Holmes DE, Nevin KP, Lovley DR.. In situ expression of Geobacteraceae nifD in subsurface sediments. Appl Environ Microbiol 70: 7251-7259

Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA.
Applied and Environmental Microbiology (Impact Factor: 3.67). 01/2005; 70(12):7251-9. DOI: 10.1128/AEM.70.12.7251-7259.2004
Source: PubMed


In order to determine whether the metabolic state of Geobacteraceae involved in bioremediation of subsurface sediments might be inferred from levels of mRNA for key genes, in situ expression
of nifD, a highly conserved gene involved in nitrogen fixation, was investigated. When Geobacter sulfurreducens was grown without a source of fixed nitrogen in chemostats with acetate provided as the limiting electron donor and Fe(III)
as the electron acceptor, levels of nifD transcripts were 4 to 5 orders of magnitude higher than in chemostat cultures provided with ammonium. In contrast, the number
of transcripts of recA and the 16S rRNA gene were slightly lower in the absence of ammonium. The addition of acetate to organic- and nitrogen-poor
subsurface sediments stimulated the growth of Geobacteraceae and Fe(III) reduction, as well as the expression of nifD in Geobacteraceae. Levels of nifD transcripts in Geobacteraceae decreased more than 100-fold within 2 days after the addition of 100 μM ammonium, while levels of recA and total bacterial 16S rRNA in Geobacteraceae remained relatively constant. Ammonium amendments had no effect on rates of Fe(III) reduction in acetate-amended sediments
or toluene degradation in petroleum-contaminated sediments, suggesting that other factors, such as the rate that Geobacteraceae could access Fe(III) oxides, limited Fe(III) reduction. These results demonstrate that it is possible to monitor one aspect
of the in situ metabolic state of Geobacteraceae species in subsurface sediments via analysis of mRNA levels, which is the first step toward a more global analysis of in
situ gene expression related to nutrient status and stress response during bioremediation by Geobacteraceae.

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    • "Reverse transcription (RT) was performed with the Enhanced Avian First Strand synthesis kit (Sigma–Aldrich Co., St Louis, MO, USA) using random nonamers as described previously (Holmes et al., 2004). Two negative controls lacking reverse transcriptase or RNA were included. "
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    ABSTRACT: Microbial processes such as nitrification and anaerobic ammonium oxidation (anammox) are important for nitrogen cycling in marine sediments. Seasonal variations of archaeal and bacterial ammonia oxidizers (AOA and AOB) and anammox bacteria, as well as the environmental factors affecting these groups, are not well studied. We have examined the seasonal and depth distribution of the abundance and potential activity of these microbial groups in coastal marine sediments of the southern North Sea. This was achieved by quantifying specific intact polar lipids as well as the abundance and gene expression of their 16S rRNA gene, the ammonia monooxygenase subunit A (amoA) gene of AOA and AOB, and the hydrazine synthase (hzsA) gene of anammox bacteria. AOA, AOB, and anammox bacteria were detected and transcriptionally active down to 12 cm sediment depth. In all seasons, the abundance of AOA was higher compared to the AOB abundance suggesting that AOA play a more dominant role in aerobic ammonia oxidation in these sediments. Anammox bacteria were abundant and active even in oxygenated and bioturbated parts of the sediment. The abundance of AOA and AOB was relatively stable with depth and over the seasonal cycle, while anammox bacteria abundance and transcriptional activity were highest in August. North Sea sediments thus seem to provide a common, stable, ecological niche for AOA, AOB, and anammox bacteria. Keywords: Thaumarchaeota, anammox bacteria, ammonia oxidizing bacteria (AOB), ammonia oxidizing Archaea (AOA), amoA gene, hzsA gene, intact polar lipids (IPLs) INTRODUCTION
    Frontiers in Microbiology 09/2014; DOI:10.3389/fmicb.2014.00472 · 3.99 Impact Factor
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    • "Understanding in situ physiological status is key to bioremediation and other environmental biotechnological applications, in order to monitor and rationally engineer the activities of Geobacter species. Methods to quantify key gene transcripts and proteins, and relating their abundances to growth rates and metabolic rates, oxidative stress, acetate availability, and limitations in iron, ammonium, and phosphate, have been developed (Chin et al. 2004; Elifantz et al. 2010; Holmes et al. 2004b, 2008; Mouser et al. 2009; N'Guessan et al. 2010; O'Neil et al. 2008). The development of these indicators of in situ physiological status took optimally advantage of the availability of pure Geobacter isolates that are closely related to those in metal-reducing environments and the ability to grow these Geobacter species in chemostats under environmental relevant conditions. "
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    ABSTRACT: Geobacteraceae, a family within the order Desulfuromonadales, is in this chapter described as consisting of the genus Geobacter and the sole species Pelobacter propionicus. The genus Geobacter comprises anaerobic, non-fermenting chemoorganotrophic mesophiles. Their hallmark feature is the ability to reduce insoluble Fe(III) and Mn(IV) for which they can employ several mechanisms, of which the most notably is extracellular electron transfer via electric conductive nanowires. Geobacter species are physiological versatile. They all oxidize acetate and mainly use small organic acids and alcohols. Several species also oxidize monoaromatic hydrocarbons, such as toluene and benzene. Next to Fe(III), a range of other electron acceptors can be used, and Geobacter species can transfer electron directly to other microbial species or graphite electrodes. Sulfur is often respired, and some species respire organohalides. P. propionicus is phylogenetically located within the Geobacter clade, but several of its physiological characteristics are distinct from Geobacter properties: it can ferment but does not utilize acetate as an electron donor nor oxidizes organic compounds completely. Furthermore, it does not contain the c-type cytochromes that are involved in electron transfer to Fe(III) in Geobacter species. Members of the genus Geobacter often dominate in iron-reducing settings, in particular in environments that have been subject to anthropogenic influences. They represent a rare case of environmental dominant species that are relatively easy to enrich and isolate. Genome-scale metabolic models have strongly contributed to understanding their physiology and ecology. The physiological characteristics of Geobacter species are employed in environmental biotechnology, such as the natural attenuation of organic matter, bioremediation of aromatic hydrocarbons, heavy metals and organohalides, and generating bioenergy in microbial fuel cells and microbial electrolysis cells.
    The Prokaryotes, 01/2014: pages 157-172; , ISBN: 978-3-642-39043-2
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    • "Potential DNA contamination was checked by PCR using RNA as a template. 2.7 Reverse transcription (RT)-PCR Reverse transcription (RT) was performed with an Enhanced Avian First Strand synthesis kit (Sigma-Aldrich Co., St Louis, MO, USA) as described previously (Holmes et al., 2004 "
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    ABSTRACT: We have examined the spatial and seasonal dis- tribution of Thaumarchaeota in the water column and sedi- ment of the southern North Sea using the specific intact polar lipid (IPL) hexose-phosphohexose (HPH) crenarchaeol, as well as thaumarchaeotal 16S rRNA gene abundances and ex- pression. In the water column, a higher abundance of Thau- marchaeota was observed in the winter season than in the summer, which is in agreement with previous studies, but this was not the case in the sediment where Thaumarchaeota were most abundant in spring and summer. This observation corresponds well with the idea that ammonia availability is a key factor in thaumarchaeotal niche determination. In the surface waters of the southern North Sea, we observed a spa- tial variability in HPH crenarchaeol, thaumarchaeotal 16S rRNA gene abundance and transcriptional activity that cor- responded well with the different water masses present. In bottom waters, a clear differentiation based on water masses was not observed; instead, we suggest that observed differ- ences in thaumarchaeotal abundance with depth may be re- lated to resuspension from the sediment. This could be due to suspension of benthic Thaumarchaeota to the water column or due to delivery of e.g. resuspended sediment or ammo- nium to the water column, which could be utilized by pelagic Thaumarchaeota. This study has shown that the seasonality of Thaumarchaeota in water and sediment is different and highlights the importance of water masses, currents and sed- imentary processes in determining the spatial abundance of Thaumarchaeota in the southern North Sea.
    Biogeosciences 11/2013; 10:7195– 7206. DOI:10.5194/bg-10-7195-2013 · 3.98 Impact Factor
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