Identification of Oxygen-Responsive Transcripts in the Silage Inoculant Lactobacillus buchneri CD034 by RNA Sequencing

Eikmeyer FG, Heinl S, Marx H, Pühler A, Grabherr R, Schlüter A. Identification of Oxygen-Responsive Transcripts in the Silage Inoculant Lactobacillus buchneri CD034 by RNA Sequencing. PLoS ONE. 2015;10(7): e0134149.The Lactobacillus buchneri CD034 strain, known to improve the ensiling process of green fodder and the quality of the silage itself was transcriptionally analyzed by sequencing of transcriptomes isolated under anaerobic vs. aerobic conditions. L. buchneri CD034 was first cultivated under anaerobic conditions and then shifted to aerobic conditions by aeration with 21% oxygen. Cultivations already showed that oxygen was consumed by L. buchneri CD034 after aeration of the culture while growth of L. buchneri CD034 was still observed. RNA sequencing data revealed that irrespective of the oxygen status of the culture, the most abundantly transcribed genes are required for basic cell functions such as protein biosynthesis, energy metabolism and lactic acid fermentation. Under aerobic conditions, 283 genes were found to be transcriptionally up-regulated while 198 genes were found to be down-regulated (p-value < 0.01). Up-regulated genes i. a. play a role in oxygen consumption via oxidation of pyruvate or lactate (pox, lctO). Additionally, genes encoding proteins required for decomposition of reactive oxygen species (ROS) such as glutathione reductase or NADH peroxidase were also found to be up-regulated. Genes related to pH homeostasis and redox potential balance were found to be down-regulated under aerobic conditions. Overall, genes required for lactic acid fermentation were hardly affected by the growth conditions applied. Genes identified to be differentially transcribed depending on the aeration status of the culture are suggested to specify the favorable performance of the strain in silage formation

Comparative metagenomics of biogas-producing microbial communities from production-scale biogas plants operating under wet or dry fermentation conditions

Stolze Y, Zakrzewski M, Maus I, et al. Comparative metagenomics of biogas-producing microbial communities from production-scale biogas plants operating under wet or dry fermentation conditions. Biotechnology for Biofuels. 2015;8(1): 14.Background Decomposition of biomass for biogas production can be practiced under wet and dry fermentation conditions. In contrast to the dry fermentation technology, wet fermentation is characterized by a high liquid content and a relatively low total solid content. In this study, the composition and functional potential of a biogas-producing microbial community in an agricultural biogas reactor operating under wet fermentation conditions was analyzed by a metagenomic approach applying 454-pyrosequencing. The obtained metagenomic dataset and corresponding 16S rRNA gene amplicon sequences were compared to the previously sequenced comparable metagenome from a dry fermentation process, meeting explicitly identical boundary conditions regarding sample and community DNA preparation, sequencing technology, processing of sequence reads and data analyses by bioinformatics tools. Results High-throughput metagenome sequencing of community DNA from the wet fermentation process applying the pyrosequencing approach resulted in 1,532,780 reads, with an average read length of 397 bp, accounting for approximately 594 million bases of sequence information in total. Taxonomic comparison of the communities from wet and dry fermentation revealed similar microbial profiles with Bacteria being the predominant superkingdom, while the superkingdom Archaea was less abundant. In both biogas plants, the bacterial phyla Firmicutes, Bacteroidetes, Spirochaetes and Proteobacteria were identified with descending frequencies. Within the archaeal superkingdom, the phylum Euryarchaeota was most abundant with the dominant class Methanomicrobia. Functional profiles of the communities revealed that environmental gene tags representing methanogenesis enzymes were present in both biogas plants in comparable frequencies. 16S rRNA gene amplicon high-throughput sequencing disclosed differences in the sub-communities comprising methanogenic Archaea between both processes. Fragment recruitments of metagenomic reads to the reference genome of the archaeon Methanoculleus bourgensis MS2T revealed that dominant methanogens within the dry fermentation process were highly related to the reference. Conclusions Although process parameters, substrates and technology differ between the wet and dry biogas fermentations analyzed in this study, community profiles are very similar at least at higher taxonomic ranks, illustrating that core community taxa perform key functions in biomass decomposition and methane synthesis. Regarding methanogenesis, Archaea highly related to the type strain M. bourgensis MS2T dominate the dry fermentation process, suggesting the adaptation of members belonging to this species to specific fermentation process parameters

Effect of the strain Bacillus amyloliquefaciens FZB42 on the microbial community in the rhizosphere of lettuce under field conditions analyzed by whole metagenome sequencing

Kröber M, Wibberg D, Grosch R, et al. Effect of the strain Bacillus amyloliquefaciens FZB42 on the microbial community in the rhizosphere of lettuce under field conditions analyzed by whole metagenome sequencing. Frontiers in Microbiology. 2014;5: 252.Application of the plant associated bacterium Bacillus amyloliquefaciens FZB42 on lettuce (Lactuca sativa) confirmed its capability to promote plant growth and health by reducing disease severity (DS) caused by the phytopathogenic fungus Rhizoctonia solani. Therefore this strain is commercially applied as an eco-friendly plant protective agent. It is able to produce cyclic lipopeptides (CLP) and polyketides featuring antifungal and antibacterial properties. Production of these secondary metabolites led to the question of a possible impact of strain FZB42 on the composition of microbial rhizosphere communities after its application. Rating of DS and lettuce growth during a field trial confirmed the positive impact of strain FZB42 on the health of the host plant. To verify B. amyloliquefaciens as an environmentally compatible plant protective agent, its effect on the indigenous rhizosphere community was analyzed by metagenome sequencing. Rhizosphere microbial communities of lettuce treated with B. amyloliquefaciens FZB42 and non-treated plants were profiled by high-throughput metagenome sequencing of whole community DNA. Fragment recruitments of metagenome sequence reads on the genome sequence of B. amyloliquefaciens FZB42 proved the presence of the strain in the rhizosphere over 5 weeks of the field trial. Comparison of taxonomic community profiles only revealed marginal changes after application of strain FZB42. The orders Burkholderiales, Actinomycetales and Rhizobiales were most abundant in all samples. Depending on plant age a general shift within the composition of the microbial communities that was independent of the application of strain FZB42 was observed. In addition to the taxonomic profiling, functional analysis of annotated sequences revealed no major differences between samples regarding application of the inoculant strain

Deeply sequenced metagenome and metatranscriptome of a biogas-producing microbial community from an agricultural production-scale biogas plant

Bremges A, Maus I, Belmann P, et al. Deeply sequenced metagenome and metatranscriptome of a biogas-producing microbial community from an agricultural production-scale biogas plant. GigaScience. 2015;4(1): 33.Background The production of biogas takes place under anaerobic conditions and involves microbial decomposition of organic matter. Most of the participating microbes are still unknown and non-cultivable. Accordingly, shotgun metagenome sequencing currently is the method of choice to obtain insights into community composition and the genetic repertoire. Findings Here, we report on the deeply sequenced metagenome and metatranscriptome of a complex biogas-producing microbial community from an agricultural production-scale biogas plant. We assembled the metagenome and, as an example application, show that we reconstructed most genes involved in the methane metabolism, a key pathway involving methanogenesis performed by methanogenic Archaea. This result indicates that there is sufficient sequencing coverage for most downstream analyses. Conclusions Sequenced at least one order of magnitude deeper than previous studies, our metagenome data will enable new insights into community composition and the genetic potential of important community members. Moreover, mapping of transcripts to reconstructed genome sequences will enable the identification of active metabolic pathways in target organisms

Untersuchung der Rolle von Lactobacillus buchneri CD034 bei der Bildung von Grassilage mittels nukleinsäurebasierter Methoden

Eikmeyer FG. Untersuchung der Rolle von Lactobacillus buchneri CD034 bei der Bildung von Grassilage mittels nukleinsäurebasierter Methoden. Bielefeld; 2015.Stämme der Spezies Lactobacillus buchneri werden als Siliermittel verwendet, da sie insbesondere die aerobe Stabilität der Silage erhöhen können. Um einen Einblick in einen solchen L. buchneri Stamm und seine Anwendung als mikrobielles Siliermittel in Grassilage zu untersuchen, wurden Genom-, Metagenom und Transkriptom-Analysen mit dem Stamm L. buchneri CD034 durchgeführt. Die Genomsequenzierung und anschließende Genannotation erlaubte die Rekonstruktion der Stoffwechselwege der heterofermentativen Milchsäuregärung und zeigte, dass L. buchneri CD034 eine Vielzahl, insbesondere pflanzlicher, Kohlehydrate verstoffwechseln kann. Zur Untersuchung der Effekte bei der Anwendung von L. buchneri CD034 als Inokulum für Grassilagen wurden physiko-chemische sowie mikrobielle Eigenschaften der Silage als auch die Zusammensetzung der mikrobiellen Gemeinschaften im Verlauf des Silierens mittels Metagenomanalysen bestimmt. Die Inokulation der Silage mit L. buchneri CD034 führte zu höheren Gehalten an Essigsäure, Ethanol und 1,2-Propandiol und niedrigeren Gehalten an Milchsäure und Hefen. Taxonomische Klassifizierungen auf Basis von 16S rRNA Gen Amplikons und Metagenomsequenzierungen zeigten in den inokulierten Silagen auch höhere Anteile von Laktobazillen in den jeweiligen bakteriellen Gemeinschaften. Fragment Recruitments konnten schließlich deutlich machen, dass L. buchneri CD034 in bakteriellen Gemeinschaften der Grassilage wettbewerbsfähig ist und sich am Ende des Silierens gegen andere Bakterien durchsetzen kann. Der Einfluss von Sauerstoff – als kennzeichnender Parameter der finalen aeroben Phase der Silierung – auf die Genexpression in L. buchneri CD034 wurde durch RNA-Sequencing und anschließende Transkriptomanalyse untersucht. Obwohl L. buchneri CD034 ein fermentativer Organismus ist, verbraucht er dennoch Sauerstoff. Dies geschieht vermutlich durch Pyruvat- und Lactat-Oxidasen, deren Gene unter Sauerstoffanwesenheit stärker transkribiert waren. Gene, die für die Milchsäurebildung benötigt werden, werden kaum durch Sauerstoff in ihrer Transkription beeinflusst. Im Gegensatz dazu werden Gene, die Enzyme für die Bildung der weiteren Gärungsprodukte wie Essigsäure oder Ethanol kodieren, unter Sauerstoffanwesenheit durchaus differentiell transkribiert. L. buchneri CD034 verfügt damit über weitere Eigenschaften, die die aerobe Stabilität der Silage positiv beeinflussen können

The IncF plasmid pRSB225 isolated from a municipal wastewater treatment plant’s on-site preflooder combining antibiotic resistance and putative virulence functions is highly related to virulence plasmids identified in pathogenic E. coli isolates

Wibberg D, Szczepanowski R, Eikmeyer FG, Pühler A, Schlüter A. The IncF plasmid pRSB225 isolated from a municipal wastewater treatment plant’s on-site preflooder combining antibiotic resistance and putative virulence functions is highly related to virulence plasmids identified in pathogenic E. coli isolates. Plasmid. 2013;69(2):127-137

Taxonomic Profiling and Metagenome Analysis of a Microbial Community from a Habitat Contaminated with Industrial Discharges

Shah V, Zakrzewski M, Wibberg D, Eikmeyer FG, Schlüter A, Madamwar D. Taxonomic Profiling and Metagenome Analysis of a Microbial Community from a Habitat Contaminated with Industrial Discharges. Microbial Ecology. 2013;66(3):533-550

Sequence of Two Plasmids from Clostridium perfringens Chicken Necrotic Enteritis Isolates and Comparison with C. perfringens Conjugative Plasmids

Parreira VR, Costa M, Eikmeyer FG, Blom J, Prescott JF. Sequence of Two Plasmids from Clostridium perfringens Chicken Necrotic Enteritis Isolates and Comparison with C. perfringens Conjugative Plasmids. PLoS ONE. 2012;7(11): e49753.Twenty-six isolates of Clostridium perfringens of different MLST types from chickens with necrotic enteritis (NE) (15 netB-positive) or from healthy chickens (6 netB-positive, 5 netB-negative) were found to contain 1–4 large plasmids, with most netB-positive isolates containing 3 large and variably sized plasmids which were more numerous and larger than plasmids in netB-negative isolates. NetB and cpb2 were found on different plasmids consistent with previous studies. The pathogenicity locus NELoc1, which includes netB, was largely conserved in these plasmids whereas NeLoc3, present in the cpb2 containing plasmids, was less well conserved. A netB-positive and a cpb2-positive plasmid were likely to be conjugative, and the plasmids were completely sequenced. Both plasmids possessed the intact tcp conjugative region characteristic of C. perfringens conjugative plasmids. Comparative genomic analysis of nine CpCPs, including the two plasmids described here, showed extensive gene rearrangements including pathogenicity locus and accessory gene insertions around rather than within the backbone region. The pattern that emerges from this analysis is that the major toxin-containing regions of the variety of virulence-associated CpCPs are organized as complex pathogenicity loci. How these different but related CpCPs can co-exist in the same host has been an unanswered question. Analysis of the replication-partition region of these plasmids suggests that this region controls plasmid incompatibility, and that CpCPs can be grouped into at least four incompatibility groups

Complete sequence of broad-host-range plasmid pNOR-2000 harbouring the metallo-beta-lactamase gene blaVIM-2 from Pseudomonas aeruginosa

Bonnin RA, Poirel L, Nordmann P, et al. Complete sequence of broad-host-range plasmid pNOR-2000 harbouring the metallo-beta-lactamase gene blaVIM-2 from Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy. 2013;68(5):1060-1065

Sequencing and comparative analysis of IncP-1 alpha antibiotic resistance plasmids reveal a highly conserved backbone and differences within accessory regions

Szczepanowski R, Eikmeyer FG, Harfmann J, et al. Sequencing and comparative analysis of IncP-1 alpha antibiotic resistance plasmids reveal a highly conserved backbone and differences within accessory regions. Journal of Biotechnology. 2011;155(1):95-103.Although IncP-1 plasmids are important for horizontal gene transfer among bacteria, in particular antibiotic resistance spread, so far only three plasmids from the subgroup IncP-1 alpha have been completely sequenced. In this study we doubled this number. The three IncP-1 alpha plasmids pB5, pB11 and pSP21 were isolated from bacteria of two different sewage treatment plants and sequenced by a combination of next-generation and capillary sequencing technologies. A comparative analysis including the previously analysed IncP-1 alpha plasmids RK2, pTB11 and pBS228 revealed a highly conserved plasmid backbone (at least 99.9% DNA sequence identity) comprising 54 core genes. The accessory elements of the plasmid pB5 constitute a class 1 integron interrupting the parC gene and an IS6100 copy inserted into the integron. In addition, the tetracycline resistance genes tetAR and the ISTB11-like element are located between the klc operon and the trfA-ssb operon. Plasmid pB11 is loaded with a Tn5053-like mercury resistance transposon between the parCBA and parDE operons and contains tetAR that are identical to those identified in plasmid pB5 and the insertion sequence ISSP21. Plasmid pSP21 harbours an ISPa7 element in a Tn402 transposon including a class 1 integron between the partitioning genes parCBA and parDE. The IS-element ISSP21 (99.89% DNA sequence identity to ISSP21 from pB11), inserted downstream of the tetR gene and a copy of ISTB11 (identical to ISTB11 on pTB11) inserted between the genes pncA and pinR. On all three plasmids the accessory genes are almost always located between the backbone modules confirming the importance of the backbone functions for plasmid maintenance. The striking backbone conservation among the six completely sequenced IncP-1 alpha plasmids is in contrast to the much higher diversity within the IncP-1 alpha subgroup. (C) 2010 Elsevier B. V. All rights reserved