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Sigma factors are transcriptional regulators that are part of complex regulatory networks for major cellular processes, as well as for growth phase-dependent regulation and stress response. Actinoplanes sp. SE50/110 is the natural producer of acarbose, an α-glucosidase inhibitor that is used in diabetes type 2 treatment. Acarbose biosynthesis is dependent on growth, making sigma factor engineering a promising tool for metabolic engineering. ACSP50_0507 is a homolog of the developmental and osmotic-stress-regulating Streptomyces coelicolor σH Sc . Therefore, the protein encoded by ACSP50_0507 was named σH As . Here, an Actinoplanes sp. SE50/110 expression strain for the alternative sigma factor gene ACSP50_0507 ( sigH As ) achieved a two-fold increased acarbose yield with acarbose production extending into the stationary growth phase. Transcriptome sequencing revealed upregulation of acarbose biosynthesis genes during growth and at the late stationary growth phase. Genes that are transcriptionally activated by σH As frequently code for secreted or membrane-associated proteins. This is also mirrored by the severely affected cell morphology, with hyperbranching, deformed and compartmentalized hyphae. The dehydrated cell morphology and upregulation of further genes point to a putative involvement in osmotic stress response, similar to its S. coelicolor homolog. The DNA-binding motif of σH As was determined based on transcriptome sequencing data and shows high motif similarity to that of its homolog. The motif was confirmed by in vitro binding of recombinantly expressed σH As to the upstream sequence of a strongly upregulated gene. Autoregulation of σH As was observed, and binding to its own gene promoter region was also confirmed.

Dwindling water sources increase the need for efficient wastewater treatment. Solar-driven algal turf scrubber (ATS) system may remediate wastewater by supporting the development and growth of periphytic microbiomes that function and interact in a highly dynamic manner through symbiotic interactions. Using ITS and 16S rRNA gene amplicon sequencing, we profiled the microbial communities of four microbial biofilms from ATS systems operated with municipal wastewater (mWW), diluted cattle and pig manure (CattleM and PigM), and biogas plant effluent supernatant (BGE) in comparison to the initial inocula and the respective wastewater substrates. The wastewater-driven biofilms differed significantly in their biodiversity and structure, exhibiting an inocula-independent but substrate-dependent establishment of the microbial communities. The prokaryotic communities were comparable among themselves and with other microbiomes of aquatic environments and were dominated by metabolically flexible prokaryotes such as nitrifiers, polyphosphate-accumulating and algicide-producing microorganisms, and anoxygenic photoautotrophs. Striking differences occurred in eukaryotic communities: While the mWW biofilm was characterized by high biodiversity and many filamentous (benthic) microalgae, the agricultural wastewater-fed biofilms consisted of less diverse communities with few benthic taxa mainly inhabited by unicellular chlorophytes and saprophytes/parasites. This study advances our understanding of the microbiome structure and function within the ATS-based wastewater treatment process.

Bacteria of the genus Streptomyces produce various specialized metabolites. Single biosynthetic gene clusters (BGCs) can give rise to different products that can vary in terms of their biological activities. For example, for alnumycin and the shunt product K115, antimicrobial activity was described, while no antimicrobial activity was detected for the shunt product 1,6-dihydro 8-propylanthraquinone. To investigate the antibacterial activity of 1,6-dihydro 8-propylanthraquinone, we produced alnumycin and 1,6-dihydro 8-propylanthraquinone from a Streptomyces isolate containing the alnumycin BGC. The strain was cultivated in liquid glycerol-nitrate-casein medium (GN), and both compounds were isolated using an activity and mass spectrometry-guided purification. The structures were validated via nuclear magnetic resonance (NMR) spectroscopy. A minimal inhibitory concentration (MIC) test revealed that 1,6-dihydro 8-propylanthraquinone exhibits antimicrobial activity against E. coli Δ tolC , B. subtilis , an S. aureus type strain, and a vancomycin intermediate-resistance S. aureus strain (VISA). Activity of 1,6-dihydro 8-propylanthraquinone against E. coli Δ tolC was approximately 10-fold higher than that of alnumycin. We were unable to confirm gyrase inhibition for either compound and believe that the modes of action of both compounds are worth reinvestigating.

The global characterization of transcriptional regulatory networks almost exclusively uses in vivo conditions, thereby providing a snapshot on multiple regulatory interactions at the same time. To complement these approaches, we developed and applied a method for characterizing bacterial promoters genome-wide by in vitro transcription coupled to transcriptome sequencing specific for native 5'-ends of transcripts. This method, called ROSE (run-off transcription/RNA-sequencing), only requires chromosomal DNA, ribonucleotides, RNA polymerase (RNAP) core enzyme, and a specific sigma factor, recognizing the corresponding promoters, which have to be analyzed. ROSE was performed on E. coli K-12 MG1655 genomic DNA using Escherichia coli RNAP holoenzyme (including σ70) and yielded 3226 transcription start sites, 2167 of which were also identified in in vivo studies, and 598 were new. Many new promoters not yet identified by in vivo experiments might be repressed under the tested conditions. Complementary in vivo experiments with E. coli K-12 strain BW25113 and isogenic transcription factor gene knockout mutants of fis , fur , and hns were used to test this hypothesis. Comparative transcriptome analysis demonstrated that ROSE could identify bona fide promoters that were apparently repressed in vivo. In this sense, ROSE is well-suited as a bottom-up approach for characterizing transcriptional networks in bacteria and ideally complementary to top-down in vivo transcriptome studies.

In household washing machines, opportunistic pathogens such as Pseudomonas aeruginosa are present, which represent the household as a possible reservoir for clinical pathogens. Here, four novel P. aeruginosa strains, isolated from different sites of household appliances, were investigated regarding their biofilm formation. Only two isolates showed strong surface-adhered biofilm formation. In consequence of these phenotypic differences, we performed whole genome sequencing using Oxford Nanopore Technology together with Illumina MiSeq. Whole genome data were screened for the prevalence of 285 virulence- and biofilm-associated genes as well as for prophages. Linking biofilm phenotypes and parallelly appearing gene compositions, we assume a relevancy of the las quorum sensing system and the phage-encoded bacteriophage control infection gene bci , which was found on integrated phi297 DNA in all biofilm-forming isolates. Additionally, only the isolates revealing strong biofilm formation harbored the ϕCTX-like prophage Dobby, implicating a role of this prophage on biofilm formation. Investigations on clinically relevant pathogens within household appliances emphasize their adaptability to harsh environments, with high concentrations of detergents, providing greater insights into pathogenicity and underlying mechanisms. This in turn opens the possibility to map and characterize potentially relevant strains even before they appear as pathogens in society.

The gilled mushroom Clitopilus passeckerianus (Entolomataceae, Agaricales, Basidiomycota) is well known to produce the terpenoid pleuromutilin, which is the biotechnological basis for medically important antibiotics such as lefamulin and retapamulin. Their unique mode of action and good tolerance entails an increasing demand of pleuromutilin-derived antibiotics in veterinary and human health care. Surprisingly, despite their pharmaceutical importance, no genome sequence is available of any pleuromutilin-producing fungus. Here, we present the high-quality draft genome sequence of the pleuromutilin-producer C. passeckerianus DSM1602 including functional genome annotation. More precisely, we employed a hybrid assembly strategy combining Illumina sequencing and Nanopore sequencing to assemble the mitochondrial genome as well as the nuclear genome. In accordance with the dikaryotic state of the fungus, the nuclear genome has a diploid character. Interestingly, the mitochondrial genome appears duplicated. Bioinformatic analysis revealed a versatile secondary metabolism with an emphasis on terpenoid biosynthetic enzymes in C. passeckerianus and also in related strains. Two alleles of biosynthetic gene clusters for pleuromutilin were found in the genome of C. passeckerianus . The pleuromutilin genes were reassembled with yeast-specific elements for heterologous expression in Saccharomyces cerevisiae . Our work lays the foundation for metabolic strain engineering towards higher yields of the valuable compound pleuromutilin.

The family of Ustilaginaceae belongs to the order of Basidiomycetes. Despite their plant pathogenicity causing, e.g., corn smut disease, they are also known as natural producers of value-added chemicals such as extracellular glycolipids, organic acids, and polyols. Here, we present 17 high-quality draft genome sequences (N50 > 1 Mb) combining third-generation nanopore and second-generation Illumina sequencing. The data were analyzed with taxonomical genome-based bioinformatics methods such as Percentage of Conserved Proteins (POCP), Average Nucleotide Identity (ANI), and Average Amino Acid Identity (AAI) analyses indicating that a reclassification of the Ustilaginaceae family might be required. Further, conserved core genes were determined to calculate a phylogenomic core genome tree of the Ustilaginaceae that also supported the results of the other phylogenomic analysis. In addition, to genomic comparisons, secondary metabolite clusters (e.g., itaconic acid, mannosylerythritol lipids, and ustilagic acid) of biotechnological interest were analyzed, whereas the sheer number of clusters did not differ much between species.

The microbial biogas network is complex and intertwined, and therefore relatively stable in its overall functionality. However, if key functional groups of microorganisms are affected by biotic or abiotic factors, the entire efficacy may be impaired. Bacteriophages are hypothesized to alter the steering process of the microbial network. In this study, an enriched fraction of virus-like particles was extracted from a mesophilic biogas reactor and sequenced on the Illumina MiSeq and Nanopore GridION sequencing platforms. Metagenome data analysis resulted in identifying 375 metagenome-assembled viral genomes (MAVGs). Two-thirds of the classified sequences were only assigned to the superkingdom Viruses and the remaining third to the family Siphoviridae , followed by Myoviridae , Podoviridae , Tectiviridae , and Inoviridae . The metavirome showed a close relationship to the phage genomes that infect members of the classes Clostridia and Bacilli . Using publicly available biogas metagenomic data, a fragment recruitment approach showed the widespread distribution of the MAVGs studied in other biogas microbiomes. In particular, phage sequences from mesophilic microbiomes were highly similar to the phage sequences of this study. Accordingly, the virus particle enrichment approach and metavirome sequencing provided additional genome sequence information for novel virome members, thus expanding the current knowledge of viral genetic diversity in biogas reactors.

Many bacteria encode so-called cold shock proteins. These proteins are characterized by a conserved protein domain. Often, the bacteria have multiple cold shock proteins that are expressed either constitutively or at low temperatures. In the Gram-positive model bacterium Bacillus subtilis , two of three cold shock proteins, CspB and CspD, belong to the most abundant proteins suggesting a very important function. To get insights into the role of these highly abundant proteins, we analyzed the phenotypes of single and double mutants, tested the expression of the csp genes and the impact of CspB and CspD on global gene expression in B. subtilis . We demonstrate that the simultaneous loss of both CspB and CspD results in a severe growth defect, in the loss of genetic competence, and the appearance of suppressor mutations. Overexpression of the third cold shock protein CspC could compensate for the loss of CspB and CspD. The transcriptome analysis revealed that the lack of CspB and CspD affects the expression of about 20% of all genes. In several cases, the lack of the cold shock proteins results in an increased read-through at transcription terminators suggesting that CspB and CspD might be involved in the control of transcription termination.

The aromatic heterocyclic compound indole is widely spread in nature. Due to its floral odor indole finds application in dairy, flavor, and fragrance products. Indole is an inter- and intracellular signaling molecule influencing cell division, sporulation, or virulence in some bacteria that synthesize it from tryptophan by tryptophanase. Corynebacterium glutamicum that is used for the industrial production of amino acids including tryptophan lacks tryptophanase. To test if indole is metabolized by C. glutamicum or has a regulatory role, the physiological response to indole by this bacterium was studied. As shown by RNAseq analysis, indole, which inhibited growth at low concentrations, increased expression of genes involved in the metabolism of iron, copper, and aromatic compounds. In part, this may be due to iron reduction as indole was shown to reduce Fe 3+ to Fe 2+ in the culture medium. Mutants with improved tolerance to indole were selected by adaptive laboratory evolution. Among the mutations identified by genome sequencing, mutations in three transcriptional regulator genes were demonstrated to be causal for increased indole tolerance. These code for the regulator of iron homeostasis DtxR, the regulator of oxidative stress response RosR, and the hitherto uncharacterized Cg3388. Gel mobility shift analysis revealed that Cg3388 binds to the intergenic region between its own gene and the iolT2-rhcM2D2 operon encoding inositol uptake system IolT2, maleylacetate reductase, and catechol 1,2-dioxygenase. Increased RNA levels of rhcM2 in a cg3388 deletion strain indicated that Cg3388 acts as repressor. Indole, hydroquinone, and 1,2,4-trihydroxybenzene may function as inducers of the iolT2-rhcM2D2 operon in vivo as they interfered with DNA binding of Cg3388 at physiological concentrations in vitro. Cg3388 was named IhtR.