The use of RNAseq to resolve the transcriptional organization of an organism was established in recent years and also showed the complexity and dynamics of bacterial transcriptomes. The aim of this ...study was to comprehensively investigate the transcriptome of the industrially relevant amino acid producer and model organism Corynebacterium glutamicum by RNAseq in order to improve its genome annotation and to describe important features for transcription and translation.
RNAseq data sets were obtained by two methods, one that focuses on 5'-ends of primary transcripts and another that provides the overall transcriptome with an improved resolution of 3'-ends of transcripts. Subsequent data analysis led to the identification of more than 2,000 transcription start sites (TSSs), the definition of 5'-UTRs (untranslated regions) for annotated protein-coding genes, operon structures and many novel transcripts located between or in antisense orientation to protein-coding regions. Interestingly, a high number of mRNAs (33%) is transcribed as leaderless transcripts. From the data, consensus promoter and ribosome binding site (RBS) motifs were identified and it was shown that the majority of genes in C. glutamicum are transcribed monocistronically, but operons containing up to 16 genes are also present.
The comprehensive transcriptome map of C. glutamicum established in this study represents a major step forward towards a complete definition of genetic elements (e.g. promoter regions, gene starts and stops, 5'-UTRs, RBSs, transcript starts and ends) and provides the ideal basis for further analyses on transcriptional regulatory networks in this organism. The methods developed are easily applicable for other bacteria and have the potential to be used also for quantification of transcriptomes, replacing microarrays in the near future.
Tropolone sesquiterpenoids (TS) are an intriguing family of biologically active fungal meroterpenoids that arise through a unique intermolecular hetero Diels–Alder (hDA) reaction between humulene and ...tropolones. Here, we report on the combinatorial biosynthesis of a series of unprecedented analogs of the TS pycnidione 1 and xenovulene A 2. In a systematic synthetic biology driven approach, we recombined genes from three TS biosynthetic gene clusters (pycnidione 1, xenovulene A 2 and eupenifeldin 3) in the fungal host Aspergillus oryzae NSAR1. Rational design of the reconstituted pathways granted control over the number of hDA reactions taking place, the chemical nature of the fused polyketide moiety (tropolono‐ vs. monobenzo‐pyranyl) and the degree of hydroxylation. Formation of unexpected monobenzopyranyl sesquiterpenoids was investigated using isotope‐feeding studies to reveal a new and highly unusual oxidative ring contraction rearrangement.
New bis‐ and mono‐ tropolono‐ and benzo‐pyranyl humulene meroterpenoids, priviledged structures with diverse potent bioactivities, were synthesised using a rational synthetic biology platform based on the heterologous expression of genes from three different natural product pathways in the fungal host Aspergillus oryzae. Control over number of additions to humulene, ring contractions and humulene hydroxylation was achieved.
Understanding Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve ...large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains.
During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed "Hamburg clone") started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance.
Our findings suggest that WGS is superior to conventional genotyping for Mtb pathogen tracing and investigating micro-epidemics. WGS provides a measure of Mtb genome evolution over time in its natural host context.
By investigating wet and dry age-related ripening of beef,
strains V3/3/4/13
and V3/K/3/5
were isolated. Strain V3/3/4/13
exhibited more than 99 % 16S rRNA gene-based similarity to
and other members ...of this group, while isolate V3/K/3/5
was very close to
and a number of relatives within the
group. Additional comparisons of complete
sequences and draft genomes allowed us to place isolate V3/3/4/13
close to
DSM 26521
. In the case of V3/K/3/5
the closest relative was
DSM 11331
. Average nucleotide identity (ANIb) and digital DNA-DNA hybridization (dDDH) values calculated from the draft genomes of V3/3/4/13
and
DSM 26521
were 88.5 and 39.8 %, respectively. For V3/K/3/5
and its closest relative
DSM 11331
, the ANIb value was 95.1 % and the dDDH value was 60.7 %. The DNA G+C contents of V3/3/4/13
and V3/K/3/5
were 57.4 and 60.8 mol%, respectively. Predominant fatty acids were C
, C
ω7
, C
cyclo and summed feature C
ω7
/C
iso 2OH. The main respiratory quinones were Q9, with minor proportions of Q8 and, in the case of V3/K/3/5
, additional Q10. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and, in the case of V3/K/3/5
, additional phosphatidylcholine. Based on the combined data, isolates V3/3/4/13
and V3/K/3/5
should be considered as representatives of two novel
species. The type strain of the newly proposed
sp. nov. is V3/3/4/13
(=DSM 113654
=LMG 32520
), a second strain belonging to the same species is FLM 004-28 (=DSM 113604=LMG 32521); the type strain for the newly proposed
sp. nov. is V3/K/3/5
(=DSM 113573
=LMG 32518
) with a second isolate FLM 11 (=DSM 113572=LMG 32519).
Biosensors have emerged as a valuable tool with high specificity and sensitivity for fast and reliable detection of hazardous substances in drinking water. Numerous substances have been addressed ...using synthetic biology approaches. However, many proposed biosensors are based on living, genetically modified organisms and are therefore limited in shelf life, usability and biosafety. We addressed these issues by the construction of an extensible, cell-free biosensor. Storage is possible through freeze drying on paper. Following the addition of an aqueous sample, a highly efficient cell-free protein synthesis (CFPS) reaction is initiated. Specific allosteric transcription factors modulate the expression of 'superfolder' green fluorescent protein (sfGFP) depending on the presence of the substance of interest. The resulting fluorescence intensities are analyzed with a conventional smartphone accompanied by simple and cheap light filters. An ordinary differential equitation (ODE) model of the biosensors was developed, which enabled prediction and optimization of performance. With an optimized cell-free biosensor based on the Shigella flexneri MerR transcriptional activator, detection of 6 μg/L Hg(II) ions in water was achieved. Furthermore, a completely new biosensor for the detection of gamma-hydroxybutyrate (GHB), a substance used as date-rape drug, was established by employing the naturally occurring transcriptional repressor BlcR from Agrobacterium tumefaciens.
Flavin-dependent halogenases catalyse halogenation of aromatic compounds. In most cases, this reaction proceeds with high regioselectivity and requires only the presence of FADH2, oxygen, and halide ...salts. Since marine habitats contain high concentrations of halides, organisms populating the oceans might be valuable sources of yet undiscovered halogenases. A new Hidden-Markov-Model (HMM) based on the PFAM tryptophan halogenase model was used for the analysis of marine metagenomes. Eleven metagenomes were screened leading to the identification of 254 complete or partial putative flavin-dependent halogenase genes. One predicted halogenase gene (brvH) was selected, codon optimised for E. coli, and overexpressed. Substrate screening revealed that this enzyme represents an active flavin-dependent halogenase able to convert indole to 3-bromoindole. Remarkably, bromination prevails also in a large excess of chloride. The BrvH crystal structure is very similar to that of tryptophan halogenases but reveals a substrate binding site that is open to the solvent instead of being covered by a loop.
Corynebacterium glutamicum is able to utilize vanillate, the product of lignin degradation, as the sole carbon source. The vanillate utilization components are encoded by the vanABK operon. The vanA ...and vanB genes encode the subunits of vanillate O -demethylase, converting vanillate to protocatechuate, while VanK is the specific vanillate transporter. The vanABK operon is regulated by a PadR-type repressor, VanR. Heterologous gene expression and variations of the vanR open reading frame revealed that the functional VanR contains 192 residues (21 kDa) and forms a dimer, as analyzed by size exclusion chromatography. In vivo , ferulate, vanillin, and vanillate induced P ᵥₐₙABK in C. glutamicum , while only vanillate induced the activity of P ᵥₐₙABK in Escherichia coli lacking the ferulate catabolic system. Differential scanning fluorimetry verified that vanillate is the only effector of VanR. Interaction between the P ᵥₐₙABK DNA fragment and the VanR protein had an equilibrium dissociation constant (K D) of 15.1 ± 1.7 nM. The VanR-DNA complex had a dissociation rate constant (K d) of (267 ± 23) × 10 ⁻⁶ s ⁻¹, with a half-life of 43.5 ± 3.6 min. DNase I footprinting localized the VanR binding site at P ᵥₐₙABK, extending from +9 to +45 on the coding strand. Deletion of the nucleotides +18 to +27 inside the VanR binding site rendered P ᵥₐₙABK constitutive. Fusion of the T7 promoter and the wild-type VanR operator, as well as its shortened versions, indicated that the inverted repeat AACTAACTAA(N ₄)TTAGGTATTT is the specific VanR binding site. It is proposed that the VanR-DNA complex contains two VanR dimers at the VanR operator.
MarR-family transcription factors often control antioxidant enzymes, multidrug efflux pumps or virulence factors in bacterial pathogens and confer resistance towards oxidative stress and antibiotics. ...In this study, we have characterized the function and redox-regulatory mechanism of the MarR-type regulator HypS in Mycobacterium smegmatis. RNA-seq transcriptomics and qRT-PCR analyses of the hypS mutant revealed that hypS is autoregulated and represses transcription of the co-transcribed hypO gene which encodes a multidrug efflux pump. DNA binding activity of HypS to the 8-5-8 bp inverted repeat sequence upstream of the hypSO operon was inhibited under NaOCl stress. However, the HypSC58S mutant protein was not impaired in DNA-binding under NaOCl stress in vitro, indicating an important role of Cys58 in redox sensing of NaOCl stress. HypS was shown to be inactivated by Cys58-Cys58’ intersubunit disulfide formation under HOCl stress, resulting in derepression of hypO transcription. Phenotype results revealed that the HypS regulon confers resistance towards HOCl, rifampicin and erythromycin stress. In conclusion, HypS was identified as a novel redox-sensitive repressor that contributes to mycobacterial resistance towards HOCl stress and antibiotics.
Display omitted
•MarR-type repressor HypS senses HOCl stress in M. smegmatis.•HypS dimer is oxidized to intersubunit disulfides by HOCl.•HypS controls the multidrug efflux pump HypO.•HypO confers resistance to HOCl, rifampicin and erythromycin.
Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such 'talented' producers represent only a minute fraction of the entire, ...mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus 'Entotheonella' with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. 'Entotheonella' spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum 'Tectomicrobia'. The pronounced bioactivities and chemical uniqueness of 'Entotheonella' compounds provide significant opportunities for ecological studies and drug discovery.
PDF
