The MerR-family regulator NmlR is involved in the defense against oxidative stress in Streptococcus pneumoniae.

Streptococcus pneumoniae has to cope with the strong oxidant hypochlorous acid (HOCl), during host-pathogen interactions. Thus, we analyzed the global gene expression profile of S. pneumoniae D39 towards HOCl stress. In the RNA-seq transcriptome, the NmlR, SifR, CtsR, HrcA, SczA and CopY regulons and the etrx1-ccdA1-msrAB2 operon were most strongly induced under HOCl stress, which participate in the oxidative, electrophile and metal stress response in S. pneumoniae. The MerR-family regulator NmlR harbors a conserved Cys52 and controls the alcohol dehydrogenase-encoding adhC gene under carbonyl and NO stress. We demonstrated that NmlR senses also HOCl stress to activate transcription of the nmlR-adhC operon. HOCl-induced transcription of adhC required Cys52 of NmlR in vivo. Using mass spectrometry, NmlR was shown to be oxidized to intersubunit disulfides or S-glutathionylated under oxidative stress in vitro. A broccoli-FLAP-based assay further showed that both NmlR disulfides significantly increased transcription initiation at the nmlR promoter by RNAP in vitro, which depends on Cys52. Phenotype analyses revealed that NmlR functions in the defense against oxidative stress and promotes survival of S. pneumoniae during macrophage infections. In conclusion, NmlR was characterized as HOCl-sensing transcriptional regulator, which activates transcription of adhC under oxidative stress by thiol switches in S. pneumoniae.

Hunting for pigments in bacterial settlers of the Great Pacific Garbage Patch.

The Great Pacific Garbage Patch, a significant collection of plastic introduced by human activities, provides an ideal environment to study bacterial lifestyles on plastic substrates. We proposed that bacteria colonizing the floating plastic debris would develop strategies to deal with the ultraviolet-exposed substrate, such as the production of antioxidant pigments. We observed a variety of pigmentation in 67 strains that were directly cultivated from plastic pieces sampled from the Garbage Patch. The genomic analysis of four representative strains, each distinct in taxonomy, revealed multiple pathways for carotenoid production. These pathways include those that produce less common carotenoids and a cluster of photosynthetic genes. This cluster appears to originate from a potentially new species of the Rhodobacteraceae family. This represents the first report of an aerobic anoxygenic photoheterotrophic bacterium from plastic biofilms. Spectral analysis showed that the bacteria actively produce carotenoids, such as beta-carotene and beta-cryptoxanthin, and bacteriochlorophyll a. Furthermore, we discovered that the genetic ability to synthesize carotenoids is more common in plastic biofilms than in the surrounding water communities. Our findings suggest that plastic biofilms could be an overlooked source of bacteria-produced carotenoids, including rare forms. It also suggests that photoreactive molecules might play a crucial role in bacterial biofilm communities in surface water.

Pseudomonas kulmbachensis sp. nov. and Pseudomonas paraveronii sp. nov., originating from chilled beef and chicken breast.

By investigating wet and dry age-related ripening of beef, Pseudomonas strains V3/3/4/13T and V3/K/3/5T were isolated. Strain V3/3/4/13T exhibited more than 99 % 16S rRNA gene-based similarity to Pseudomonas fragi and other members of this group, while isolate V3/K/3/5T was very close to Pseudomonas veronii and a number of relatives within the Pseudomonas fluorescens group. Additional comparisons of complete rpoB sequences and draft genomes allowed us to place isolate V3/3/4/13T close to Pseudomonas deceptionensis DSM 26521T. In the case of V3/K/3/5T the closest relative was P. veronii DSM 11331T. Average nucleotide identity (ANIb) and digital DNA-DNA hybridization (dDDH) values calculated from the draft genomes of V3/3/4/13T and P. deceptionensis DSM 26521T were 88.5 and 39.8 %, respectively. For V3/K/3/5T and its closest relative P. veronii DSM 11331T, the ANIb value was 95.1 % and the dDDH value was 60.7 %. The DNA G+C contents of V3/3/4/13T and V3/K/3/5T were 57.4 and 60.8 mol%, respectively. Predominant fatty acids were C16 : 0, C18 : 1 ω7c, C17 : 0 cyclo and summed feature C16 : 1 ω7ct/C15 : 0 iso 2OH. The main respiratory quinones were Q9, with minor proportions of Q8 and, in the case of V3/K/3/5T, additional Q10. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and, in the case of V3/K/3/5T, additional phosphatidylcholine. Based on the combined data, isolates V3/3/4/13T and V3/K/3/5T should be considered as representatives of two novel Pseudomonas species. The type strain of the newly proposed Pseudomonas kulmbachensis sp. nov. is V3/3/4/13T (=DSM 113654T=LMG 32520T), a second strain belonging to the same species is FLM 004-28 (=DSM 113604=LMG 32521); the type strain for the newly proposed Pseudomonas paraveronii sp. nov. is V3/K/3/5T (=DSM 113573T=LMG 32518T) with a second isolate FLM 11 (=DSM 113572=LMG 32519).

Lacticaseibacillus parahuelsenbergensis sp. nov., Lacticaseibacillus styriensis sp. nov. and Lacticaseibacillus zeae subsp. silagei subsp. nov., isolated from different grass and corn silage.

Four rod-shaped, non-motile, non-spore-forming, facultative anaerobic, Gram-stain-positive lactic acid bacteria, designated as EB0058T, SCR0080, LD0937T and SCR0063T, were isolated from different corn and grass silage samples. The isolated strains were characterized using a polyphasic approach and EB0058T and SCR0080 were identified as Lacticaseibacillus zeae by 16S rRNA gene sequence analysis. Based on whole-genome sequence-based characterization, EB0058T and SCR0080 were separated into a distinct clade from Lacticaseibacillus zeae DSM 20178T, together with CECT9104 and UD2202, whose genomic sequences are available from NCBI GenBank. The average nucleotide identity (ANI) values within the new subgroup are 99.9 % and the digital DNA-DNA hybridization (dDDH) values are 99.3-99.9 %, respectively. In contrast, comparison of the new subgroup with publicly available genomic sequences of L. zeae strains, including the type strain DSM 20178T, revealed dDDH values of 70.2-72.5 % and ANI values of 96.2-96.6 %. Based on their chemotaxonomic, phenotypic and phylogenetic characteristics, EB0058T and SCR0080 represent a new subspecies of L. zeae. The name Lacticaseibacillus zeae subsp. silagei subsp. nov. is proposed with the type strain EB0058T (=DSM 116376T=NCIMB 15474T). According to the results of 16S rRNA gene sequencing, LD0937T and SCR0063T are members of the Lacticaseibacillus group. The dDDH value between the isolates LD0937T and SCR0063T was 67.6 %, which is below the species threshold of 70 %, clearly showing that these two isolates belong to different species. For both strains, whole genome-sequencing revealed that the closest relatives within the Lacticaseibacillus group were Lacticaseibacillus huelsenbergensis DSM 115425 (dDDH 66.5 and 65.9 %) and Lacticaseibacillus casei DSM 20011T (dDDH 64.1 and 64.9 %). Based on the genomic, chemotaxonomic and morphological data obtained in this study, two novel species, Lacticaseibacillus parahuelsenbergensis sp. nov. and Lacticaseibacillus styriensis sp. nov. are proposed and the type strains are LD0937T (=DSM 116105T=NCIMB 15471T) and SCR0063T (=DSM 116297T=NCIMB 15473T), respectively.

Itaconate Production from Crude Substrates with U. maydis: Scale-up of an Industrially Relevant Bioprocess.

BACKGROUND: Industrial by-products accrue in most agricultural or food-related production processes, but additional value chains have already been established for many of them. Crude glycerol has a 60% lower market value than commercial glucose, as large quantities are produced in the biodiesel industry, but its valorisation is still underutilized. Due to its high carbon content and the natural ability of many microorganisms to metabolise it, microbial upcycling is a suitable option for this waste product. RESULTS: In this work, the use of crude glycerol for the production of the value-added compound itaconate is demonstrated using the smut fungus Ustilago maydis. Starting with a highly engineered strain, itaconate production from an industrial glycerol waste stream was quickly established on a small scale, and the resulting yields were already competitive with processes using commercial sugars. Adaptive laboratory evolution resulted in an evolved strain with a 72% increased growth rate on glycerol. In the subsequent development and optimisation of a fed-batch process on a 1.5-2 L scale, the use of molasses, a side stream of sugar beet processing, eliminated the need for other expensive media components such as nitrogen or vitamins for biomass growth. The optimised process was scaled up to 150 L, achieving an overall titre of 72 g L- 1, a yield of 0.34 g g- 1, and a productivity of 0.54 g L- 1 h- 1. CONCLUSIONS: Pilot-scale itaconate production from the complementary waste streams molasses and glycerol has been successfully established. In addition to achieving competitive performance indicators, the proposed dual feedstock strategy offers lower process costs and carbon footprint for the production of bio-based itaconate.

Enhanced protein secretion in reduced genome strains of Streptomyces lividans.

BACKGROUND: S. lividans TK24 is a popular host for the production of small molecules and the secretion of heterologous protein. Within its large genome, twenty-nine non-essential clusters direct the biosynthesis of secondary metabolites. We had previously constructed ten chassis strains, carrying deletions in various combinations of specialized metabolites biosynthetic clusters, such as those of the blue actinorhodin (act), the calcium-dependent antibiotic (cda), the undecylprodigiosin (red), the coelimycin A (cpk) and the melanin (mel) clusters, as well as the genes hrdD, encoding a non-essential sigma factor, and matAB, a locus affecting mycelial aggregation. Genome reduction was aimed at reducing carbon flow toward specialized metabolite biosynthesis to optimize the production of secreted heterologous protein. RESULTS: Two of these S. lividans TK24 derived chassis strains showed ~ 15% reduction in biomass yield, 2-fold increase of their total native secretome mass yield and enhanced abundance of several secreted proteins compared to the parental strain. RNAseq and proteomic analysis of the secretome suggested that genome reduction led to cell wall and oxidative stresses and was accompanied by the up-regulation of secretory chaperones and of secDF, a Sec-pathway component. Interestingly, the amount of the secreted heterologous proteins mRFP and mTNFα, by one of these strains, was 12 and 70% higher, respectively, than that secreted by the parental strain. CONCLUSION: The current study described a strategy to construct chassis strains with enhanced secretory abilities and proposed a model linking the deletion of specialized metabolite biosynthetic clusters to improved production of secreted heterologous proteins.

Manipulation of topoisomerase expression inhibits cell division but not growth and reveals a distinctive promoter structure in Synechocystis.

In cyanobacteria DNA supercoiling varies over the diurnal cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knockdown of gyrase subunits and overexpression of topoisomerase I (TopoI). Cell division was blocked but cell growth continued in all strains. The small endogenous plasmids were only transiently relaxed, then became strongly supercoiled in the TopoI overexpression strain. Transcript abundances showed a pronounced 5'/3' gradient along transcription units, incl. the rRNA genes, in the gyrase knockdown strains. These observations are consistent with the basic tenets of the homeostasis and twin-domain models of supercoiling in bacteria. TopoI induction initially led to downregulation of G+C-rich and upregulation of A+T-rich genes. The transcriptional response quickly bifurcated into six groups which overlap with diurnally co-expressed gene groups. Each group shows distinct deviations from a common core promoter structure, where helically phased A-tracts are in phase with the transcription start site. Together, our data show that major co-expression groups (regulons) in Synechocystis all respond differentially to DNA supercoiling, and suggest to re-evaluate the long-standing question of the role of A-tracts in bacterial promoters.

Discovery and overproduction of novel highly bioactive pamamycins through transcriptional engineering of the biosynthetic gene cluster.

BACKGROUND: Pamamycins are a family of highly bioactive macrodiolide polyketides produced by Streptomyces alboniger as a complex mixture of derivatives with molecular weights ranging from 579 to 705 Daltons. The large derivatives are produced as a minor fraction, which has prevented their isolation and thus studies of chemical and biological properties. RESULTS: Herein, we describe the transcriptional engineering of the pamamycin biosynthetic gene cluster (pam BGC), which resulted in the shift in production profile toward high molecular weight derivatives. The pam BGC library was constructed by inserting randomized promoter sequences in front of key biosynthetic operons. The library was expressed in Streptomyces albus strain with improved resistance to pamamycins to overcome sensitivity-related host limitations. Clones with modified pamamycin profiles were selected and the properties of engineered pam BGC were studied in detail. The production level and composition of the mixture of pamamycins was found to depend on balance in expression of the corresponding biosynthetic genes. This approach enabled the isolation of known pamamycins and the discovery of three novel derivatives with molecular weights of 663 Da and higher. One of them, homopamamycin 677A, is the largest described representative of this family of natural products with an elucidated structure. The new pamamycin 663A shows extraordinary activity (IC50 2 nM) against hepatocyte cancer cells as well as strong activity (in the one-digit micromolar range) against a range of Gram-positive pathogenic bacteria. CONCLUSION: By employing transcriptional gene cluster refactoring, we not only enhanced the production of known pamamycins but also discovered novel derivatives exhibiting promising biological activities. This approach has the potential for broader application in various biosynthetic gene clusters, creating a sustainable supply and discovery platform for bioactive natural products.

Coupling of the engineered DNA "mutator" to a biosensor as a new paradigm for activation of silent biosynthetic gene clusters in Streptomyces.

DNA replication fidelity in Streptomyces bacteria, prolific producers of many medically important secondary metabolites, is understudied, while in Escherichia coli it is controlled by DnaQ, the ϵ subunit of DNA polymerase III (DNA PolIII). Manipulation of dnaQ paralogues in Streptomyces lividans TK24, did not lead to increased spontaneous mutagenesis in this bacterium suggesting that S. lividans DNA PolIII uses an alternative exonuclease activity for proofreading. In Mycobacterium tuberculosis, such activity is attributed to the DnaE protein representing α subunit of DNA PolIII. Eight DnaE mutants designed based on the literature data were overexpressed in S. lividans, and recombinant strains overexpressing two of these mutants displayed markedly increased frequency of spontaneous mutagenesis (up to 1000-fold higher compared to the control). One of these 'mutators' was combined in S. lividans with a biosensor specific for antibiotic coelimycin, which biosynthetic gene cluster is present but not expressed in this strain. Colonies giving a positive biosensor signal appeared at a frequency of ca 10-5, and all of them were found to produce coelimycin congeners. This result confirmed that our approach can be applied for chemical- and radiation-free mutagenesis in Streptomyces leading to activation of orphan biosynthetic gene clusters and discovery of novel bioactive secondary metabolites.

The transcriptomic landscape of Magnetospirillum gryphiswaldense during magnetosome biomineralization.

BACKGROUND: One of the most complex prokaryotic organelles are magnetosomes, which are formed by magnetotactic bacteria as sensors for navigation in the Earth's magnetic field. In the alphaproteobacterium Magnetospirillum gryphiswaldense magnetosomes consist of chains of magnetite crystals (Fe3O4) that under microoxic to anoxic conditions are biomineralized within membrane vesicles. To form such an intricate structure, the transcription of > 30 specific structural genes clustered within the genomic magnetosome island (MAI) has to be coordinated with the expression of an as-yet unknown number of auxiliary genes encoding several generic metabolic functions. However, their global regulation and transcriptional organization in response to anoxic conditions most favorable for magnetite biomineralization are still unclear. RESULTS: Here, we compared transcriptional profiles of anaerobically grown magnetosome forming cells with those in which magnetosome biosynthesis has been suppressed by aerobic condition. Using whole transcriptome shotgun sequencing, we found that transcription of about 300 of the > 4300 genes was significantly enhanced during magnetosome formation. About 40 of the top upregulated genes are directly or indirectly linked to aerobic and anaerobic respiration (denitrification) or unknown functions. The mam and mms gene clusters, specifically controlling magnetosome biosynthesis, were highly transcribed, but constitutively expressed irrespective of the growth condition. By Cappable-sequencing, we show that the transcriptional complexity of both the MAI and the entire genome decreased under anaerobic conditions optimal for magnetosome formation. In addition, predominant promoter structures were highly similar to sigma factor σ70 dependent promoters in other Alphaproteobacteria. CONCLUSIONS: Our transcriptome-wide analysis revealed that magnetite biomineralization relies on a complex interplay between generic metabolic processes such as aerobic and anaerobic respiration, cellular redox control, and the biosynthesis of specific magnetosome structures. In addition, we provide insights into global regulatory features that have remained uncharacterized in the widely studied model organism M. gryphiswaldense, including a comprehensive dataset of newly annotated transcription start sites and genome-wide operon detection as a community resource (GEO Series accession number GSE197098).

Biosynthetic Potential of the Endophytic Fungus Helotiales sp. BL73 Revealed via Compound Identification and Genome Mining.

Endophytic fungi have been recognized as prolific producers of chemically diverse secondary metabolites. In this work, we describe a new representative of the order Helotiales isolated from the medicinal plant Bergenia pacumbis. Several bioactive secondary metabolites were produced by this Helotiales sp. BL 73 isolate grown on rice medium, including cochlioquinones and isofusidienols. Sequencing and analysis of the approximately 59-Mb genome revealed at least 77 secondary metabolite biosynthesis gene clusters, of which several could be associated with detected compounds or linked to previously reported molecules. Four terpene synthase genes identified in the BL73 genome were codon optimized and expressed, together with farnesyl-, geranyl-, and geranylgeranyl-pyrophosphate synthases, in Streptomyces spp. An analysis of recombinant strains revealed the production of linalool and its oxidized form, terpenoids typically associated with plants, as well as a yet unidentified terpenoid. This study demonstrates the importance of a complex approach to the investigation of the biosynthetic potential of endophytic fungi using both conventional methods and genome mining. IMPORTANCE Endophytic fungi represent an as yet underexplored source of secondary metabolites, of which some may have industrial and medical applications. We isolated a slow-growing fungus belonging to the order Helotiales from the traditional medicinal plant Bergenia pacumbis and characterized its potential to biosynthesize secondary metabolites. We used cultivation of the isolate with a subsequent analysis of compounds produced, bioinformatics-based mining of the genome, and heterologous expression of several terpene synthase genes. Our study revealed that this Helotiales isolate has enormous potential to produce structurally diverse natural products, including polyketides, nonribosomally synthesized peptides, terpenoids, and ribosomally synthesized and posttranslationally modified peptides (RiPPs). Identification of meroterpenoids and xanthones, along with establishing a link between these molecules and their putative biosynthetic genes, sets the stage for investigation of the respective biosynthetic pathways. The heterologous production of terpenoids suggests that this approach can be used for the discovery of new compounds belonging to this chemical class using Streptomyces bacteria as hosts.

Anaeropeptidivorans aminofermentans gen. nov., sp. nov., a mesophilic proteolytic salt-tolerant bacterium isolated from a laboratory-scale biogas fermenter, and emended description of Clostridium colinum.

An anaerobic bacterial strain, designated strain M3/9T, was isolated from a laboratory-scale biogas fermenter fed with maize silage supplemented with 5 % wheat straw. Cells were straight, non-motile rods, which stained Gram-negative. Optimal growth occurred between 30 and 40°C, at pH 7.5-8.5, and up to 3.9 % (w/v) NaCl was tolerated. When grown on peptone from casein and soymeal, strain M3/9T produced mainly acetic acid, ethanol, and isobutyric acid. The major cellular fatty acids of the novel strain were C16 : 0 and C16 : 0 DMA. The genome of strain M3/9T is 3757  330 bp in size with a G+C content of 38.45 mol%. Phylogenetic analysis allocated strain M3/9T within the family Lachnospiraceae with Clostridium colinum DSM 6011T and Anaerotignum lactatifermentans DSM 14214T being the most closely related species sharing 57.86 and 56.99% average amino acid identity and 16S rRNA gene sequence similarities of 91.58 and 91.26 %, respectively. Based on physiological, chemotaxonomic and genetic data, we propose the description of a novel species and genus Anaeropeptidivorans aminofermentans gen. nov., sp. nov., represented by the type strain M3/9T (=DSM 100058T=LMG 29527T). In addition, an emended description of Clostridium colinum is provided.

Bioassay-Guided Fractionation Leads to the Detection of Cholic Acid Generated by the Rare Thalassomonas sp.

Bacterial symbionts of marine invertebrates are rich sources of novel, pharmaceutically relevant natural products that could become leads in combatting multidrug-resistant pathogens and treating disease. In this study, the bioactive potential of the marine invertebrate symbiont Thalassomonas actiniarum was investigated. Bioactivity screening of the strain revealed Gram-positive specific antibacterial activity as well as cytotoxic activity against a human melanoma cell line (A2058). The dereplication of the active fraction using HPLC-MS led to the isolation and structural elucidation of cholic acid and 3-oxo cholic acid. T. actiniarum is one of three type species belonging to the genus Thalassomonas. The ability to generate cholic acid was assessed for all three species using thin-layer chromatography and was confirmed by LC-MS. The re-sequencing of all three Thalassomonas type species using long-read Oxford Nanopore Technology (ONT) and Illumina data produced complete genomes, enabling the bioinformatic assessment of the ability of the strains to produce cholic acid. Although a complete biosynthetic pathway for cholic acid synthesis in this genus could not be determined based on sequence-based homology searches, the identification of putative penicillin or homoserine lactone acylases in all three species suggests a mechanism for the hydrolysis of conjugated bile acids present in the growth medium, resulting in the generation of cholic acid and 3-oxo cholic acid. With little known currently about the bioactivities of this genus, this study serves as the foundation for future investigations into their bioactive potential as well as the potential ecological role of bile acid transformation, sterol modification and quorum quenching by Thalassomonas sp. in the marine environment.

A Second Gamma-Glutamylpolyamine Synthetase, GlnA2, Is Involved in Polyamine Catabolism in Streptomyces coelicolor.

Streptomyces coelicolor is a soil bacterium living in a habitat with very changeable nutrient availability. This organism possesses a complex nitrogen metabolism and is able to utilize the polyamines putrescine, cadaverine, spermidine, and spermine and the monoamine ethanolamine. We demonstrated that GlnA2 (SCO2241) facilitates S. coelicolor to survive under high toxic polyamine concentrations. GlnA2 is a gamma-glutamylpolyamine synthetase, an enzyme catalyzing the first step in polyamine catabolism. The role of GlnA2 was confirmed in phenotypical studies with a glnA2 deletion mutant as well as in transcriptional and biochemical analyses. Among all GS-like enzymes in S. coelicolor, GlnA2 possesses the highest specificity towards short-chain polyamines (putrescine and cadaverine), while its functional homolog GlnA3 (SCO6962) prefers long-chain polyamines (spermidine and spermine) and GlnA4 (SCO1613) accepts only monoamines. The genome-wide RNAseq analysis in the presence of the polyamines putrescine, cadaverine, spermidine, or spermine revealed indication of the occurrence of different routes for polyamine catabolism in S. coelicolor involving GlnA2 and GlnA3. Furthermore, GlnA2 and GlnA3 are differently regulated. From our results, we can propose a complemented model of polyamine catabolism in S. coelicolor, which involves the gamma-glutamylation pathway as well as other alternative utilization pathways.

Exposure to 1-Butanol Exemplifies the Response of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius to Solvent Stress.

Sulfolobus acidocaldarius is a thermoacidophilic crenarchaeon with optimal growth at 80°C and pH 2 to 3. Due to its unique physiological properties, allowing life at environmental extremes, and the recent availability of genetic tools, this extremophile has received increasing interest for biotechnological applications. In order to elucidate the potential of tolerating process-related stress conditions, we investigated the response of S. acidocaldarius toward the industrially relevant organic solvent 1-butanol. In response to butanol exposure, biofilm formation of S. acidocaldarius was enhanced and occurred at up to 1.5% (vol/vol) 1-butanol, while planktonic growth was observed at up to 1% (vol/vol) 1-butanol. Confocal laser-scanning microscopy revealed that biofilm architecture changed with the formation of denser and higher tower-like structures. Concomitantly, changes in the extracellular polymeric substances with enhanced carbohydrate and protein content were determined in 1-butanol-exposed biofilms. Using scanning electron microscopy, three different cell morphotypes were observed in response to 1-butanol. Transcriptome and proteome analyses were performed comparing the response of planktonic and biofilm cells in the absence and presence of 1-butanol. In response to 1% (vol/vol) 1-butanol, transcript levels of genes encoding motility and cell envelope structures, as well as membrane proteins, were reduced. Cell division and/or vesicle formation were upregulated. Furthermore, changes in immune and defense systems, as well as metabolism and general stress responses, were observed. Our findings show that the extreme lifestyle of S.acidocaldarius coincided with a high tolerance to organic solvents. This study provides what may be the first insights into biofilm formation and membrane/cell stress caused by organic solvents in S. acidocaldariusIMPORTANCEArchaea are unique in terms of metabolic and cellular processes, as well as the adaptation to extreme environments. In the past few years, the development of genetic systems and biochemical, genetic, and polyomics studies has provided deep insights into the physiology of some archaeal model organisms. In this study, we used S. acidocaldarius, which is adapted to the two extremes of low pH and high temperature, to study its tolerance and robustness as well as its global cellular response toward organic solvents, as exemplified by 1-butanol. We were able to identify biofilm formation as a primary cellular response to 1-butanol. Furthermore, the triggered cell/membrane stress led to significant changes in culture heterogeneity accompanied by changes in central cellular processes, such as cell division and cellular defense systems, thus suggesting a global response for the protection at the population level.

Identification and elimination of genomic regions irrelevant for magnetosome biosynthesis by large-scale deletion in Magnetospirillum gryphiswaldense.

BACKGROUND: Magnetosome formation in the alphaproteobacterium Magnetospirillum gryphiswaldense is controlled by more than 30 known mam and mms genes clustered within a large genomic region, the 'magnetosome island' (MAI), which also harbors numerous mobile genetic elements, repeats, and genetic junk. Because of the inherent genetic instability of the MAI caused by neighboring gene content, the elimination of these regions and their substitution by a compact, minimal magnetosome expression cassette would be important for future analysis and engineering. In addition, the role of the MAI boundaries and adjacent regions are still unclear, and recent studies indicated that further auxiliary determinants for magnetosome biosynthesis are encoded outside the MAI. However, techniques for large-scale genome editing of magnetic bacteria are still limited, and the full complement of genes controlling magnetosome formation has remained uncertain. RESULTS: Here we demonstrate that an allelic replacement method based on homologous recombination can be applied for large-scale genome editing in M. gryphiswaldense. By analysis of 24 deletion mutants covering about 167 kb of non-redundant genome content, we identified genes and regions inside and outside the MAI irrelevant for magnetosome biosynthesis. A contiguous stretch of ~ 100 kb, including the scattered mam and mms6 operons, could be functionally substituted by a compact and contiguous ~ 38 kb cassette comprising all essential biosynthetic gene clusters, but devoid of interspersing irrelevant or problematic gene content. CONCLUSIONS: Our results further delineate the genetic complement for magnetosome biosynthesis and will be useful for future large-scale genome editing and genetic engineering of magnetosome biosynthesis.

Classification of three corynebacterial strains isolated from a small paddock in North Rhine-Westphalia: proposal of Corynebacterium kalinowskii sp. nov., Corynebacterium comes sp. nov. and Corynebacterium occultum sp. nov.

Three novel corynebacterial species were isolated from soil sampled at a paddock in Vilsendorf, North Rhine-Westphalia, Germany. The strains were coccoid or irregular rod-shaped, catalase-positive and pale white to yellow-orange in colour. By whole genome sequencing and comparison of the 16S rRNA genes as well as the whole genome structure, it was shown that all three strains represent novel species of the family Corynebacteriaceae, order Corynebacteriales, class Actinobacteria. This project describes the isolation, identification, sequencing, and phenotypic characterization of the three novel Corynebacterium species. We propose the names Corynebacterium kalinowskii sp. nov. (DSM 110639T=LMG 31801T), Corynebacterium comes sp. nov. (DSM 110640T=LMG 31802T), and Corynebacterium occultum sp. nov. (DSM 110642T=LMG 31803T).

Towards a 'chassis' for bacterial magnetosome biosynthesis: genome streamlining of Magnetospirillum gryphiswaldense by multiple deletions.

BACKGROUND: Because of its tractability and straightforward cultivation, the magnetic bacterium Magnetospirillum gryphiswaldense has emerged as a model for the analysis of magnetosome biosynthesis and bioproduction. However, its future use as platform for synthetic biology and biotechnology will require methods for large-scale genome editing and streamlining. RESULTS: We established an approach for combinatory genome reduction and generated a library of strains in which up to 16 regions including large gene clusters, mobile genetic elements and phage-related genes were sequentially removed, equivalent to ~ 227.6 kb and nearly 5.5% of the genome. Finally, the fragmented genomic magnetosome island was replaced by a compact cassette comprising all key magnetosome biosynthetic gene clusters. The prospective 'chassis' revealed wild type-like cell growth and magnetosome biosynthesis under optimal conditions, as well as slightly improved resilience and increased genetic stability. CONCLUSION: We provide first proof-of-principle for the feasibility of multiple genome reduction and large-scale engineering of magnetotactic bacteria. The library of deletions will be valuable for turning M. gryphiswaldense into a microbial cell factory for synthetic biology and production of magnetic nanoparticles.

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum.

BACKGROUND: The demand for biobased polymers is increasing steadily worldwide. Microbial hosts for production of their monomeric precursors such as glutarate are developed. To meet the market demand, production hosts have to be improved constantly with respect to product titers and yields, but also shortening bioprocess duration is important. RESULTS: In this study, adaptive laboratory evolution was used to improve a C. glutamicum strain engineered for production of the C5-dicarboxylic acid glutarate by flux enforcement. Deletion of the L-glutamic acid dehydrogenase gene gdh coupled growth to glutarate production since two transaminases in the glutarate pathway are crucial for nitrogen assimilation. The hypothesis that strains selected for faster glutarate-coupled growth by adaptive laboratory evolution show improved glutarate production was tested. A serial dilution growth experiment allowed isolating faster growing mutants with growth rates increasing from 0.10 h-1 by the parental strain to 0.17 h-1 by the fastest mutant. Indeed, the fastest growing mutant produced glutarate with a twofold higher volumetric productivity of 0.18 g L-1 h-1 than the parental strain. Genome sequencing of the evolved strain revealed candidate mutations for improved production. Reverse genetic engineering revealed that an amino acid exchange in the large subunit of L-glutamic acid-2-oxoglutarate aminotransferase was causal for accelerated glutarate production and its beneficial effect was dependent on flux enforcement due to deletion of gdh. Performance of the evolved mutant was stable at the 2 L bioreactor-scale operated in batch and fed-batch mode in a mineral salts medium and reached a titer of 22.7 g L-1, a yield of 0.23 g g-1 and a volumetric productivity of 0.35 g L-1 h-1. Reactive extraction of glutarate directly from the fermentation broth was optimized leading to yields of 58% and 99% in the reactive extraction and reactive re-extraction step, respectively. The fermentation medium was adapted according to the downstream processing results. CONCLUSION: Flux enforcement to couple growth to operation of a product biosynthesis pathway provides a basis to select strains growing and producing faster by adaptive laboratory evolution. After identifying candidate mutations by genome sequencing causal mutations can be identified by reverse genetics. As exemplified here for glutarate production by C. glutamicum, this approach allowed deducing rational metabolic engineering strategies.

Recombinant expression and characterization of novel P450s from Actinosynnema mirum.

Cytochrome P450 monooxygenases (P450s) are the major contributor in the metabolism of xenobiotics, including therapeutic agents. Thus, P450s find broad application in the pharmaceutical industry to synthesize metabolites of new active pharmaceutical ingredients in order to evaluate toxicity and pharmacokinetics. As an alternative to human hepatic P450s, microbial P450s offer several advantages, such as an easier and more efficient heterologous expression as well as higher stability under process conditions. Recently, the wild-type strain Actinosynnema mirum has been reported to catalyze hydroxylation reactions with high activity on a broad range of substrates. In this study, one of these substrates, ritonavir, was used to analyze the transcriptional response of the wild-type strain. Analysis of the differential gene expression pattern allowed the assignment of genes potentially responsible for ritonavir conversion. Heterologous expression of these candidates and activity testing led to the identification of a novel P450 that efficiently converts ritonavir resembling the activity of the human CYP3A4.