluster analysis shows that the bacterial communities in water blooming sites were similar. Three genes were employed to characterize bacteria in the two parts of Dianchi Lake. The 16S rRNA gene was used to analyze the total bacterial community, the ammonia monooxygenase (amoA) gene for detecting ammonia-oxidizing bacteria, and the nitrous oxide reductase (nosZ) gene for identifying denitrifying bacteria. The clone library results demonstrate that Proteobacteria, Bacteroidetes, and Cyanobacteria were the dominant bacteria in both parts of the lake. The communities of ammonia-oxidizing bacteria were very different in the two parts of the lake and belonged to Nitrospira in the north and Nitrosomonas in the south part, respectively. Denitrifying bacteria in the Dianchi lakeshore were related to several cultured denitrifiers such as Pseudomonas, Paracoccus, Achromobacter, and Rubrivivax.

Dynamics of bacterial community exposed to hydrocarbons and oleophilic fertilizer in high-Arctic intertidal beach

Exposure of pristine microbial environments to hydrocarbon contamination stimulates growth of the initially small fraction of indigenous hydrocarbon-degrading bacteria. Custom-made oleophilic fertilizers have been demonstrated to promote oil bioremediation by boosting this proliferation. In the present study, the temporal dynamics of the bacterial community structure and the individual influences of hydrocarbons and an oleophilic fertilizer in shaping the community structure was explored during a 78 days bioremediation experiment in a high-Arctic intertidal beach environment. A combination of cultivation-independent 16S rRNA gene length-heterogeneity polymerase chain reaction (LH-PCR) profiling and identification of hydrocarbon-degrading isolates based on partial 16S rRNA gene sequences was employed. LH-PCR community profiles in the fertilizer alone and fertilized kerosene plots were largely indistinguishable throughout the experimental period, while kerosene alone plots showed a markedly different composition of dominant groups. This pointed to the fertilizer as the more decisive factor in shaping the community structure. Most prominent LH-PCR fragments which emerged after kerosene or fertilizer addition could be provisionally assigned to bacterial taxa through coinciding LH-PCR fragment lengths with hydrocarbon-degrading isolates obtained from the same type of experimental units. However, a few quantitatively significant LH-PCR groups had no counterparts among the cultivated bacteria. One of these was affiliated to a hitherto unspeciated subgroup within the Alkanindiges/Acinetobacter clade of Moraxellaceae by a 16S rRNA gene cloning approach.

Removal of pyridine and quinoline by bio-zeolite composed of mixed degrading bacteria and modified zeolite

In the process of the biodegradation of pyridine and quinoline, ammonium is often generated because of the transformation of N from pyridine and quinoline. Zeolite has been proven to be an effective sorbent for the removal of the ammonium. The natural zeolite can be modified to be the macroporous carrier in the biological wastewater treatment process. In this study, a specific bio-zeolite composed of mixed bacteria (a pyridine-degrading bacterium and a quinoline-degrading bacterium) and modified zeolite was used for biodegradation and adsorption in two types of wastewater: sterile synthetic and coking wastewater. The experimental results indicated that pyridine and quinoline could be degraded simultaneously by the mixed bacteria. Furthermore, NH 4 +-N transformed from pyridine and quinoline could be removed by the modified zeolite. In addition, the bacterial community structures of the coking wastewater and the bio-zeolite were monitored by the amplicon length heterogeneity polymerase-chain reaction (LH-PCR) technique. Both LH-PCR results and scanning electron microscope (SEM) observations indicated that the microorganisms, including BW001 and BW003, could be easily attached on the surface of the modified zeolite and that the bio-zeolite could be used in the treatment of wastewater containing pyridine and/or quinoline.

Bioaugmentation treatment for coking wastewater containing pyridine and quinoline in a sequencing batch reactor

Two pyridine-degrading bacteria and two quinoline-degrading bacteria were introduced for bioaugmentation to treat the coking wastewater. Sequencing batch reactors (SBRs) were used for a comparative study on the treatment efficiency of pyridine, quinoline, and chemical oxygen demand. Results showed that the treatment efficiency with coking-activated sludge plus a mixture of the four degrading bacteria was much better than that ones with coking-activated sludge only or mixed degrading bacteria only. Moreover, a 52-day continuous operation of the bioaugmented and general SBRs was investigated. The bioaugmented SBR showed better treatment efficiency and stronger capacity to treat high pyridine and quinoline shock loading. The general SBR failed to cope with the shock loading, and the biomass of the activated sludge decreased significantly. In order to monitor the microbial ecological variation during the long-term treatment, the bacterial community in both reactors was monitored by the amplicon length heterogeneity polymerase chain reaction technique. The diversity of the bacterial community decreased in both reactors, but the introduced highly efficient bacteria were dominant in the bioaugmented SBR. Our experiment showed clearly that the use of highly efficient bacteria in SBR process could be a feasible method to treat wastewater containing pyridine or/and quinoline.

Quantitative assessment of the human gut microbiome using multitag pyrosequencing.

Recent advances in molecular techniques have now made it possible to interrogate the human microbiome in depth to better understand the interactions with the host organism and its role in diseases. We now report the utility of Length Heterogeneity Polymerase Chain Reaction (LH-PCR) to survey samples and a proprietary Multitagged Pyrosequencing (MTPS) methodology to interrogate the gut microbiome in healthy and disease states. We present an overview of our studies demonstrating the application of these molecular-biology techniques to an example disease state such as Inflammatory Bowel Disease (IBD). The findings show that there is a core mucosal bacterial microbiome (i.e., a mucosal biofilm) that is distinct from the luminal microbiome in health, and that the mucosal microbiome appears to be dysbiotic in IBD. We propose that the mucosal microbiome forms a synergistic and stable interaction with the host immune system, while the lumen microbiome varies based on diet or other environmental factors. We define this composite ecosystem of the human microbiome and human host as the Human Metabiome.

Combination of 16S rRNA variable regions provides a detailed analysis of bacterial community dynamics in the lungs of cystic fibrosis patients

Chronic bronchopulmonary bacterial infections remain the most common cause of morbidity and mortality among patients with cystic fibrosis (CF). Recent community sequencing work has now shown that the bacterial community in the CF lung is polymicrobial. Identifying bacteria in the CF lung through sequencing can be costly and is not practical for many laboratories. Molecular techniques such as terminal restriction fragment length polymorphism or amplicon length heterogeneity-polymerase chain reaction (LH-PCR) can provide many laboratories with the ability to study CF bacterial communities without costly sequencing. The aim of this study was to determine if the use of LH-PCR with multiple hypervariable regions of the 16S rRNA gene could be used to identify organisms found in sputum DNA. This work also determined if LH-PCR could be used to observe the dynamics of lung infections over a period of time. Nineteen samples were analysed with the V1 and the V1_V2 region of the 16S rRNA gene. Based on the amplicon size present in the V1_V2 region, Pseudomonas aeruginosa was confirmed to be in all 19 samples obtained from the patients. The V1 region provided a higher power of discrimination between bacterial profiles of patients. Both regions were able to identify trends in the bacterial population over a period of time. LH profiles showed that the CF lung community is dynamic and that changes in the community may in part be driven by the patient's antibiotic treatment. LH-PCR is a tool that is well suited for studying bacterial communities and their dynamics.

Natural whey starter for Parmigiano Reggiano: culture-independent approach

The aim of this work was to obtain a deeper insight into the knowledge of microbial composition of Parmigiano Reggiano natural whey starters through different culture-independent methods. Eighteen different Parmigiano Reggiano natural whey starters sampled from three different provinces of this cheese production area and the nonacidified wheys from which they arose have been studied by length heterogeneity polymerase chain reaction (LH-PCR) and fluorescent in situ hybridization (FISH). A high microbial composition variability between different samples has been observed. Revealing different images of the same community, LH-PCR and FISH have given a more accurate view of the not well-known Parmigiano Reggiano whey starter ecosystem. New lights have been shed on Parmigiano Reggiano natural whey starters microbial composition, highlighting how culture-independent approach could be used and improved to study this and other food ecosystems.

Soil bacterial community structure and physicochemical properties in mitigation wetlands created in the Piedmont region of Virginia (USA)

Wetland creation is a common practice for compensatory mitigation in the United States. Vegetation attributes have been used as a quick measure of mitigation success in most post-creation monitoring, while little attention has been paid to soils that provide the substrate for flora and fauna to establish and develop. Created wetland soils are often found not indicative of ‘hydric soil’ with a lack of development of physicochemical properties (i.e., bulk density, moisture content, and carbon and nitrogen contents) comparable to those in natural wetlands. Moreover, soil bacterial communities are rarely examined though they are integrally involved in biogeochemical functions that are critical for ecosystem development in created wetlands. We analyzed soil physicochemistry and profiled soil bacterial community structure using amplicon length heterogeneity polymerase chain reaction (LH-PCR) of 16S ribosomal DNA in three relatively young wetlands (<10 years old) created in the Piedmont region of Virginia. We examined the data by site and by specific conditions of each site (i.e., induced microtopography and hydrologic regime). Multidimensional scaling (MDS) and analysis of similarity (ANOSIM) showed clear clustering and significant differences both in soil physicochemistry (Global R = 0.70, p = 0.001) and in soil bacterial community profiles (Global R = 0. 77, p = 0.001) between sites. Soil physicochemistry (Global R = 1, p = 0.005) and bacterial community structure (Global R = 0.79, p = 0.005) of soils significantly differed by hydrologic regime within a wetland, but not by microtopography treatment. A significant association was found between physicochemistry and bacterial community structure in wetland soils, revealing a close link between two attributes ( ρ = 0.39, p = 0.002) . C/N (carbon to nitrogen) ratio was the best predictor of soil bacterial community patterns ( ρ = 0.56, p = 0.001). The diversity of soil bacterial community (Shannon's H′) differed between sites with a slightly higher diversity observed in a relatively older created wetland, and seemed also fairly determined by hydrologic regime of a site, with a relatively dry site being more diverse.

Simultaneous biodegradation of pyridine and quinoline by two mixed bacterial strains

Experiments were conducted to provide data on the effectiveness of bioaugmentation in the removal of pyridine and quinoline from different wastewaters. A pyridine-degrading bacterial strain (Paracoccus sp. BW001) and a quinoline-degrading strain (Pseudomonas sp. BW003) were isolated from the activated sludge of a coking wastewater treatment plant. In this study, a consortium of these two bacterial strains was used as inoculum to simultaneously degrade pyridine and quinoline in three types of wastewaters: sterile synthetic, domestic, and industrial. In addition, variation of the bacterial community structures during degradation was monitored by denaturing gradient gel electrophoresis and amplicon length heterogeneity polymerase chain reaction techniques. The results of our experiments indicate that pyridine and quinoline can be removed efficiently using this inoculum but that the degradation process results in the production of ammonium as a by-product. Also, in the two actual wastewaters investigated, we observed that several autochthonous strains of bacteria in both the domestic and industrial wastewater were tolerant of pyridine and quinoline and grew rapidly.

Bacterial biofilm formation, pathogenicity, diagnostics and control: An overview

Bacterial biofilms are complex, mono- or poly-microbialn communities adhering to biotic or abiotic surfaces. This adaptation has been implicated as a survival strategy. The formation of biofilms is mediated by mechanical, biochemical and genetical factors. The biofilms enhance the virulence of the pathogen and have their potential role in various infections, such as dental caries, cystic fibrosis, osteonecrosis, urinary tract infection and eye infections. A number of diagnostic techniques, viz., bright-field microscopy, epifluorescence microscopy, scanning electron microscopy, confocal laser scanning microscopy and amplicon length heterogeneity polymerase chain reaction, have been employed for detection of these communities. Researchers have worked on applications of catheter lock solutions, a fish protein coating, acid shock treatment, susceptibility to bacteriophages, etc., for biofilm control. However, we need to rearrange our strategies to have thorough insight and concentrate on priority basis to develop new accurate, precise and rapid diagnostic protocols for detection and evaluation of biofilm. Above all, the strict compliance to these techniques is required for accurate diagnosis and control.

The response of microbial communities to diverse organic matter sources in the Arctic Ocean

Abstract The response of Arctic microbial communities to a variety of natural organic matter substrates, including peat, ice algae and ice-rafted debris was examined using bacterial regrowth experiments and compared to unamended controls. Bacterial growth and production were followed together with the phylogenetic composition using length-heterogeneity polymerase chain reaction (LH-PCR), and 16S rRNA gene cloning and sequencing. Intact phospholipids (IPLs) and fatty acids evaluated the relationship between lipids and bacterial community structure and the impact of varied organic substrates on microbial lipid synthesis. Differential responses to organic matter sources were observed, with ice algae supporting both higher bacterial growth and production than terrestrial-derived peat. In spite of disparate growth kinetics, the community composition remained similar in all amended incubations as was confirmed by automated ribosomal intergenic spacer analysis (ARISA). Gammaproteobacteria dominated the initial incubations, whereas in extended incubations with terrestrial peat Alphaproteobacteria dominated; in particular Sulfitobacter phylotypes closely related (>99%) to an Arctic sea-ice-associated member of the Roseobacter clade (ARK10278). Arctic bacterioplankton preferentially synthesized two phospholipids, phosphatidylethanolamine (PE) and phosphatidylglygerol (PG), with 18:0n, 18:1Δ11, 16:0n and 16:1Δ9 as the primary fatty acids. Overall, results show that organic matter source plays an important role in structuring bacterioplankton community composition, with similar IPL and fatty acid lipid distributions observed among phylogenetically distinct bacteria.

Comparison of the Diversity of the Vaginal Microbiota in HIV‐Infected and HIV‐Uninfected Women with or without Bacterial Vaginosis

Whether human immunodeficiency virus (HIV) infection is associated with a change in the diversity of genital microbiota in women was investigated. Amplicon length heterogeneity polymerase chain reaction (LH-PCR) analysis and pyrosequencing of the 16S ribosomal RNA gene were used to analyze the diversity of the microbiota in HIV-positive (HIV(+)) and HIV-negative (HIV(-)) women with or without bacterial vaginosis (BV). LH-PCR analysis revealed significantly more microbiota diversity in BV-positive (BV(+)) women than in BV-negative (BV(-)) women, but no significant difference was noted between HIV(+) women and HIV(-) women. Pyrosequencing revealed that Lactobacillus organisms constituted a median of 96% of the bacteria in BV(-) women. BV(+) women had a significantly higher number of taxa found at > or =1% of the total genital microbiota (median, 11 taxa). Common taxa in BV(+) women were Prevotella, Megasphaera, Gardnerella, Coriobacterineae, Lachnospira, and Sneathia. There was a trend (P = .07) toward the presence of a higher number of taxa in HIV(+)BV(+) women than in HIV(-)BV(+) women. Propionibacterineae, Citrobacter, and Anaerococcus were the taxa found only in HIV(+) women (P < .05). The present study demonstrated that both LH-PCR analysis and pyrosequencing differentiated microbiota in BV(+) women from that in BV(-) women and that pyrosequencing indicated a trend toward increased diversity in BV(+)HIV(+) women, suggesting that HIV infection is associated with changes in the diversity of genital microbiota.

An assessment of soil bacterial community structure and physicochemistry in two microtopographic locations of a palustrine forested wetland

We studied redoximorphic features, field indicators and bacterial communities of soils in hummocks and hollows of a palustrine forested wetland in Virginia. We hypothesized that presence of hydric soils, soil physicochemistry and soil bacterial community structure would differ between hummocks and hollows. We fingerprinted soils collected from different microtopographic locations using Length Heterogeneity Polymerase Chain Reaction (LH-PCR) to study their bacterial community structures. Two hummocks had silty/sandy loam soils with mean chroma values of > 4, showing no indication of ‘hydric soils’ (i.e., wetland soils). Two hollows, however, had clay loam soils with mean chroma values of 2 with gleying and redox concentrations observed, indicative of seasonally inundated wetlands. The soils of hollows also had higher organic matter content and soil moisture compared to the soils of hummocks (P < 0.05). Multidimensional scaling (MDS) and Analysis of similarity (ANOSIM) of the fingerprints revealed differences in soil microbial community structures between hummocks and hollows (Global R = 0.30, P < 0.01). The diversity measures of the fingerprints (Shannon’s H′) were also different by microtopography with higher diversity in hollows relative to hummocks (P < 0.05). LH-PCR proves to be a useful tool in examining bacterial community composition of wetland soils in this study. However, cloning and sequencing of specific community LH-PCR profiles of interest is necessary to fully characterize the community down to genus/species level. With species identities we should be able to not only better explain differences observed in the community profiles, but study their relations to hydrologic and/or physicochemical conditions of wetlands.