TY  - JOUR
AB  - Methanobactins (MBs) are ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The post-translational modification that define MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b, mbnA, which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC, is found in all MB operons, and in conjunction with mbnB, is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC, a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the ΔmbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry and solution NMR spectroscopy. MB-OB3b from ΔmbnC was missing the C-terminal Met and also found to contain a Pro and a Cys in place of the pyrrolidiny-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. IMPORTANCE A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals, nanoparticle formation, as well as for the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined. The discovery that mbnC is involved in the formation of the C-terminal oxazolone group with associated thioamide but not for the formation of the N-terminal oxazolone group with associated thioamide in M. trichosporium OB3b suggests the enzymes responsible for post-translational modification(s) of the two oxazolone groups are not identical.
AU  - Dershwitz, P.*
AU  - Gu, W.*
AU  - Roche, J.*
AU  - Kang-Yun, C.S.*
AU  - Semrau, J.D.*
AU  - Bobik, T.A.*
AU  - Fulton, B.*
AU  - Zischka, H.
AU  - DiSpirito, A.A.*
C1  - 63418
C2  - 51525
CY  - 1752 N St Nw, Washington, Dc 20036-2904 Usa
TI  - MbnC is not required for the formation of the N-terminal oxazolone in the methanobactin from Methylosinus trichosporium OB3b.
JO  - Appl. Environ. Microbiol.
VL  - 88
IS  - 2
PB  - Amer Soc Microbiology
PY  - 2022
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Methanobactins (MBs) are small (<1,300 Da) post-translationally modified copper-binding peptides and represent the extracellular component of a copper acquisition system in some methanotrophs. Interestingly, MBs can bind a range of metal ions, with some reduced after binding, e.g., Cu2+ reduced to Cu+ Other metal ions, however, are bound but not reduced, e.g., K+ The source of electrons for selective metal ion reduction has been speculated to be water but never empirically shown. Here, using H218O, we show that when MB from Methylocystis sp strain SB2 (MB-SB2) and Methylosinus trichosporium OB3b (MB-OB3) were incubated in the presence of either Au3+, Cu2, and Ag+, 18,18O2 and free protons were released. No 18,18O2 production was observed either in presence of MB-SB2 or MB-OB3b alone, gold alone, copper alone, silver alone or when K+ or Mo2+ was incubated with MB-SB2.In contrast to MB-OB3b, MB-SB2 binds Fe3+ with an N2S2 coordination and will also reduce Fe3+ to Fe2+ Iron reduction was also found to be coupled to oxidation of 2H2O and generation of O2 MB-SB2 will also couple Hg2+, Ni2+ and Co2+ reduction to the oxidation of 2H2O and generation of O2, but MB-OB3b will not, ostensibly as MB-OB3b binds but does not reduce these metal ions.To determine if the O2 generated during metal ion reduction by MB could be coupled to methane oxidation, 13CH4 oxidation by Methylosinus trichosporium OB3b was monitored under anoxic conditions. The results demonstrate O2 generation from metal ion reduction by MB-OB3b can support methane oxidation.IMPORTANCEThe discovery that MB will couple the oxidation of H2O to metal ion reduction and the release of O2 suggests that methanotrophs expressing MB may be able to maintain their activity in hypoxic/anoxic conditions through "self-generation" of dioxygen required for the initial oxidation of methane to methanol. Such an ability may be an important factor in enabling methanotrophs to not only colonize the oxic-anoxic interface where methane concentrations are highest, but also tolerate significant temporal fluctuations of this interface. Given that genomic surveys often show evidence of aerobic methanotrophs within anoxic zones, the ability to express MB (and thereby generate dioxygen) may be an important parameter in facilitating their ability to remove methane, a potent greenhouse gas, before it enters the atmosphere.
AU  - Dershwitz, P.*
AU  - Bandow, N.L.*
AU  - Yang, J.*
AU  - Semrau, J.D.*
AU  - McEllistrem, M.T.*
AU  - Heinze, R.A.*
AU  - Fonseca, M.*
AU  - Ledesma, J.C.*
AU  - Jennett, J.R.*
AU  - DiSpirito, A.M.*
AU  - Athwal, N.S.*
AU  - Hargrove, M.S.*
AU  - Bobik, T.A.*
AU  - Zischka, H.
AU  - DiSpirito, A.A.*
C1  - 61926
C2  - 50509
CY  - 1752 N St Nw, Washington, Dc 20036-2904 Usa
TI  - Oxygen generation via water splitting by a novel biogenic metal ion binding compound.
JO  - Appl. Environ. Microbiol.
VL  - 87
IS  - 14
PB  - Amer Soc Microbiology
PY  - 2021
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Caries development is associated with shifts in the oral biofilm microbiota and primarily linked to frequent simple carbohydrate consumption. Different nutritional ingredients can either promote or prevent caries development. To investigate the effects of selected ingredients on the oral biofilm microbiota in situ, 11 study participants underwent 3-month-long dietary phases with intake of a regular diet (PI), additional frequent sucrose (PII), milk and yoghurt (PIII), and a diet rich in dietary fiber (PIV) and then returned to their regular diet (PV). Oral biofilm was sampled and analyzed applying 16S rRNA Illumina MiSeq sequencing. Additionally, the effect on the enamel was analyzed by measuring enamel surface roughness with laser scanning microscopy. The beta-diversity results showed that the microbiota in all the following phases differed significantly from PI and that the microbial community in PII was significantly different from all other phases. The abundance of the genus Streptococcus fluctuated over the course of the five phases, with a significant increase in PII (P = 0.01), decreasing in PIII and PIV (PIII and PIV versus PII: P < 0.00001) and increasing again toward PV. Other taxa showed various fluctuations of their abundances, with PV returning approximately to the levels of PI. In conclusion, while elevated sucrose consumption favored caries-promoting non-mutans streptococci, frequent milk and yoghurt intake caused a significant decrease in the abundance of these microbial taxa and in addition reduced enamel surface roughness. These results indicate that modulations of the oral biofilm microbiota can be attained even in adults through dietary changes and corresponding recommendations can be made for the prevention of caries development.IMPORTANCE Caries affects a large proportion of the population worldwide, resulting in high treatment costs. Its etiology can be ascribed to shifts of the microbiota in dental biofilms primarily driven by dietary factors. It is unclear how diet affects the microbial community of plaque biofilm in situ and whether it can be modulated to help prevent caries development. To address these issues, we analyzed changes of the in situ plaque microbiota following 3-month-long dietary changes involving elevated sucrose, dairy, and dietary fiber consumption over a period of 15 months. Applying high-throughput sequencing, we found non-mutans streptococci, a taxonomic group involved in the beginning stages toward microbial dysbiosis, in decreased abundance with elevated dairy and dietary fiber intake. Through analysis of the enamel surface roughness, these effects were confirmed. Therefore, correspondent dietary measures can be recommended for children as well as adults for caries prevention.
AU  - Anderson, A.C.*
AU  - Rothballer, M.
AU  - Altenburger, M.J.*
AU  - Woelber, J.P.*
AU  - Karygianni, L.*
AU  - Vach, K.*
AU  - Hellwig, E.*
AU  - Al-Ahmad, A.*
C1  - 60522
C2  - 49336
CY  - 1752 N St Nw, Washington, Dc 20036-2904 Usa
TI  - Long-term fluctuation of oral biofilm microbiota following different dietary phases.
JO  - Appl. Environ. Microbiol.
VL  - 86
IS  - 20
PB  - Amer Soc Microbiology
PY  - 2020
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Anaerobic degradation of polycyclic aromatic hydrocarbons has been investigated mostly with naphthalene as a model compound. Naphthalene degradation by sulfate-reducing bacteria proceeds via carboxylation to 2-naphthoic acid, formation of a coenzyme A thioester, and subsequent reduction to 5,6,7,8-tetrahydro-2-naphthoylcoenzyme A (THNCoA), which is further reduced to hexahydro-2-naphthoyl-CoA (HHNCoA) by tetrahydronaphthoyl-CoA reductase (THNCoA reductase), an enzyme similar to class I benzoyl-CoA reductases. When analyzing THNCoA reductase assays with crude cell extracts and NADH as electron donor via liquid chromatography-mass spectrometry (LC-MS), scanning for putative metabolites, we found that small amounts of the product of an HHNCoA hydratase were formed in the assays, but the downstream conversion by an NAD(+)-dependent beta-hydroxyacyl-CoA dehydrogenase was prevented by the excess of NADH in those assays. Experiments with alternative electron donors indicated that 2-oxoglutarate can serve as an indirect electron donor for the THNCoA-reducing system via a 2-oxoglutarate:ferredoxin oxidoreductase. With 2-oxoglutarate as electron donor, THNCoA was completely converted and further metabolites resulting from subsequent beta-oxidation-like reactions and hydrolytic ring cleavage were detected. These metabolites indicate a downstream pathway with water addition to HHNCoA and ring fission via a hydrolase acting on a beta'-hydroxy-beta-oxo-decahydro-2-naphthoyl-CoA intermediate. Formation of the downstream intermediate cis-2-carboxycyclohexylacetyl-CoA, which is the substrate for the previously described lower degradation pathway leading to the central metabolism, completes the anaerobic degradation pathway of naphthalene.IMPORTANCE Anaerobic degradation of polycyclic aromatic hydrocarbons is poorly investigated despite its significance in anoxic sediments. Using alternative electron donors for the 5,6,7,8-tetrahydro-2-naphthoyl-CoA reductase reaction, we observed intermediary metabolites of anaerobic naphthalene degradation via in vitro enzyme assays with cell extracts of anaerobic naphthalene degraders. The identified metabolites provide evidence that ring reduction terminates at the stage of hexahydro-2-naphthoyl-CoA and a sequence of beta-oxidation-like degradation reactions starts with a hydratase acting on this intermediate. The final product of this reaction sequence was identified as cis-2-carboxycyclohexylacetyl-CoA, a compound for which a further downstream degradation pathway has recently been published (P. Weyrauch, A. V. Zaytsev, S. Stephan, L. Kocks, et al., Environ Microbiol 19:2819-2830, 2017, https://doi .org/10.1111/1462-2920.13806). Our study reveals the first ring-cleaving reaction in the anaerobic naphthalene degradation pathway. It closes the gap between the reduction of the first ring of 2-naphthoyl-CoA by 2-napthoyl-CoA reductase and the lower degradation pathway starting from cis-2-carboxycyclohexylacetyl-CoA, where the second ring cleavage takes place.
AU  - Weyrauch, P.
AU  - Heker, I.*
AU  - Zaytsev, A.V.*
AU  - von Hagen, C.A.*
AU  - Arnold, M.E.*
AU  - Golding, B.T.*
AU  - Meckenstock, R.U.*
C1  - 59193
C2  - 48663
CY  - 1752 N St Nw, Washington, Dc 20036-2904 Usa
TI  - The 5,6,7,8-tetrahydro-2-naphthoyl-CoA reductase reaction in the anaerobic degradation of naphthalene and identification of downstream metabolites.
JO  - Appl. Environ. Microbiol.
VL  - 86
IS  - 15
PB  - Amer Soc Microbiology
PY  - 2020
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - The oxygenic dismutation of NO into N2 and O2 has recently been suggested for the anaerobic methanotrophic Candidatus Methylomirabilis oxyfera and the alkane-oxidizing gammaproteobacterium HdN1. It represents a new pathway in microbial nitrogen cycling and is catalyzed by a putative NO dismutase (Nod). The formed O2 enables microbes to employ aerobic catabolic pathways in anoxic habitats, suggesting an ecophysiological niche space of substantial appeal for bioremediation and water treatment. However, it is still unknown whether this physiology is limited to M. oxyfera and HdN1, and whether it can be coupled to the oxidation of electron donors other than alkanes. Here, we report first insights into an unexpected diversity and remarkable abundance of nod genes in natural and engineered water systems. Phylogenetically diverse nod genes were recovered from a range of contaminated aquifers and N-removing wastewater treatment systems. Together with nod genes from M. oxyfera and HdN1, the novel environmental nod sequences formed no less than 6 well-supported phylogenetic clusters, clearly distinct from canonical NO-reductase (qNor and cNor) genes. The abundance of nod genes in the investigated samples ranged from 1.6 * 107 to 5.2 * 1010 copies g-1 wet sediment or sludge biomass, accounting for up to 10% of total bacterial 16S rRNA gene counts. In essence, NO dismutation could be a much more widespread physiology than currently perceived. Understanding the controls of this emergent microbial capacity could offer new routes for nitrogen elimination or pollutant remediation in natural and engineered water systems.
AU  - Zhu, B.
AU  - Bradford, L.
AU  - Huang, S.
AU  - Szalay, A.A.
AU  - Leix, C.*
AU  - Weissbach, M.*
AU  - Táncsics, A.*
AU  - Drewes, J.E.*
AU  - Lueders, T.
C1  - 50528
C2  - 42317
CY  - Washington
TI  - Unexpected diversity and high abundance of putative nitric oxide dismutase (Nod) genes in contaminated aquifers and wastewater treatment systems.
JO  - Appl. Environ. Microbiol.
VL  - 83
IS  - 4
PB  - Amer Soc Microbiology
PY  - 2017
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Methanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding for the polypeptide precursor of methanobactin, mbnA, is part of a gene cluster that also includes several genes encoding for proteins of unknown function (but speculated to be involved in methanobactin formation), as well as mbnT, encoding for a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine if mbnT was truly responsible for methanobactin uptake, a knock-out was constructed in Methylosinus trichosporium OB3b using marker exchange mutagenesis. The resulting M. trichosporium mbnT::Gm(R) mutant was found to be able to produce methanobactin, but unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression of mmoX and pmoA, encoding for polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, indicating that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, was not affected in M. trichosporium OB3b wildtype, indicating that the TonB-dependent transporter encoded by mbnT is responsible for methanobactin uptake, and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When the mbnT::Gm(R) mutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from M. trichosporium OB3b wildtype, indicating that this methanotroph has multiple mechanisms for copper uptake.
AU  - Gu, W.*
AU  - Farhan Ul Haque, M.*
AU  - Baral, B.S.*
AU  - Turpin, E.A.*
AU  - Bandow, N.L.*
AU  - Kremmer, E.
AU  - Flatley, A.
AU  - Zischka, H.
AU  - DiSpirito, A.A.*
AU  - Semrau, J.D.*
C1  - 47700
C2  - 39565
CY  - Washington
SP  - 1917-1923
TI  - A TonB-dependent transporter is responsible for methanobactin uptake by Methylosinus trichosporium OB3b.
JO  - Appl. Environ. Microbiol.
VL  - 82
IS  - 6
PB  - Amer Soc Microbiology
PY  - 2016
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Newcastle disease (ND), caused by the virulent Newcastle disease virus (NDV), is one of the most important viral diseases of birds globally, but little is currently known regarding enzootic trends of NDV in Northeastern China, especially for class I viruses. Thus, we performed a surveillance study for NDV in Northeastern China from 2013 to 2015. A total 755 samples from wild and domestic birds in wetlands and live bird markets (LBMs) were collected and ten isolates of NDV were identified. Genetic and phylogenetic analyses showed that five isolates from LBMs belong to class I subgenotype 1b, two (one from wild birds and one from LBMs) belong to the vaccine-like class II genotype II, and three (all from wild birds) belong to class II subgenotype Ib. Interestingly, the five class I isolates had epidemiological connections with viruses from Southern, Eastern and Southeastern China. Our findings, together with recent prevalent trends of class I and virulent class II NDV in China, suggest possible virus transmission between wild and domestic birds and the potential for a NDV epidemic in the future.
AU  - Zhang, P.*
AU  - Xie, G.*
AU  - Liu, X.*
AU  - Ai, L.*
AU  - Chen, Y.*
AU  - Meng, X.*
AU  - Bi, Y.*
AU  - Chen, J.*
AU  - Sun, Y.*
AU  - Stöger, T.
AU  - Ding, Z.*
AU  - Yin, R.*
C1  - 47604
C2  - 39432
CY  - Washington
SP  - 1530-1536
TI  - High genetic diversity of Newcastle disease virus in wild and domestic birds in Northeastern China from 2013 to 2015 reveals potential epidemic trends.
JO  - Appl. Environ. Microbiol.
VL  - 82
IS  - 5
PB  - Amer Soc Microbiology
PY  - 2016
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Beech (Fagus sylvatica), a dominant forest species in Central Europe, competes for nitrogen with soil microbes and suffers from N limitation under dry conditions. We hypothesized that ectomycorrhizal communities and the free living rhizosphere microbes from beech trees of two contrasting climatic conditions exhibit differences in N acquisition that contribute to differences in host N uptake and are related to differences in host below-ground carbon allocation. To test these hypotheses young trees from the natural regeneration of two genetically similar populations, one from dryer conditions (SW) and the other from cooler, moist climate (NE) were transplanted into a homogeneous substrate in the same environment and labelled with (13)CO2 and (15)NH4 (+). Free living rhizosphere microbes were characterized by marker genes for the N cycle, but no differences between the rhizosphere of SW or NE trees were found. Lower (15)N enrichment was found in the ectomycorrhizal communities of NE compared with the SW communities, whereas no significant differences were observed for non-mycorrhizal root tips of SW and NE trees. Neither ectomycorrhizal communities nor non-mycorhizal root tips showed differences in (13)C signatures between the NE and SW origins. Because (15)N accumulation in fine roots and transfer to leaves were lower in NE compared to SW trees, our data support that ectomycorrhizal community influence N transfer to their host and demonstrate that the fungal community from the dry condition was more efficient in N acquisition when environmental constraints were relieved. These findings highlight the importance of adapted ectomycorrhizal communities for forest nutrition in a changing climate.
AU  - Leberecht, M.*
AU  - Dannenmann, M.*
AU  - Gschwendtner, S.
AU  - Bilela, S.*
AU  - Meier, R.*
AU  - Simon, J.*
AU  - Rennenberg, H.*
AU  - Schloter, M.
AU  - Polle, A.*
C1  - 45364
C2  - 37300
CY  - Washington
SP  - 5957-5967
TI  - Ectomycorrhizal communities on the roots of two beech (Fagus sylvatica) populations from contrasting climate differ in nitrogen acquisition in a common environment.
JO  - Appl. Environ. Microbiol.
VL  - 81
IS  - 17
PB  - Amer Soc Microbiology
PY  - 2015
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - During the last two decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single cell level using green fluorescent protein as a model product revealed a cell culture heterogeneity characterized by a significant proportion of low-producing bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time-lapse microscopy and flow cytometry. Cell culture heterogeneity was not simply caused by plasmid loss: Instead, an asymmetric distribution of plasmids during cell division was detected during the exponential growth phase. Multi-copy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was mainly achieved by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multi-copy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species.
AU  - Münch, K.M.*
AU  - Müller, J.
AU  - Wienecke, S.*
AU  - Bergmann, S.*
AU  - Heyber, S.*
AU  - Biedendieck, R.*
AU  - Münch, R.*
AU  - Jahn, D.*
C1  - 45493
C2  - 37349
CY  - Washington
SP  - 5976-5986
TI  - Polar fixation of plasmids during recombinant protein production in Bacillus megaterium results in population heterogeneity.
JO  - Appl. Environ. Microbiol.
VL  - 81
IS  - 17
PB  - Amer Soc Microbiology
PY  - 2015
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Azospirillum is a rhizobacterial genus containing plant growth-promoting species associated with different crops worldwide. Azospirillum brasilense strains exhibit a growth-promoting effect by means of phytohormone production and possibly by N2 fixation. However, one of the most important factors for increase in crop yield by plant growth-promoting rhizobacteria is the survival of the inoculant in the rhizosphere, which is not always achieved. The objective of this study was to develop quantitative PCR protocols for strain-specific quantification of A. brasilense FP2. A novel approach was applied to identify strain-specific DNA sequences based on comparison of genomic sequences within the same species. The draft-genome sequence of A. brasilense FP2 and Sp245 were aligned, FP2-specific regions were filtered and checked for other possible matches in public databases, Strain-specific regions were then selected to design and evaluate strain-specific primer pairs. The primer pairs AzoR2.1, AzoR2.2, AzoR5.1, AzoR5.2, and AzoR5.3 were strain-specific for A. brasilense FP2. These primer pairs were used to monitor quantitatively the population of A. brasilense in wheat roots under sterile and non-sterile growth conditions. In addition, co-inoculations with other plant-growth promoting bacteria in wheat were performed under non-sterile conditions. Results showed that A. brasilense FP2 inoculated to wheat roots is highly competitive and achieve high cell numbers (∼10(7) CFU/g of fresh weight root) in the rhizosphere even under non-sterile conditions and when co-inoculated with other rhizobacteria, maintaining the population rather stable for at least up to 13 days after inoculation. The strategy used here can be applied for other organisms whose genome sequences are available.
AU  - Stets, M.I.
AU  - Alqueres, S.
AU  - Souza, E.M.*
AU  - Pedrosa, F.O.*
AU  - Schmid, M.
AU  - Hartmann, A.
AU  - Cruz, L.M.*
C1  - 46376
C2  - 37557
SP  - 6700-6709
TI  - Quantification of Azospirillum brasilense FP2 in wheat roots by strain-specific qPCR.
JO  - Appl. Environ. Microbiol.
VL  - 81
IS  - 19
PY  - 2015
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - The oral microbiome plays a key role for caries, periodontitis and systemic diseases. A method for rapid, high resolution, robust taxonomic profiling of subgingival bacterial communities for early detection of periodontitis biomarkers would therefore be a useful tool for individualized medicine. Here we used Illumina sequencing of the V1-V2 and V5-V6 hypervariable regions of the 16S ribosomal RNA gene. A sample stratification pipeline was developed in a pilot study of 19 individuals, of which 9 had been diagnosed with chronic periodontitis. 523 operational taxonomic units (OTUs) were obtained from the V1-V2 region and 432 OTUs from the V5-V6 region. Key periodontal pathogens like Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia could be identified at the species level with both primer sets. Principal Coordinate Analysis identified two outliers consistently independent of the hypervariable region and method of DNA extraction used. The linear discriminant analysis (LDA) effect size algorithm (LEfSe) identified 80 OTU-level biomarkers of periodontitis and 17 of health. Health and periodontitis related clusters of OTUs were identified using a connectivity analysis and confirmed previous studies with several thousands of samples. A machine learning algorithm was developed which was trained on all but one sample and then predicted the diagnosis of the left-out sample (jack-knife method). Using a combination of the ten best biomarkers, 15 out of 17 samples were correctly diagnosed. Training the algorithm on time-resolved community profiles might provide a highly sensitive tool to detect the onset of periodontitis.
AU  - Szafranski, S.P.*
AU  - Wos-Oxley, M.L.*
AU  - Vilchez-Vargas, R.*
AU  - Jáuregui, R.*
AU  - Plumeier, I.*
AU  - Klawonn, F.*
AU  - Tomasch, J.*
AU  - Meisinger, C.
AU  - Kühnisch, J.*
AU  - Sztajer, H.*
AU  - Pieper, D.H.*
AU  - Wagner-Döbler, I.*
C1  - 42890
C2  - 35728
CY  - Washington
SP  - 1047-1058
TI  - High resolution taxonomic profiling of the subgingival microbiome for biomarker discovery and periodontitis diagnosis.
JO  - Appl. Environ. Microbiol.
VL  - 81
IS  - 3
PB  - Amer Soc Microbiology
PY  - 2015
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Populations of genetically identical Sinorhizobium fredii NGR234 cells differ significantly in their expression profiles of autoinducer (AI)-dependent and AI-independent genes. Promoter fusions of the NGR234 AI synthase genes traI and ngrI showed high levels of phenotypic heterogeneity during growth in TY medium on a single cell level. However, adding very high concentrations of N-(3-oxooctanoyl-)-L-homoserine lactone resulted in a more homogeneous expression profile. Similarly, the lack of internally synthesized AIs in the background of the NGR234-ΔtraI or the NGR234-ΔngrI mutant resulted in a highly homogenous expression of the corresponding promoter fusions in the population. Expression studies with reporter fusions of the promoter regions of the quorum quenching genes dlhR, qsdR1 and the pNGR234b encoded type IV pilus gene cluster suggested that other factors than AI molecules may affect NGR234 phenotypic heterogeneity. Further studies with root exudates and developing Arabidopsis thaliana seedlings provide first evidence that plant root exudates have strong impact on the heterogeneity of AI synthase and quorum quenching genes in NGR234. Thereby, plant-released octopine appears to play a key role in modulation of heterogeneous gene expression.
AU  - Grote, J.*
AU  - Krysciak, D.*
AU  - Schorn, A.*
AU  - Dahlke, R.I.*
AU  - Soonvald, L.*
AU  - Müller, J.
AU  - Hense, B.A.
AU  - Schwarzfischer, M.
AU  - Sauter, M.*
AU  - Schmeisser, C.*
AU  - Streit, W.R.*
C1  - 31744
C2  - 34702
CY  - Washington
SP  - 5572-5582
TI  - Evidence of autoinducer-dependent and autoinducer-independent heterogeneous gene expression in Sinorhizobium fredii NGR234.
JO  - Appl. Environ. Microbiol.
VL  - 80
IS  - 18
PB  - Amer Soc Microbiology
PY  - 2014
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses revealed multiple alkB gene copies/cell in soil bacterial isolates and an apparently high genetic mobility among various phylogenetic groups. Identifying alkane degraders by alkB terminal restriction fragments (T-RFs) and sequences is strongly biased, as the phylogenetic trees based on 16S rRNA and alkB gene sequences were highly inconsistent.
AU  - Giebler, J.*
AU  - Wick, L.Y.*
AU  - Schloter, M.
AU  - Harms, H.*
AU  - Chatzinotas, A.*
C1  - 24314
C2  - 31501
SP  - 3129-3132
TI  - Evaluating the assignment of alkB terminal restriction fragments and sequence types to distinct bacterial taxa.
JO  - Appl. Environ. Microbiol.
VL  - 79
IS  - 9
PB  - Amer. Soc. Microbiology
PY  - 2013
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Two 4-chloro-2-methylphenoxyacetic acid (MCPA)-degrading enrichment cultures selected from an aquifer on low (0.1 mg liter(-1)) or high (25 mg liter(-1)) MCPA concentrations were compared in terms of metabolic activity, community composition, population growth, and single cell physiology. Different community compositions and major shifts in community structure following exposure to different MCPA concentrations were observed using both 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and pyrosequencing. The communities also differed in their MCPA-mineralizing activities. The enrichments selected on low concentrations mineralized MCPA with shorter lag phases than those selected on high concentrations. Flow cytometry measurements revealed that mineralization led to cell growth. The presence of low-nucleic acid-content bacteria (LNA bacteria) was correlated with mineralization activity in cultures selected on low herbicide concentrations. This suggests that LNA bacteria may play a role in degradation of low herbicide concentrations in aquifers impacted by agriculture. This study shows that subpopulations of herbicide-degrading bacteria that are adapted to different pesticide concentrations can coexist in the same environment and that using a low herbicide concentration enables enrichment of apparently oligotrophic subpopulations.
AU  - Gözdereliler, E.
AU  - Boon, N.*
AU  - Aamand, J.*
AU  - de Roy, K.*
AU  - Granitsiotis, M.S.
AU  - Albrechtsen, H.J.*
AU  - Sørensen, S.R.*
C1  - 11813
C2  - 30822
SP  - 367-375
TI  - Comparing metabolic functionalities, community structures, and dynamics of herbicide-degrading communities cultivated with different substrate concentrations.
JO  - Appl. Environ. Microbiol.
VL  - 79
IS  - 1
PB  - American Society of Microbiology
PY  - 2013
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Histone modifications are crucial for the regulation of secondary metabolism in various filamentous fungi. Here we studied the involvement of histone deacetylases (HDACs) in secondary metabolism in the phytopathogenic fungus Fusarium fujikuroi, a known producer of several secondary metabolites including phytohormones, pigments and mycotoxins. Deletion of three Zn(2+)-dependent HDAC-encoding genes, ffhda1, ffhda2 and ffhda4, indicated that FfHda1 and FfHda2 regulate secondary metabolism, whereas FfHda4 is involved in developmental processes but dispensable for secondary metabolite production in F. fujikuroi. Single deletions of ffhda1 and ffhda2 resulted not only in an increase or decrease, but also in derepression of metabolite biosynthesis under normally repressing conditions. Moreover, double deletion of both genes ffhda1 and ffhda2 shows additive but also distinct phenotypes with regard to secondary metabolite biosynthesis and both genes are required for GA-induced bakanae disease on the preferred host-plant rice, as ΔΔffhda1/ffhda2 mutants resemble the uninfected control plant. Microarray analysis with a Δffhda1 mutant that has lost the major HDAC revealed differential expression of secondary metabolite gene clusters subsequently verified by a combination of chemical and biological approaches. These results indicate that HDACs are not only involved in gene silencing but also in activation of some genes. Chromatin immunoprecipitation with Δffhda1 revealed significant alterations in the acetylation state of secondary metabolite gene clusters compared to the wild type thereby providing insights into the regulatory mechanism on chromatin level. Altogether, manipulation of HDAC-encoding genes constitutes a powerful tool to control secondary metabolism in filamentous fungi.
AU  - Studt, L.*
AU  - Schmidt, F.J.*
AU  - Jahn, L.*
AU  - Sieber, C.M.K.
AU  - Connolly, L.R.*
AU  - Niehaus, E.M.*
AU  - Freitag, M.*
AU  - Humpf, H.U.*
AU  - Tudzynski, B.*
C1  - 27776
C2  - 32807
SP  - 7719-7734
TI  - Two histone deacetylases, FfHda1 and FfHda2, are important for secondary metabolism and virulence in Fusarium fujikuroi.
JO  - Appl. Environ. Microbiol.
VL  - 79
IS  - 24
PB  - Amer. Soc. Microbiology
PY  - 2013
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - The detection of anaerobic hydrocarbon degrader populations via catabolic gene markers is important for the understanding of processes at contaminated sites. Fumarate-adding enzymes (FAEs; i.e., benzylsuccinate and alkylsuccinate synthases) have already been established as specific functional marker genes for anaerobic hydrocarbon degraders. Several recent studies based on pure cultures and laboratory enrichments have shown the existence of new and deeply branching FAE gene lineages, such as clostridial benzylsuccinate synthases and homologues, as well as naphthylmethylsuccinate synthases. However, established FAE gene detection assays were not designed to target these novel lineages, and consequently, their detectability in different environments remains obscure. Here, we present a new suite of parallel primer sets for detecting the comprehensive range of FAE markers known to date, including clostridial benzylsuccinate, naphthylmethylsuccinate, and alkylsuccinate synthases. It was not possible to develop one single assay spanning the complete diversity of FAE genes alone. The enhanced assays were tested with a range of hydrocarbon-degrading pure cultures, enrichments, and environmental samples of marine and terrestrial origin. They revealed the presence of several, partially unexpected FAE gene lineages not detected in these environments before: distinct deltaproteobacterial and also clostridial bssA homologues as well as environmental nmsA homologues. These findings were backed up by dual-digest terminal restriction fragment length polymorphism diagnostics to identify FAE gene populations independently of sequencing. This allows rapid insights into intrinsic degrader populations and degradation potentials established in aromatic and aliphatic hydrocarbon-impacted environmental systems.
AU  - von Netzer, F.
AU  - Pilloni, G.
AU  - Kleindienst, S.*
AU  - Krüger, M.*
AU  - Knittel, K.*
AU  - Gründger, F.*
AU  - Lueders, T.
C1  - 22407
C2  - 30894
SP  - 543-552
TI  - Enhanced gene detection assays for fumarate-adding enzymes allow uncovering of anaerobic hydrocarbon degraders in terrestrial and marine systems.
JO  - Appl. Environ. Microbiol.
VL  - 79
IS  - 2
PB  - American Society of Microbiology
PY  - 2013
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - The plant-beneficial bacterium Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonization under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as noncoding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small RNA (sRNA) genes rsmX, rsmY, and rsmZ. Naturally occurring mutations in the gacS-gacA system accounted for phenotypic switching, which was characterized by downregulation of antifungal secondary metabolites (2,4-diacetylphloroglucinol and cyanide), indoleacetate, exoenzymes (lipase and protease), and three different N-acyl-homoserine lactone molecules. Moreover, in addition to abrogating these biocontrol traits, gacS and gacA mutations resulted in reduced expression of the type VI secretion machinery, alginate biosynthesis, and biofilm formation. In a gacA mutant, the expression of rsmX was completely abolished, unlike that of rsmY and rsmZ. Overexpression of any of the three sRNAs in the gacA mutant overruled the pleiotropic changes and restored the wild-type phenotypes, suggesting functional redundancy of these sRNAs. In conclusion, our data show that phenotypic switching in P. brassicacearum results from mutations in the gacS-gacA system.
AU  - Lalaouna, D.*
AU  - Fochesato, S.*
AU  - Sanchez, L.*
AU  - Schmitt-Kopplin, P.
AU  - Haas, D.*
AU  - Heulin, T.*
AU  - Achouak, W.*
C1  - 7278
C2  - 29638
SP  - 1658-1665
TI  - Phenotypic switching in Pseudomonas brassicacearum involves GacS- and GacA-dependent Rsm small RNAs.
JO  - Appl. Environ. Microbiol.
VL  - 78
IS  - 6
PB  - Amer. Soc. Microbiology
PY  - 2012
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - In many areas of China, tidal wetlands have been converted into agricultural land for rice cultivation. However, the consequences of land use changes for soil microbial communities are poorly understood. Therefore, we investigated bacterial and archaeal communities involved in inorganic nitrogen turnover (nitrogen fixation, nitrification, and denitrification) based on abundances and relative species richness of the corresponding functional genes along a soil chronosequence ranging between 50 and 2,000 years of paddy soil management compared to findings for a tidal wetland. Changes in abundance and diversity of the functional groups could be observed, reflecting the different chemical and physical properties of the soils, which changed in terms of soil development. The tidal wetland was characterized by a low microbial biomass and relatively high abundances of ammonia-oxidizing microbes. Conversion of the tidal wetlands into paddy soils was followed by a significant increase in microbial biomass. Fifty years of paddy management resulted in a higher abundance of nitrogen-fixing microbes than was found in the tidal wetland, whereas dominant genes of nitrification and denitrification in the paddy soils showed no differences. With ongoing rice cultivation, copy numbers of archaeal ammonia oxidizers did not change, while that of their bacterial counterparts declined. The nirK gene, coding for nitrite reductase, increased with rice cultivation time and dominated its functionally redundant counterpart, nirS, at all sites under investigation. Relative species richness showed significant differences between all soils with the exception of the archaeal ammonia oxidizers in the paddy soils cultivated for 100 and 300 years. In general, changes in diversity patterns were more pronounced than those in functional gene abundances.
AU  - Bannert, A.
AU  - Kleineidam, K.
AU  - Wissing, L.*
AU  - Mueller-Niggemann, C.*
AU  - Vogelsang, V.*
AU  - Welzl, G.
AU  - Cao, Z.*
AU  - Schloter, M.
C1  - 6168
C2  - 29236
SP  - 6109-6116
TI  - Changes in diversity and functional gene abundances of microbial communities involved in nitrogen fixation, nitrification, and denitrification in a tidal wetland versus paddy soils cultivated for different time periods.
JO  - Appl. Environ. Microbiol.
VL  - 77
IS  - 17
PB  - American Society for Microbiology
PY  - 2011
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [(13)C]acetate and H(2)-(13)CO(2), were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [(13)C]methane was linked to acetoclastic methanogenesis in both the [(13)C]acetate- and the H(2)-(13)CO(2)-amended cultures of coal and timber. H(2)-(13)CO(2) was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H(2) conditions found in coal mines.
AU  - Beckmann, S.*
AU  - Lüders, T.
AU  - Krüger, M.*
AU  - von Netzer, F.
AU  - Engelen, B.*
AU  - Cypionka, H.*
C1  - 6474
C2  - 28750
CY  - Washington, USA
SP  - 3749-3756
TI  - Acetogens and acetoclastic methanosarcinales govern methane formation in abandoned coal mines.
JO  - Appl. Environ. Microbiol.
VL  - 77
IS  - 11
PB  - American Society for Microbiology
PY  - 2011
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B(12) biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.
AU  - Hemme, C.L.*
AU  - Fields, M.W.*
AU  - He, Q.*
AU  - Deng, Y.*
AU  - Lin, L.*
AU  - Tu, Q.*
AU  - Mouttaki, H.
AU  - Zhou, A.*
AU  - Feng, X.*
AU  - Zuo, Z.*
AU  - Ramsay, B.D.*
AU  - He, Z.*
AU  - Wu, L.*
AU  - van Nostrand, J.*
AU  - Xu, J.*
AU  - Tang, Y.J.*
AU  - Wiegel, J.*
AU  - Phelps, T.J.*
AU  - Zhou, J.*
C1  - 6671
C2  - 29088
SP  - 7998-8008
TI  - Correlation of genomic and physiological traits of thermoanaerobacter species with biofuel yields.
JO  - Appl. Environ. Microbiol.
VL  - 77
IS  - 22
PB  - American Society for Microbiology
PY  - 2011
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Two soils were amended three times with pig manure. The abundance of sulfonamide resistance genes was determined by quantitative PCR 2 months after each application. In both soils treated with sulfadiazine-containing manure, the numbers of copies of sul1 and sul2 significantly increased compared to numbers after treatments with antibiotic-free manure or a control and accumulated with repeated applications.
AU  - Heuer, H.*
AU  - Solehati, Q.*
AU  - Zimmerling, U.*
AU  - Kleineidam, K.
AU  - Schloter, M.
AU  - Müller, T.*
AU  - Focks, A.*
AU  - Thiele-Bruhn, S.*
AU  - Smalla, K.
C1  - 5705
C2  - 28444
CY  - Washington, USA
SP  - 2527-2530
TI  - Accumulation of sulfonamide resistance genes in arable soils due to repeated application of manure containing sulfadiazine.
JO  - Appl. Environ. Microbiol.
VL  - 77
IS  - 7
PB  - Amer. Soc. Microbiology
PY  - 2011
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Microbial iron reduction is considered to be a significant subsurface process. The rate-limiting bioavailability of the insoluble iron oxyhydroxides, however, is a topic for debate. Surface area and mineral structure are recognized as crucial parameters for microbial reduction rates of bulk, macroaggregate iron minerals. However, a significant fraction of iron oxide minerals in the subsurface is supposed to be present as nanosized colloids. We therefore studied the role of colloidal iron oxides in microbial iron reduction. In batch growth experiments with Geobacter sulfurreducens, colloids of ferrihydrite (hydrodynamic diameter, 336 nm), hematite (123 nm), goethite (157 nm), and akaganeite (64 nm) were added as electron acceptors. The colloidal iron oxides were reduced up to 2 orders of magnitude more rapidly (up to 1,255 pmol h(-1) cell(-1)) than bulk macroaggregates of the same iron phases (6 to 70 pmol h(-1) cell(-1)). The increased reactivity was not only due to the large surface areas of the colloidal aggregates but also was due to a higher reactivity per unit surface. We hypothesize that this can be attributed to the high bioavailability of the nanosized aggregates and their colloidal suspension. Furthermore, a strong enhancement of reduction rates of bulk ferrihydrite was observed when nanosized ferrihydrite aggregates were added.
AU  - Bosch, J.
AU  - Heister, K.*
AU  - Hofmann, T.*
AU  - Meckenstock, R.U.
C1  - 192
C2  - 27099
SP  - 184-189
TI  - Nanosized iron oxide colloids strongly enhance microbial iron reduction.
JO  - Appl. Environ. Microbiol.
VL  - 76
IS  - 1
PB  - American Society for Microbiology
PY  - 2010
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Seven different actA subtypes forming two phylogenetic lineages could be distinguished by sequencing the actA gene of Listeria seeligeri isolates from different habitats. Isolates of the two lineages differ in hemolytic as well as phospholipase activities and in the arrangement of the virulence gene cluster. The presence of a serine protease gene resembling orf2110 of L. monocytogenes in some isolates further supports the hypothesis that L. seeligeri is subject to ongoing adaptation to changing environments.
AU  - Müller, A.A.*
AU  - Schmid, M.W.
AU  - Meyer, O.*
AU  - Meussdoerffer, F.G.*
C1  - 6079
C2  - 27988
SP  - 3044-3047
TI  - Listeria seeligeri isolates from food processing environments form two phylogenetic lineages.
JO  - Appl. Environ. Microbiol.
VL  - 76
IS  - 9
PB  - American Society for Microbiology
PY  - 2010
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - The antibiotic sulfadiazine (SDZ) can enter the environment by application of manure from antibiotic-treated animals to arable soil. Because antibiotics are explicitly designed to target microorganisms, they likely affect microbes in the soil ecosystem, compromising important soil functions and disturbing processes in nutrient cycles. In a greenhouse experiment, we investigated the impact of sulfadiazine-contaminated pig manure on functional microbial communities involved in key processes of the nitrogen cycle in the root-rhizosphere complexes (RRCs) of maize (Zea mays) and clover (Trifolium alexandrinum). At both the gene and transcript level, we performed real-time PCR using nifH, amoA (in both ammonia-oxidizing bacteria and archaea), nirK, nirS, and nosZ as molecular markers for nitrogen fixation, nitrification, and denitrification. Sampling was performed 10, 20, and 30 days after the application. SDZ affected the abundance pattern of all investigated genes in the RRCs of both plant species (with stronger effects in the RRC of clover) 20 and 30 days after the addition. Surprisingly, effects on the transcript level were less pronounced, which might indicate that parts of the investigated functional groups were tolerant or resistant against SDZ or, as in the case of nifH and clover, have been protected by the nodules.
AU  - Ollivier, J.
AU  - Kleineidam, K.
AU  - Reichel, R.*
AU  - Thiele-Bruhn, S.*
AU  - Kotzerke, A.*
AU  - Kindler, R.*
AU  - Wilke, B.-M.*
AU  - Schloter, M.
C1  - 6074
C2  - 27927
SP  - 7903-7909
TI  - Effect of sulfadiazine-contaminated pig manure on the abundances of genes and transcripts involved in nitrogen transformation in the root-rhizosphere complexes of maize and clover.
JO  - Appl. Environ. Microbiol.
VL  - 76
IS  - 24
PB  - American Society for Microbiology
PY  - 2010
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - The root disease take-all, caused by Gaeumannomyces graminis var. tritici, can be managed by monoculture-induced take-all decline (TAD). This natural biocontrol mechanism typically occurs after a take-all outbreak and is believed to arise from an enrichment of antagonistic populations in the rhizosphere. However, it is not known whether these changes are induced by the monoculture or by ecological rhizosphere conditions due to a disease outbreak and subsequent attenuation. This question was addressed by comparing the rhizosphere microflora of barley, either inoculated with the pathogen or noninoculated, in a microcosm experiment in five consecutive vegetation cycles. TAD occurred in soil inoculated with the pathogen but not in noninoculated soil. Bacterial community analysis using terminal restriction fragment length polymorphism of 16S rRNA showed pronounced population shifts in the successive vegetation cycles, but pathogen inoculation had little effect. To elucidate rhizobacterial dynamics during TAD development, a 16S rRNA-based taxonomic microarray was used. Actinobacteria were the prevailing indicators in the first vegetation cycle, whereas the third cycle-affected most severely by take-all-was characterized by Proteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes, and Acidobacteria. Indicator taxa for the last cycle (TAD) belonged exclusively to Proteobacteria, including several genera with known biocontrol traits. Our results suggest that TAD involves monoculture-induced enrichment of plant-beneficial taxa.
AU  - Schreiner, K.
AU  - Hagn, A.
AU  - Kyselkova, M.*
AU  - Moenne-Loccoz, Y.*
AU  - Welzl, G.
AU  - Munch, J.-C.
AU  - Schloter, M.
C1  - 1399
C2  - 27282
SP  - 4703-4712
TI  - Comparison of barley succession and take-all disease as environmental factors shaping the rhizobacterial community during take-all decline.
JO  - Appl. Environ. Microbiol.
VL  - 76
IS  - 14
PB  - American Society for Microbiology
PY  - 2010
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - The effect of agricultural management practices on geochemical cycles in moderate ecosystems is by far better understood than in semiarid regions, where fertilizer availability and climatic conditions are less favorable. We studied the impact of different fertilizer regimens in an agricultural long-term observatory in Burkina Faso at three different plant development stages ( early leaf development, flowering, and senescence) of sorghum cultivars. Using real-time PCR, we investigated functional microbial communities involved in key processes of the nitrogen cycle ( nitrogen fixation, ammonia oxidation, and denitrification) in the rhizosphere. The results indicate that fertilizer treatments and plant development stages combined with environmental factors affected the abundance of the targeted functional genes in the rhizosphere. While nitrogen-fixing populations dominated the investigated communities when organic fertilizers ( manure and straw) were applied, their numbers were comparatively reduced in urea-treated plots. In contrast, ammonia-oxidizing bacteria (AOB) increased not only in absolute numbers but also in relation to the other bacterial groups investigated in the urea-amended plots. Ammonia-oxidizing archaea exhibited higher numbers compared to AOB independent of fertilizer application. Similarly, denitrifiers were also more abundant in the urea-treated plots. Our data imply as well that, more than in moderate regions, water availability might shape microbial communities in the rhizosphere, since low gene abundance data were obtained for all tested genes at the flowering stage, when water availability was very limited.
AU  - Hai, B.
AU  - Diallo, N.H.*
AU  - Sall, S.*
AU  - Haesler, F.
AU  - Schauss, K.
AU  - Bonzi, M.*
AU  - Assigbetse, K.*
AU  - Chotte, J.L.*
AU  - Munch, J.-C.
AU  - Schloter, M.
C1  - 1232
C2  - 26281
SP  - 4993-5000
TI  - Quantification of key genes steering the microbial nitrogen cycle in the rhizosphere of sorghum cultivars in tropical agroecosystems.
JO  - Appl. Environ. Microbiol.
VL  - 75
IS  - 15
PB  - Amer Soc Microbiology
PY  - 2009
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - The effects of genetically modified (GM), zeaxanthin-accumulating potato plants on microbial communities in the rhizosphere were compared to the effects of different potato cultivars. Two GM lines and their parental cultivar, as well as four other potato cultivars, were grown in randomized field plots at two sites and in different years. Rhizosphere samples were taken at three developmental stages during plant growth and analyzed using denaturing gradient gel electrophoresis (DGGE) fingerprints of Bacteria, Actinobacteria, Alpha- and Betaproteobacteria, Bacillus, Streptomycetaceae, Pseudomonas, gacA, Fungi, and Ascomycetes. In the bacterial DGGE gels analyzed, significant differences between the parental cultivar and the two GM lines were detected mainly for Actinobacteria but also for Betaproteobacteria and Streptomycetaceae, yet these differences occurred only at one site and in one year. Significant differences occurred more frequently for Fungi, especially Ascomycetes, than for bacteria. When all seven plant genotypes were compared, DGGE analysis revealed that different cultivars had a greater effect on both bacterial and fungal communities than genetic modification. The effects of genetic modification were detected mostly at the senescence developmental stage of the plants. The site was the overriding factor affecting microbial community structure compared to the plant genotype. In general, the fingerprints of the two GM lines were more similar to that of the parental cultivar, and the differences observed did not exceed natural cultivar-dependent variability.
AU  - Weinert, N.*
AU  - Meincke, R.*
AU  - Gottwald, C.*
AU  - Heuer, H.*
AU  - Gomes, N.C.M.*
AU  - Schloter, M.
AU  - Berg, G.*
AU  - Smalla, K.
C1  - 2463
C2  - 26169
SP  - 3859-3865
TI  - Rhizosphere communities of genetically modified zeaxanthin-accumulating potato plants and their parent cultivar differ less than those of different potato cultivars.
JO  - Appl. Environ. Microbiol.
VL  - 75
IS  - 12
PB  - Amer Soc Microbiology
PY  - 2009
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Microbial degradation is the only sustainable component of natural attenuation in contaminated groundwater environments, yet its controls, especially in anaerobic aquifers, are still poorly understood. Hence, putative spatial correlations between specific populations of key microbial players and the occurrence of respective degradation processes remain to be unraveled. We therefore characterized microbial community distribution across a high-resolution depth profile of a tar oil-impacted aquifer where benzene, toluene, ethylbenzene, and xylene (BTEX) degradation depends mainly on sulfate reduction. We conducted depth-resolved terminal restriction fragment length polymorphism fingerprinting and quantitative PCR of bacterial 16S rRNA and benzylsuccinate synthase genes (bssA) to quantify the distribution of total microbiota and specific anaerobic toluene degraders. We show that a highly specialized degrader community of microbes related to known deltaproteobacterial iron and sulfate reducers (Geobacter and Desulfocapsa spp.), as well as clostridial fermenters (Sedimentibacter spp.), resides within the biogeochemical gradient zone underneath the highly contaminated plume core. This zone, where BTEX compounds and sulfate--an important electron acceptor--meet, also harbors a surprisingly high abundance of the yet-unidentified anaerobic toluene degraders carrying the previously detected F1-cluster bssA genes (C. Winderl, S. Schaefer, and T. Lueders, Environ. Microbiol. 9:1035-1046, 2007). Our data suggest that this biogeochemical gradient zone is a hot spot of anaerobic toluene degradation. These findings show that the distribution of specific aquifer microbiota and degradation processes in contaminated aquifers are tightly coupled, which may be of value for the assessment and prediction of natural attenuation based on intrinsic aquifer microbiota.
AU  - Winderl, C.
AU  - Anneser, B.
AU  - Griebler, C.
AU  - Meckenstock, R.U.
AU  - Lüders, T.
C1  - 2704
C2  - 25136
SP  - 792-801
TI  - Depth-resolved quantification of anaerobic toluene degraders and aquifer microbial community patterns in distinct redox zones of a tar oil contaminant plume.
JO  - Appl. Environ. Microbiol.
VL  - 74
IS  - 3
PB  - American Society for Microbiology
PY  - 2008
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - The occurrence and activation of glutathione S-transferase (GST) and the GST activities in biofilms in cold sulfidic spring waters were compared to the occurrence and activation of GST and the GST activities of the aquatic fungal strains EH5 and EH7 of Mucor hiemalis isolated for the first time from such waters. Using fluorescently labeled polyclonal anti-GST antibodies and GST activity measurements, we demonstrated that a high level of GST occurred in situ in natural biofilms and pure cultures of strain EH5. Measurement of microsomal and cytosolic soluble GST activities using different xenobiotic substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)propane, 1-iodo-2,4-dinitrobenzene, and fluorodifen, showed that the overall biotransforming abilities of biofilms were at least sixfold greater than that of strain EH5 alone. Increasing the level of sodium thiosulfate (STS) in the medium stimulated the microsomal and cytosolic GST activities with CDNB of strain EH5 about 44- and 94-fold, respectively, compared to the activities in the control. The induction of microsomal GST activity with fluorodifen by STS was strongly linear, but the initial strong linear increase in cytosolic GST activity with fluorodifen showed saturation-like effects at STS concentrations higher than approximately 1 mM. Using laser scanning confocal and conventional fluorescence microscopy, abundant fluorescently labeled GST proteins were identified in germinating sporangiospores of strain EH5 after activation by STS. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least two main GSTs (~27.8- and ~25.6-kDa subunits) in the cytosol of EH5, whereas the major 27.8-kDa subunit was the only GST in microsomes. We suggest that differential cellular GST expression takes place in strain EH5 depending on spore and hyphal development. Our results may contribute to our understanding of induction of GST by sulfurous compounds, as well as to the immunofluorescence visualization of GST in aquatic fungus and fungus-bacterium biofilms.
AU  - Hoque, E.
AU  - Pflugmacher, S.*
AU  - Fritscher, J.
AU  - Wolf, M.
C1  - 3937
C2  - 24469
SP  - 2697-2707
TI  - Induction of Glutathione S-Transferase in Biofilms and Germinating Spores of Mucor hiemalis Strain EH5 from Cold Sulfidic Spring Waters.
JO  - Appl. Environ. Microbiol.
VL  - 73
IS  - 8
PB  - American Society for Microbiology
PY  - 2007
SN  - 0099-2240
ER  - 

TY  - JOUR
AU  - Lüders, T.
AU  - Kindler, R.*
AU  - Miltner, A.*
AU  - Friedrich, M.W.*
C1  - 2584
C2  - 23780
SP  - 5342-5348
TI  - Identification of bacterial micropredators distinctively active in a soil microbial food web.
JO  - Appl. Environ. Microbiol.
VL  - 72
PY  - 2006
SN  - 0099-2240
ER  - 

TY  - JOUR
AU  - Penning, H.
AU  - Claus, P.*
AU  - Casper, P.*
AU  - Conrad, R.*
C1  - 2130
C2  - 23782
SP  - 5648-5652
TI  - Carbon isotope fractionation during acetoclastic methanogenesis by Methanosaeta concilii in culture and a lake sediment.
JO  - Appl. Environ. Microbiol.
VL  - 72
PY  - 2006
SN  - 0099-2240
ER  - 

TY  - JOUR
AU  - Sharma, S.
AU  - Szele, Z.
AU  - Schilling, R.
AU  - Munch, J.-C.
AU  - Schloter, M.
C1  - 4007
C2  - 23539
SP  - 2148-2154
TI  - Influence of freeze-thaw stress on the structure and function of microbial communities and denitrifying populations in soil.
JO  - Appl. Environ. Microbiol.
VL  - 72
PY  - 2006
SN  - 0099-2240
ER  - 

TY  - JOUR
AU  - Smalla, K.*
AU  - Haines, A.S.*
AU  - Jones, K.*
AU  - Krögerrecklenfort, E.*
AU  - Heuer, H.*
AU  - Schloter, M.
AU  - Thomas, C.M.*
C1  - 3869
C2  - 24070
SP  - 7253-7259
TI  - Increased abundance of IncP-1ß plasmids and mercury resistance genes in mercury-polluted river sediments: First discovery of IncP-1ß plasmids with a complex mer transposon as the sole accessory element.
JO  - Appl. Environ. Microbiol.
VL  - 72
PY  - 2006
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are widespread contaminants in groundwater. We examined the anaerobic degradation of BTEX compounds with amorphous ferric oxide as electron acceptor. Successful enrichment cultures were obtained for all BTEX substrates both in the presence and absence of AQDS (9,10-anthraquinone-2,6-disulfonic acid). The electron balances showed a complete anaerobic oxidation of the aromatic compounds to CO2. This is the first report on the anaerobic degradation of o-xylene and ethylbenzene in sediment-free iron-reducing enrichment cultures.
AU  - Jahn, M.K.*
AU  - Haderlein, S.B.*
AU  - Meckenstock, R.U.
C1  - 937
C2  - 22741
SP  - 3355-3358
TI  - Anaerobic degradation of benzene, toluene, ethylbenzene and o-xylene in sediment-free iron-reducing enrichment cultures.
JO  - Appl. Environ. Microbiol.
VL  - 71
IS  - 6
PY  - 2005
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Bacterial growth occurs in noncarbonated natural mineral waters a few days after filling and storage at room temperature, a phenomenon known for more than 40 years. Using the full-cycle rRNA approach, we monitored the development of the planktonic bacterial community in a noncarbonated natural mineral water after bottling. Seven 16S rRNA gene libraries, comprising 108 clones in total, were constructed from water samples taken at various days after bottling and from two different bottle sizes. Sequence analyses identified 11 operational taxonomic units (OTUs), all but one affiliated with the betaproteobacterial order Burkholderiales (6 OTUs) or the class Alphaproteobacteria (4 OTUs). Fluorescence in situ hybridization (FISH) was applied in combination with DAPI (4′,6′-diamidino-2-phenylindole) staining, viability staining, and microscopic counting to quantitatively monitor changes in bacterial community composition. A growth curve similar to that of a bacterium grown in a batch culture was recorded. In contrast to the current perception that Gammaproteobacteria are the most important bacterial components of natural mineral water in bottles, Betaproteobacteria dominated the growing bacterial community and accounted for 80 to 98% of all bacteria detected by FISH in the late-exponential and stationary-growth phases. Using previously published and newly designed genus-specific probes, members of the betaproteobacterial genera Hydrogenophaga, Aquabacterium, and Polaromonas were found to constitute a significant proportion of the bacterial flora (21 to 86% of all bacteria detected by FISH). For the first time, key genera responsible for bacterial growth in a natural mineral water were identified by applying molecular cultivation-independent techniques.
AU  - Loy, A.*
AU  - Beisker, W.
AU  - Meier, H.*
C1  - 3362
C2  - 23306
SP  - 3624-3632
TI  - Diversity of bacteria growning in natural mineral water after bottling.
JO  - Appl. Environ. Microbiol.
VL  - 71
IS  - 7
PY  - 2005
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - For simultaneous identification of members of the betaproteobacterial order “Rhodocyclales” in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the “Rhodocyclales.” The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent “Rhodocyclales” diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed “Rhodocyclales”-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of “Rhodocyclales” populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.
AU  - Loy, A.*
AU  - Schulz, C.*
AU  - Lücker, S.*
AU  - Schöpfer-Wendels, A.
AU  - Stöcker, K.*
AU  - Lehner, A.*
AU  - Wagner, M.*
C1  - 4501
C2  - 23329
SP  - 1373-1386
TI  - 16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales".
JO  - Appl. Environ. Microbiol.
VL  - 71
IS  - 3
PY  - 2005
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - A PCR-based approach was developed to detect ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) form I large-subunit genes (cbbL) as a functional marker of autotrophic bacteria that fix carbon dioxide via the Calvin-Benson-Bassham cycle. We constructed two different primer sets, targeting the green-like and red-like phylogenetic groups of cbbL genes. The diversity of these cbbL genes was analyzed by the use of three differently managed agricultural soils from a long-term field experiment. cbbL gene fragments were amplified from extracted soil DNAs, and PCR products were cloned and screened by restriction fragment length polymorphism analysis. Selected unique cbbL clones were sequenced and analyzed phylogenetically. The green-like cbbL sequences revealed a very low level of diversity, being closely related to the cbbL genes of Nitrobacter winogradskyi and Nitrobacter vulgaris. In contrast, the red-like cbbL gene libraries revealed a high level of diversity in the two fertilized soils and less diversity in unfertilized soil. The majority of environmental red-like cbbL genes were only distantly related to already known cbbL sequences and even formed separate clusters. In order to extend the database of available red-like cbbL sequences, we amplified cbbL sequences from bacterial type culture strains and from bacterial isolates obtained from the investigated soils. Bacterial isolates harboring the cbbL gene were analyzed phylogenetically on the basis of their 16S rRNA gene sequences. These analyses revealed that bacterial genera such as Bacillus, Streptomyces, and Arthrobacter harbor red-like cbbL genes which fall into the cbbL gene clusters retrieved from the investigated soils.
AU  - Selesi, D.
AU  - Schmid, M.
AU  - Hartmann, A.
C1  - 2857
C2  - 22898
SP  - 175-184
TI  - Diversity of Green-Like and Red-like ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbL) in differently managed agricultural soils.
JO  - Appl. Environ. Microbiol.
VL  - 71
IS  - 1
PY  - 2005
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Transcription of the nirK and nirS genes coding for dissimilatory bacterial nitrite reductases was analyzed by reverse transcription PCR (RT-PCR) of mRNA isolated from rhizosphere samples of three economically important grain legumes at maturity: Vicia faba, Lupinus albus, and Pisum sativum. The nirK gene and transcripts could be detected in all the rhizosphere samples. In contrast, nirS could not be detected. Sampling variations were analyzed by comparing denaturing gradient gel electrophoresis profiles derived from nirK RT-PCR products. High similarity was observed between the replicates, and so one representative product per legume was cloned. Clones with the correct insert size were screened by restriction fragment length polymorphism by using the restriction enzyme MspI. The clones could be distributed into 12 different patterns. Patterns 1, 3, 4, 5, and 7 were common in clone libraries of the three rhizosphere types under study. Patterns 2, 9, 10, and 11 were absent from Pisum rhizospheres, while patterns 6, 8, and 12 were absent from the Vicia library. Pattern 1, which was the most dominant in the Vicia and Lupinus libraries, constituted about 25% of all clones. The Lupinus library had clones representing all 12 patterns, indicating it to be the most diverse among the three. Clones representative of each pattern were sequenced. All patterns grouped together forming a distinct cluster, which was divergent from previously described nirK sequences in the database. The study revealed a hitherto unknown diversity of denitrifiers in legume rhizospheres. A plant-dependent rhizosphere effect on the transcripts of a gene was evident.
AU  - Sharma, S.
AU  - Aneja, M.K.
AU  - Mayer, J.*
AU  - Munch, J.-C.
AU  - Schloter, M.
C1  - 1880
C2  - 22603
SP  - 2001-2007
TI  - Diversity of transcripts of nitrite reductase genes (nirK and nirS) in rhizospheres of grain legumes.
JO  - Appl. Environ. Microbiol.
VL  - 71
IS  - 4
PY  - 2005
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Denaturing gradient gel electrophoresis (DGGE) of amplified fragments of genes coding for 16S rRNA was used to study the development of bacterial communities during decomposition of crop residues in agricultural soils. Ten strains were tested, and eight of these strains produced a single band. Furthermore, a mixture of strains yielded distinguishable bands. Thus, DGGE DNA band patterns were used to estimate bacterial diversity. A field experiment performed with litter in nylon bags was used to evaluate the bacterial diversity during the decomposition of readily degradable rye and more refractory wheat material in comparable luvisols and cambisols in northern, central, and southern Germany. The amount of bacterial DNA in the fresh litter was small. The DNA content increased rapidly after the litter was added to the soil, particularly in the rapidly decomposing rye material. Concurrently, diversity indices, such as the Shannon-Weaver index, evenness, and equitability, which were calculated from the number and relative abundance (intensity) of the bacterial DNA bands amplified from genes coding for 16S rRNA, increased during the course of decomposition. This general trend was not significant for evenness and equitability at any time. The indices were higher for the more degradation-resistant wheat straw than for the more easily decomposed rye grass. Thus, the DNA band patterns indicated that there was increasing bacterial diversity as decomposition proceeded and substrate quality decreased. The bacterial diversity differed for the sites in northern, central, and southern Germany, where the same litter material was buried in the soil. This shows that in addition to litter type climate, vegetation, and indigenous microbes in the surrounding soil affected the development of the bacterial communities in the litter.
AU  - Dilly, O.*
AU  - Bloem, J.*
AU  - Vos, A.*
AU  - Munch, J.-C.
C1  - 1015
C2  - 21564
SP  - 468-474
TI  - Bacterial diversity in agricultural soils during litter decomposition.
JO  - Appl. Environ. Microbiol.
VL  - 70
IS  - 1
PB  - American Society for Microbiology
PY  - 2004
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H2-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [13C]propionate (<10 mM) and the oxidation of ∼30 μmol of 13C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were 13C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in “heavy” 13C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of 13C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [13C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.
AU  - Lüders, T.
AU  - Pommerenke, B.*
AU  - Friedrich, M.W.*
C1  - 1661
C2  - 21990
SP  - 5778-5786
TI  - Stable-isotope probing of microorganisms thriving at thermodynamic limits: Syntropic propionate oxidation in flooded soil.
JO  - Appl. Environ. Microbiol.
VL  - 70
IS  - 10
PY  - 2004
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - table isotope fractionation was studied during the degradation of m-xylene, o-xylene, m-cresol, and p-cresol with two pure cultures of sulfate-reducing bacteria. Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation. The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors (ε) of −1.5 and −3.9, which is in the same order of magnitude as data provided before for anaerobic toluene degradation. Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms. In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic ε (εintrinsic) were calculated. A comparison of εintrinsic at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific ε elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of ε found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average ε if no fractionation factor is available for single compounds.
AU  - Morasch, B.*
AU  - Richnow, H.H.*
AU  - Vieth, A.*
AU  - Schink, B.*
AU  - Meckenstock, R.U.
C1  - 605
C2  - 21822
SP  - 2935-2940
TI  - Stable isotope fractionation caused by glycyl radical enzymes during bacterial degradation of aromatic compounds.
JO  - Appl. Environ. Microbiol.
VL  - 70
IS  - 5
PY  - 2004
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - A new microarray method, the isotope array approach, for identifying microorganisms which consume a (14)C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [(14)C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of (14)C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO(2) fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [(14)C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO(2) fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by (14)C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of (14)C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.
AU  - Adamczyk, J.*
AU  - Hesselsoe, M.*
AU  - Iversen, N.*
AU  - Horn, M.*
AU  - Lehner, A.*
AU  - Nielsen, P.H.*
AU  - Schloter, M.
AU  - Roslev, P.*
AU  - Wagner, M.*
C1  - 24402
C2  - 32739
SP  - 6875-6887
TI  - The isotope array, a new tool that employs substrate-mediated labeling of rRNA for determination of microbial community structure and function.
JO  - Appl. Environ. Microbiol.
VL  - 69
IS  - 11
PB  - Amer. Soc. Microbiology
PY  - 2003
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Bacterial proliferations have recurrently been observed for the past 15 years in fermentor cultures of the ectomycorrhizal fungus Laccaria bicolor S238N, suggesting the presence of cryptic bacteria in the collection culture of this fungus. In this study, intracellular bacteria were detected by fluorescence in situ hybridization in combination with confocal laser scanning microscopy in several collection subcultures of L. bicolor S238N. They were small (0.5 micro m in diameter), rare, and heterogeneously distributed in the mycelium and were identified as Paenibacillus spp. by using a 16S rRNA-directed oligonucleotide probe initially designed for bacteria isolated from a fermentor culture of L. bicolor S238N.
AU  - Bertaux, J.*
AU  - Schmid, M.
AU  - Prevost-Boure, N.C.*
AU  - Churin, J.L.*
AU  - Hartmann, A.
AU  - Garbaye, J.*
AU  - Frey-Klett, P.*
C1  - 22943
C2  - 31074
SP  - 4243-4248
TI  - In situ identification of intracellular bacteria related to Paenibacillus spp. in the mycelium of the ectomycorrhizal fungus Laccaria bicolor S238N.
JO  - Appl. Environ. Microbiol.
VL  - 69
IS  - 7
PB  - American Society for Microbiology
PY  - 2003
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Given that a large proportion of the bacteria colonizing the roots of plants is capable of producingN-acyl-l-homoserine lactone (AHL) molecules, it appears likely that these bacterial pheromones may serve as signals for communication between cells of different species. In this study, we have developed and characterized novel Gfp-based monitor strains that allow in situ visualization of AHL-mediated communication between individual cells in the plant rhizosphere. For this purpose, three Gfp-based AHL sensor plasmids that respond to different spectra of AHL molecules were transferred into AHL-negative derivatives ofPseudomonas putida IsoF andSerratia liquefaciens MG1, two strains that are capable of colonizing tomato roots. These AHL monitor strains were used to visualize communication between defined bacterial populations in the rhizosphere of axenically grown tomato plants. Furthermore, we integrated into the chromosome of AHL-negativeP. putida strain F117 an AHL sensor cassette that responds to the presence of long-chain AHLs with the expression of Gfp. This monitor strain was used to demonstrate that the indigenous bacterial community colonizing the roots of tomato plants growing in nonsterile soil produces AHL molecules. The results strongly support the view that AHL signal molecules serve as a universal language for communication between the different bacterial populations of the rhizosphere consortium.
AU  - Steidle, A.*
AU  - Sigl, K.*
AU  - Schuhegger, R.
AU  - Ihring, A.*
AU  - Schmid, M.*
AU  - Gantner, S.
AU  - Stoffels, M.
AU  - Riedel, K.*
AU  - Givskov, M.*
AU  - Hartmann, A.
AU  - Langebartels, C.
AU  - Eberl, L.*
C1  - 21845
C2  - 20048
SP  - 5761-5770
TI  - Visualization of N-Acylhomoserine Lactone-Mediated Cell-Cell Communication between Bacteria Colonizing the Tomato Rhizosphere.
JO  - Appl. Environ. Microbiol.
VL  - 67
IS  - 12
PY  - 2001
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - A pair of primers and a gene probe for the amplification and detection of the Bacillus cereus neutral protease gene (NPRC) were developed. Specificity for the npr genes of the B. cereus group members B. cereus, B. mycoides, and B. thuringiensis was shown. Restriction polymorphism patterns of the PCR products confirmed the presence of the NPRC gene in all three species.
AU  - Bach, H.-J.
AU  - Errampalli, D.*
AU  - Leung, K.T.*
AU  - Lee, H.*
AU  - Hartmann, A.
AU  - Trevors, J.T.*
AU  - Munch, J.-C.
C1  - 23023
C2  - 31158
SP  - 3226-3228
TI  - Specific detection of the gene for the extracellular neutral protease of Bacillus cereus by PCR and blot hybridization.
JO  - Appl. Environ. Microbiol.
VL  - 65
IS  - 7
PB  - American Society of Microbiology
PY  - 1999
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Monospecific polyclonal antisera raised against Rhizobium leguminosarum bv. trifolii R39, a bacterium which was isolated originally from red clover nodules, were used to study the colonization of roots of leguminous and nonleguminous plants (Pisum sativum, Lupinus albus, Triticúm aestivum, and Zea mays) after inoculation. Eight weeks after inoculation of soil-grown plants, between 0.1 and 1% of the total bacterial population in the rhizospheres of all inoculated plants were identified as R. leguminosarum bv. trifolii R39. To characterize the associative colonization of the nonleguminous plants by R.leguminosarum bv. trifolii R39 in more detail, a time course study was performed with inoculated roots of Z. mays. R. leguminosarum bv. trifolii R39 was found almost exclusively in the rhizosphere soil and on the rhizoplane 4 weeks after inoculation. Colonization of inner root tissues was detected only occasionally at this time. During the process of attachment of R. leguminosarum bv. trifolii R39 to the rhizoplane, bacterial lipopolysaccharides were overexpressed, and this may be important for plant-microbe interaction. Fourteen weeks after inoculation, microcolonies of R. leguminosarum bv. trifolii R39 were detected in lysed cells of the root cortex as well as in intracellular space of central root cylinder cells. At the beginning of flowering (18 weeks after inoculation), the number of R. leguminosarum bv. trifolii R39 organisms decreased in the rhizosphere soil, rhizoplane, and inner root tissue.
AU  - Schloter, M.
AU  - Wiehe, W.*
AU  - Aßmus, B.
AU  - Steindl, H.
AU  - Becke, H.
AU  - Höflich, G.*
AU  - Hartmann, A.
C1  - 23139
C2  - 31044
SP  - 2038-2046
TI  - Root colonization of different plants by plant-growth-promoting Rhizobium leguminosarum bv. trifolii R39 studied with monospecific polyclonal antisera.
JO  - Appl. Environ. Microbiol.
VL  - 63
IS  - 5
PB  - American Society for Microbiology
PY  - 1997
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar- or soil-grown 10- and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species-specific probe for A. brasilense, a probe hybridizing to alpha subclass proteobacteria, and a probe specific for the domain Bacteria to identify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4(prm1),6-diamidino-2-phenylindole (DAPI) gave sufficient fluorescence signals to be detected with rRNA-targeted probes. Scanning confocal laser microscopy was used to overcome disturbing effects arising from autofluorescence of the object or narrow depth of focus in thick specimens. This technique also allowed high-resolution analysis of the spatial distribution of bacteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 and Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone. C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeatedly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scanning confocal laser microscopy provided a very suitable strategy for detailed studies of rhizosphere microbial ecology.
AU  - Aßmus, B.
AU  - Hutzler, P.
AU  - Kirchhof, G.
AU  - Amann, R.
AU  - Lawrence, J.R.*
AU  - Hartmann, A.
C1  - 23190
C2  - 31478
SP  - 1013-1019
TI  - In situ localization of Azospirillum brasilense in the rhizosphere of wheat with fluorescently labeled, rRNA-targeted oligonucleotide probes and scanning confocal laser microscopy.
JO  - Appl. Environ. Microbiol.
VL  - 61
IS  - 3
PB  - American Assoc. of Microbiology
PY  - 1995
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - The organochlorine Thiodan CE inhibited growth and nitrogenase activity of Azospirillum lipoferum. The active ingredient, Endosulfan, was nonspecifically bound to proteins and mainly adsorbed to the cell envelope with small amounts transported into cytosol. The involvement of the external membrane and cyst formation in protection against hazardous substances is discussed.
AU  - Buff, K.
AU  - Mano, D.M.S.
AU  - Langenbach, T.
C1  - 19946
C2  - 13113
SP  - 3173-3176
TI  - Effect of Endosulfan on Azospirillum lipoferum Growth, Morphology, Nitrogenase Activity, and Protein Binding.
JO  - Appl. Environ. Microbiol.
VL  - 58
IS  - 9
PY  - 1992
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro and 3-fluorocatechol and iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K2) were 1.62 x 10-3 sec-1 for 3-chlorocatechol and 2.38 x 10-3 sec-1 for 3-fluorocatechol. The inhibitor constants (K(i)) were 23 μM for 3-chlorocatechol and 17 μM for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-diendioic acid was formed from 3-chlorocatechol suggesting 5-chloroformyl-2-hydroxypenta-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoic acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation.
AU  - Bartels, I.
AU  - Knackmuß, H.J.
AU  - Reineke, W.
C1  - 41057
C2  - 38472
SP  - 500-505
TI  - Suicide inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-halocatechols.
JO  - Appl. Environ. Microbiol.
VL  - 47
IS  - 3
PY  - 1984
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - A chlorobenzene-degrading bacterium was isolated by continuous enrichment from a mixture of soil and sewage samples. This organism, strain WR1306, was grown in a chemostat on a mineral medium with chlorobenzene being supplied through the vapor phase with a critical D(c) value at a dilution rate of 0.55 h-1. Maximum growth rates in batch culture were accomplished at substrate concentrations of ≤0.5 mM in the culture medium. During growth on chlorobenzene, stoichiometric amounts of chloride were released. Respiration data and enzyme activities in cell extracts as well as the isolation of 3-chlorocatechol from the culture fluid are consistent with the degradation of chlorobenzene via 3-chloro-cis-1,2-dihydroxycyclohexa-3,5-diene, 3-chlorocatechol, 2-chloro-cis,cis-muconate, trans-4-carboxymethylenebut-2-en-4-olide, maleylacetate, and 3-oxoadipate.
AU  - Reineke, W.
AU  - Knackmuß, H.J.
C1  - 41741
C2  - 38470
SP  - 395-402
TI  - Microbial metabolism of haloaromatics: Isolation and properties of a chlorobenzene-degrading bacterium.
JO  - Appl. Environ. Microbiol.
VL  - 47
IS  - 2
PY  - 1984
SN  - 0099-2240
ER  - 

TY  - JOUR
AB  - Twenty-two strains of soil bacteria, including representatives of the genera Bacillus, Micromonospora, Mycobacterium, Nocardia, Streptomyces, Thermoactinomyces, and Pseudomonas and 10 unidentified gram-negative, motile, rod-shaped bacteria, were shown to degrade aldrin to its epoxide dieldrin. In every case, the exo-stereoisomer of dieldrin was produced exclusively.
AU  - Ferguson, J.A.
AU  - Korte, F.
C1  - 41651
C2  - 35687
SP  - 7-13
TI  - Epoxidation of aldrin to exo dieldrin by soil bacteria.
JO  - Appl. Environ. Microbiol.
VL  - 34
IS  - 1
PY  - 1977
SN  - 0099-2240
ER  - 

TY  - JOUR
AU  - Wolfe, R.S.
AU  - Pfennig, N.
C1  - 41859
C2  - 35690
SP  - 427-433
TI  - Reduction of sulfur by spirillum 5175 and syntrophism with Chlorobium.
JO  - Appl. Environ. Microbiol.
VL  - 33
IS  - 2
PY  - 1977
SN  - 0099-2240
ER  -