
Siegfried SchererTechnical University of Munich | TUM · Faculty of Biosciences
Siegfried Scherer
Diplom in Biology, PhD, Dr. habil.
About
426
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Introduction
Siegfried Scherer currently works at the Faculty of Biosciences, Technische Universität München. Siegfried does research in Evolutionary Biology, Microbiology and Molecular Biology. His current interest has a major focus on the bioinformatic and experimental characterization of overlapping genes in procaryotes.
Publications
Publications (426)
Analysis of genome wide transcription start sites (TSSs) revealed an unexpected complexity since not only canonical TSS of annotated genes are recognized by RNA polymerase. Non-canonical TSS were detected antisense to, or within, annotated genes as well new intergenic (orphan) TSS, not associated with known genes. Previously, it was hypothesized th...
Bacteria are the most abundant and diverse organisms among the kingdoms of life. Due to this excessive variance, finding a unified, comprehensive, and safe workflow for quantitative bacterial proteomics is challenging. In this study, we have systematically evaluated and optimized sample preparation, mass spectrometric data acquisition, and data ana...
The abundance of long overlapping genes in prokaryotic genomes is likely to be significantly underestimated. To date, only a few examples of such genes are fully established. Using RNA sequencing and ribosome profiling, we found expression of novel overlapping open reading frames in Escherichia coli O157:H7 EDL933 (EHEC). Indeed, the overlapping ca...
Ornithinibacillus (Or.ni.thi.ni.ba.cil'lus. N.L. neut. n. ornithinum, ornithine; L. masc. n. bacillus, a small staff, a wand; N.L. masc. n. Ornithinibacillus, a rod with ornithine).
Bacillota / Bacilli / Bacillales / Bacillaceae / Ornithinibacillus
The genus Ornithinibacillus was proposed in 2006 with the description of Ornithinibacillus (O.) bavar...
The existence of overlapping genes (OLGs) with significant coding overlaps revolutionises our understanding of genomic complexity. We report two exceptionally long (957 nt and 1536 nt), evolutionarily novel, translated antisense open reading frames (ORFs) embedded within annotated genes in the pathogenic Gram-negative bacterium Pseudomonas aerugino...
Background
Overlapping genes (OLGs) with long protein-coding overlapping sequences are disallowed by standard genome annotation programs, outside of viruses. Recently however they have been discovered in Archaea, diverse Bacteria, and Mammals. The biological factors underlying life’s ability to create overlapping genes require more study, and may h...
Powdered milk products, such as skim milk powder or whey protein powder, represent a large fraction of the dairy sector, especially with respect to export. In the past years, the contamination with aerobic endospore-forming bacteria has become one of the main factors to evaluate microbial powder quality. Besides mesophilic spore formers, thermophil...
Two strains of a Gram-staining-positive species were isolated from German bulk tank milk. On the basis of their 16S rRNA sequences they were affiliated to the genus Facklamia but could not be assigned to any species with a validly published name. Facklamia miroungae ATCC BAA-466 T (97.3 % 16S rRNA sequence similarity), Facklamia languida CCUG 37842...
The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The sele...
The existence of overlapping genes (OLGs) with significant coding overlaps revolutionises our understanding of genomic complexity. We report two exceptionally long (957 nt and 1536 nt), evolutionarily novel, translated antisense open reading frames (ORFs) embedded within annotated genes in the medically important Gram-negative bacterium Pseudomonas...
The heat-stable peptidase AprX, secreted by psychrotolerant Pseudomonas species in raw milk, is a major cause of destabilization and premature spoilage of ultra-high temperature (UHT) milk and milk products. To enable rapid detection and quantification of seven frequent and proteolytic Pseudomonas species ( P. proteolytica , P. gessardii , P. lacti...
During a study investigating the microbiota of raw milk and its semi-finished products, strains WS 5106 T and WS 5096 were isolated from cream and skimmed milk concentrate. They could be assigned to the genus Pseudomonas by their 16S rRNA sequences, but not to any validly named species. In this work, a polyphasic approach was used to characterize t...
The genetic code allows six reading frames at a double-stranded DNA locus, and many open reading frames (ORFs) overlap extensively with ORFs of annotated genes (e.g., at least 30 bp or having an embedded ORF). Currently, bacterial genome annotation systematically discards embedded overlapping ORFs of genes (OLGs) due to an assumed information-conte...
Overlapping genes (OLGs) with long protein-coding overlapping sequences are often excluded by genome annotation programs, with the exception of virus genomes. A recent study used a novel algorithm to construct OLGs from arbitrary protein domain pairs and concluded that virus genes are best suited for creating OLGs, a result which fitted with common...
During the last decades, thermophilic spore counts became a very important quality parameter for manufacturers with regard to powdered dairy products. Low-spore count powders are highly demanded but challenging to produce when high production volume and long process times are intended. In this study a detailed monitoring of microbial levels in thre...
Three strains of a Gram-stain-positive, catalase-negative, facultative anaerobic, and coccoid species were isolated from German bulk tank milk. Phylogenetic analyses based on the 16S rRNA gene sequences indicated that the three strains (WS4937 T , WS4759 and WS5303) constitute an independent phylogenetic lineage within the family Aerococcaceae with...
Many prokaryotic RNAs are transcribed from loci outside of annotated protein coding genes. Across bacterial species hundreds of short open reading frames antisense to annotated genes show evidence of both transcription and translation, for instance in ribosome profiling data. Determining the functional fraction of these protein products awaits furt...
Raw milk microbiota are complex communities with a significant impact on the hygienic, sensory and technological quality of milk products. However, there is a lack of knowledge on factors determining their composition. In the present study, four bulk tank milk samples of two farms at two different time points were analyzed in detail for their micro...
Psychrotolerant Pseudomonas species are a main cause of proteolytic spoilage of ultra-high temperature (UHT) milk products due to the secretion of the heat-resistant metallopeptidase AprX, which is encoded by the first gene of the aprX-lipA2 operon. While the proteolytic property has been characterized for many different Pseudomonas isolates, the u...
Antisense transcription is well known in bacteria. However, translation of antisense RNAs is typically not considered, as the implied overlapping coding at a DNA locus is assumed to be highly improbable. Therefore, such overlapping genes are systematically excluded in prokaryotic genome annotation. Here we report an exceptional 603 bp long open rea...
Many prokaryotic RNAs are transcribed from loci outside of annotated protein coding genes. Across bacterial species hundreds of short open reading frames antisense to annotated genes show evidence of both transcription and translation, for instance in ribosome profiling data. Determining the functional fraction of these protein products awaits furt...
Two strains, WS 5063T and WS 5067, isolated from raw cow's milk and skimmed milk concentrate, could be affiliated as members of the same, hitherto unknown, Pseudomonas species by 16S rRNA and rpoD gene sequences. Multilocus sequence and average nucleotide identity (ANIm) analyses based on draft genome sequences confirmed the discovery of a novel Ps...
Eight facultatively anaerobic rod-shaped bacteria were isolated from raw milk and two other dairy products. Results of phylogenetic analyses based on 16S rRNA gene sequences showed that the isolates are placed in a distinct lineage within the family Propionibacteriaceae with Propioniciclava sinopodophylli and Propioniciclava tarda as the closest re...
Antisense transcription is well known in bacteria. However, translation of antisense RNAs is typically not considered, as the implied overlapping coding at a DNA locus is assumed to be highly improbable. Therefore, such overlapping genes are systematically excluded in prokaryotic genome annotation. Here we report an exceptional 603 bp long open rea...
A polyphasic approach was used to investigate the taxonomic status of two bacterial strains, WS 5072T and WS 5092, isolated from skimmed milk concentrate and raw cow's milk. The 16S rRNA and rpoD gene sequences affiliated the strains to the same, hitherto unknown, Pseudomonas species. Further examinations of the draft genomes based on multilocus se...
Thermophilic spore formers are prevalent contaminants in powdered milk products. In this study, we investigated whether fouling layers formed during the heat treatment of milk have protective effects on entrapped thermophilic spores. First, we developed an experimental set-up to produce fouling layers and incorporate spores. It was possible to prod...
Internationally, there are no official guidelines for the quantification of thermophilic spores in dairy products, which leads to variations in applied methodology. In this study, we assess the heat sensitivity of thermophilic spores, vegetative cells grown under laboratory conditions and spores in German dairy powders to determine appropriate heat...
The occurrence of thermophilic spore formers in dairy powders is a major concern for producers worldwide. This study aims to investigate the resistance of thermophilic endospores towards cleaning solutions typically used for cleaning-in-place in dairy manufacturing plants. From eleven tested strains, all were able to survive an alkaline treatment (...
The bacterial strains 4284/11T and 812/17 isolated from the respiratory tract of two royal pythons in 2011 and 2017, respectively were subjected to taxonomic characterization. The 16S rRNA gene sequences of the two strains were identical and showed highest sequence similarities to Lysobacter tolerans UM1 T (97.2%) and Luteimonas aestuarii DSM 19680...
Only a few overlapping gene pairs are known in the best-analyzed bacterial model organism Escherichia coli. Automatic annotation programs usually annotate only one out of six reading frames at a locus, allowing only small overlaps between protein-coding sequences. However, both RNAseq and RIBOseq show signals corresponding to non-trivially overlapp...
Bacillus anthracis ist der Erreger des Milzbrands. In ihrer Zuschrift (DOI: 10.1002/ange.201807442) beschreiben A. Skerra et al. eine durch Protein‐Design erhaltene Variante eines körpereigenen Lipocalins, die den FeIII‐Petrocalin‐Komplex hochaffin bindet. Das Abfangen dieses für das pathogene Bakterium spezifischen Siderophors unterbindet die Vers...
Bacillus anthracis is the etiologic agent of anthrax disease. In their Communication (DOI: 10.1002/anie.201807442), A. Skerra and co‐workers report on the construction of a variant human lipocalin by protein design that binds the iron(III)⋅petrobactin complex with high affinity. Scavenging of this specific siderophore abolishes supply with the esse...
Bacillus anthracis constitutes a dangerous biohazard which, apart from specific toxins, owes its pronounced virulence to a two‐pronged import mechanism for FeIII ions, which are scarce in body fluids. This pathogenic bacterium secretes a pair of siderophores, bacillibactin (BB) and petrobactin (PB), of which only BB is bound and neutralized by the...
Bacillus anthracis constitutes a dangerous biohazard which, apart from specific toxins, owes its pronounced virulence to a two‐pronged import mechanism for FeIII ions, which are scarce in body fluids. This pathogenic bacterium secretes a pair of siderophores, bacillibactin (BB) and petrobactin (PB), of which only BB is bound and neutralized by the...
Saliva flow measurements and SDS-PAGE separation of human whole saliva freshly collected after oral stimulation with citric acid (sour), aspartame (sweet), iso-α-acids (bitter), mono sodium L-glutamate (umami), NaCl (salty), 6-gingerol (pungent), hydroxy-α-sanshool (tingling), and hydroxy-β-sanshool (numbing), followed by tryptic digestion, nano-HP...
Current notion presumes that only one protein is encoded at a given bacterial genetic locus. However, transcription and translation of an overlapping open reading frame (ORF) of 186 bp length were discovered by RNAseq and RIBOseq experiments. This ORF is almost completely embedded in the annotated L,D-transpeptidase gene ECs2385 of Escherichia coli...
Phylogenetic tree of species with ECs2385 homologs. Possible start codons are colored in green and stop codons in red (∗). Variable regions are colored in blue. The pink arrow indicates the position of ano.
Bacterial strains and plasmids used in this study.
Competitive growth of EHEC wild type against EHEC ano∗ at anaerobic conditions in the presence of several stressors. Wild type and mutant were mixed in equal numbers and after 18 h incubation at different growth conditions their abundance was determined by Sanger sequencing. Significant changes between complementation and mutant were calculated by...
Oligonucleotides used in this study. Restriction enzyme cut sites are highlighted in bold.
Phylogenetic tree of species with ECs2384 homologs. Possible start codons are colored in green and stop codons in red (∗). Variable regions are colored in blue.
Background: Due to the DNA triplet code, it is possible that the sequences of two or more protein-coding genes overlap to a large degree. However, such non-trivial overlaps are usually excluded by genome annotation pipelines and, thus, only a few overlapping gene pairs have been described in bacteria. In contrast, transcriptome and translatome sequ...
The main goals of this project are: (i) to improve the reliability of RNA sequencing on Illumina platforms; (ii) to develop a new, more sensitive, experimental pipeline for sequencing single bacterial cells; (iii) and, finally, to explore the individual transcriptome of isogenic cells. Currently used techniques need a large number of bacterial cell...
The general goal of the project is to find and verify new overlapping protein-coding DNA sequences in prokaryotes and to understand the underlying mechanisms with the help of models from information and communication theory. To reach these goals, a cooperation of three groups is necessary, namely a group performing in vivo and in vitro molecular bi...
In the past, short protein-coding genes were often disregarded by genome annotation pipelines. Transcriptome sequencing (RNAseq) signals outside of annotated genes have usually been interpreted to indicate either ncRNA or pervasive transcription. Therefore, in addition to the transcriptome, the translatome (RIBOseq) of the enteric pathogen Escheric...
Distribution of RCV for the short annotated genes, novel genes with and without annotated homologs.
(A) RCV distribution at BHI control. (B) RCV distribution at BHI COS.
(PPTX)
Properties of the 250 short annotated genes.
With bioinformatics methods the presence of a σ70 promoter, a ρ-independent terminator, a Shine-Dalgarno sequence and selection pressure (kA/kS) were predicted or estimated. The last column gives the classification of the short genes by the machine-learning algorithm.
(DOCX)
Conservation of the novel genes.
Summary of ORF conservation as represented in Fig 5.
(XLSX)
Significant transcriptional and translational regulation in LB compared to BHI control of the novel genes and the short annotated genes.
The mean value of the two biological replicates of transcriptome and translatome counts of the BHI control and the LB condition are shown. The log-fold change was calculated and differential gene expression was de...
Summary of NGS results.
The total number of reads, the number of reads mapping to the E. coli O157:H7 Sakai genome and the distribution of mapped reads to rRNA, tRNA and mRNA are shown. Only the reads mapping to mRNA were used for further analysis. Every library contains between 1.5–9.7 m. mRNA reads.
(DOCX)
RNAseq and RIBOseq results of three different growth conditions for the 465 novel genes and the 250 short annotated genes.
The novel genes are consecutively numbered after their appearance in the EHEC Sakai genome. The RPKM transcriptome, RPKM translatome, RCV, and coverage values represent mean values of the two biological replicates.
(DOCX)
Properties of the novel genes.
Annotated homologs in other strains/species were searched using blastp. Only the best hit is listed. The fourth column illustrates annotated homologs in other E. coli O157:H7 strains or duplications of annotated genes in EHEC Sakai. With bioinformatics methods the presence of a σ70 promoter, a ρ-independent terminator...
Transcriptional and translational regulation at BHI COS compared to BHI control of the novel genes and the short annotated genes.
The mean value of the two biological replicates of transcriptome and translatome counts of the BHI control and the stress condition COS are shown. The log-fold change was calculated and differential gene expression was d...
Summary of the Predict Protein results for the short annotated genes.
The first columns show the AA composition, followed by predicted cellular localization, number of transmembrane helices, disulfide bonds and binding motives. Additionally, secondary structures, disordered regions and domains are predicted.
(XLSX)
Classification into 'real' and 'pseudo' proteins by the machine-learning algorithm.
The upper part of the table shows the results for the novel genes and the lower part for the scrambled sequences.
(XLSX)
Conservation of intergenic sequences.
A similar process as used for Fig 5 was repeated on unannotated sequences upstream and downstream of the novel genes, but without removing sequences with stop codons. Many of the sequences had no tblastn hits (too short) and some others were excluded as more than one novel gene was situated between two annotate...
Custom script used for extracting intergenic sequences—for comparative conservation analysis.
(BASH)
Summary of the Predict Protein results for the putative proteins encoded by the novel genes.
The first columns show the AA composition, followed by predicted cellular localization, number of transmembrane helices, disulfide bonds and binding motives. Additionally, secondary structures, disordered regions and domains are predicted.
(XLSX)
Custom script used for reading frame determination in the sum signal of gene groups.
(TXT)
Custom script used for detecting sequence conservation.
(BASH)
The aim of this study was to analyze the adaptation of the environmental Listeria weihenstephanensis DSM 24698 to anaerobiosis. The complete circular genome sequence of this species is reported and the adaptation of L. weihenstephanensis DSM 24698 to oxygen availability was investigated by global transcriptional analyses via RNAseq at 18 and 34°C....
Bacillus cereus is a ubiquitous bacterial pathogen increasingly reported to be the causative agent of foodborne infections and intoxications. Since the enterotoxins linked to the diarrheal form of food poising are foremost produced in the human intestine, the toxic potential of enteropathogenic B. cereus strains is difficult to predict from studies...
Background
While NGS allows rapid global detection of transcripts, it remains difficult to distinguish ncRNAs from short mRNAs. To detect potentially translated RNAs, we developed an improved protocol for bacterial ribosomal footprinting (RIBOseq). This allowed distinguishing ncRNA from mRNA in EHEC. A high ratio of ribosomal footprints per transcr...
Premature spoilage and varying product quality due to microbial contamination still constitute major problems in the production of microfiltered and pasteurized extended shelf life (ESL) milk. Spoilage-associated bacteria may enter the product either as part of the raw milk microbiota or as recontaminants in the dairy plant. To identify spoilage-in...