FEMS microbesPub Date : 2023-01-01DOI: 10.1093/femsmc/xtad002
Scott J Britton, Lisa J Rogers, Jane S White, Hedwig Neven, Dawn L Maskell
{"title":"Disparity in pseudohyphal morphogenic switching response to the quorum sensing molecule 2-phenylethanol in commercial brewing strains of <i>Saccharomyces cerevisiae</i>.","authors":"Scott J Britton, Lisa J Rogers, Jane S White, Hedwig Neven, Dawn L Maskell","doi":"10.1093/femsmc/xtad002","DOIUrl":"https://doi.org/10.1093/femsmc/xtad002","url":null,"abstract":"<p><p><i>Saccharomyces cerevisiae</i> can undergo filamentous growth in response to specific environmental stressors, particularly nitrogen-limitation, whereby cells undergo pseudohyphal differentiation, a process where cells transition from a singular ellipsoidal appearance to multicellular filamentous chains from the incomplete scission of the mother-daughter cells. Previously, it was demonstrated that filamentous growth in <i>S. cerevisiae</i> is co-regulated by multiple signaling networks, including the glucose-sensing RAS/cAMP-PKA and SNF pathways, the nutrient-sensing TOR pathway, the filamentous growth MAPK pathway, and the Rim101 pathway, and can be induced by quorum-sensing aromatic alcohols, such as 2-phenylethanol. However, the prevalent research on the yeast-pseudohyphal transition and its induction by aromatic alcohols in <i>S. cerevisiae</i> has been primarily limited to the strain Σ1278b. Due to the prospective influence of quorum sensing on commercial fermentation, the native variation of yeast-to-filamentous phenotypic transition and its induction by 2-phenylethanol in commercial brewing strains was investigated. Image analysis software was exploited to enumerate the magnitude of whole colony filamentation in 16 commercial strains cultured on nitrogen-limiting SLAD medium; some supplemented with exogenous 2-phenylethanol. The results demonstrate that phenotypic switching is a generalized, highly varied response occurring only in select brewing strains. Nevertheless, strains exhibiting switching behavior altered their filamentation response to exogenous concentrations of 2-phenylethanol.</p>","PeriodicalId":73024,"journal":{"name":"FEMS microbes","volume":"4 ","pages":"xtad002"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10117810/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10036433","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
FEMS microbesPub Date : 2023-01-01DOI: 10.1093/femsmc/xtad012
Melisa Gür, Jelena Erdmann, Anke Will, Ziwei Liang, Jens Bo Andersen, Tim Tolker-Nielsen, Susanne Häussler
{"title":"Challenges in using transcriptome data to study the c-di-GMP signaling network in <i>Pseudomonas aeruginosa</i> clinical isolates.","authors":"Melisa Gür, Jelena Erdmann, Anke Will, Ziwei Liang, Jens Bo Andersen, Tim Tolker-Nielsen, Susanne Häussler","doi":"10.1093/femsmc/xtad012","DOIUrl":"https://doi.org/10.1093/femsmc/xtad012","url":null,"abstract":"<p><p>In the <i>Pseudomonas aeruginosa</i> type strain PA14, 40 genes are known to encode for diguanylate cyclases (DGCs) and/or phosphodiesterases (PDEs), which modulate the intracellular pool of the nucleotide second messenger c-di-GMP. While in general, high levels of c-di-GMP drive the switch from highly motile phenotypes towards a sessile lifestyle, the different c-di-GMP modulating enzymes are responsible for smaller and in parts nonoverlapping phenotypes. In this study, we sought to utilize previously recorded <i>P. aeruginosa</i> gene expression datasets on 414 clinical isolates to uncover transcriptional changes as a result of a high expression of genes encoding DGCs. This approach might provide a unique opportunity to bypass the problem that for many c-di-GMP modulating enzymes it is not known under which conditions their expression is activated. However, while we demonstrate that the selection of subgroups of clinical isolates with high versus low expression of sigma factor encoding genes served the identification of their downstream regulons, we were unable to confirm the predicted DGC regulons, because the high c-di-GMP associated phenotypes were rapidly lost in the clinical isolates,. Further studies are needed to determine the specific mechanisms underlying the loss of cyclase activity upon prolonged cultivation of clinical <i>P. aeruginosa</i> isolates.</p>","PeriodicalId":73024,"journal":{"name":"FEMS microbes","volume":"4 ","pages":"xtad012"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10411656/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9981114","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
FEMS microbesPub Date : 2023-01-01DOI: 10.1093/femsmc/xtad017
Punit Kumar, Khushboo, Deepanshi Rajput, Kashyap K Dubey
{"title":"Insights into the mechanism of mycelium transformation of <i>Streptomyces toxytricini</i> into pellet.","authors":"Punit Kumar, Khushboo, Deepanshi Rajput, Kashyap K Dubey","doi":"10.1093/femsmc/xtad017","DOIUrl":"https://doi.org/10.1093/femsmc/xtad017","url":null,"abstract":"<p><p>Formation of the mycelial pellet in submerged cultivation of <i>Streptomycetes</i> is unwanted in industrial fermentation processes as it imposes mass transfer limitations, changes in the rheology of a medium, and affects the production of secondary metabolites. Though detailed information is not available about the factors involved in regulating mycelial morphology, it is studied that culture conditions and the genetic information of strain play a crucial role. Moreover, the proteomic study has revealed the involvement of low molecular weight proteins such as; DivIVA, FilP, ParA, Scy, and SsgA proteins in apical growth and branching of hyphae, which results in the establishment of the mycelial network. The present study proposes the mechanism of pellet formation of <i>Streptomyces toxytricini</i> (NRRL B-5426) with the help of microscopic and proteomic analysis. The microscopic analysis revealed that growing hyphae contain a bud-like structure behind the apical tip, which follows a certain organized path of growth and branching, which was further converted into the pellet when shake flask to the shake flask inoculation was performed. Proteomic analysis revealed the production of low molecular weight proteins ranging between 20 and 95 kDa, which are involved in apical growth and hyphae branching and can possibly participate in the regulation of pellet morphology.</p>","PeriodicalId":73024,"journal":{"name":"FEMS microbes","volume":"4 ","pages":"xtad017"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10473828/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10524099","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
FEMS microbesPub Date : 2023-01-01DOI: 10.1093/femsmc/xtad016
Alessio Cavallaro, William J Rhoads, Émile Sylvestre, Thierry Marti, Jean-Claude Walser, Frederik Hammes
{"title":"<i>Legionella</i> relative abundance in shower hose biofilms is associated with specific microbiome members.","authors":"Alessio Cavallaro, William J Rhoads, Émile Sylvestre, Thierry Marti, Jean-Claude Walser, Frederik Hammes","doi":"10.1093/femsmc/xtad016","DOIUrl":"https://doi.org/10.1093/femsmc/xtad016","url":null,"abstract":"<p><p><i>Legionella</i> are natural inhabitants of building plumbing biofilms, where interactions with other microorganisms influence their survival, proliferation, and death. Here, we investigated the associations of <i>Legionella</i> with bacterial and eukaryotic microbiomes in biofilm samples extracted from 85 shower hoses of a multiunit residential building. <i>Legionella</i> spp. relative abundance in the biofilms ranged between 0-7.8%, of which only 0-0.46% was <i>L. pneumophila</i>. Our data suggest that some microbiome members were associated with high (e.g. <i>Chthonomonas, Vrihiamoeba</i>) or low (e.g. <i>Aquabacterium, Vannella</i>) <i>Legionella</i> relative abundance. The correlations of the different <i>Legionella</i> variants (30 Zero-Radius OTUs detected) showed distinct patterns, suggesting separate ecological niches occupied by different <i>Legionella</i> species. This study provides insights into the ecology of <i>Legionella</i> with respect to: (i) the colonization of a high number of real shower hoses biofilm samples; (ii) the ecological meaning of associations between <i>Legionella</i> and co-occurring bacterial/eukaryotic organisms; (iii) critical points and future directions of microbial-interaction-based-ecological-investigations.</p>","PeriodicalId":73024,"journal":{"name":"FEMS microbes","volume":"4 ","pages":"xtad016"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10496943/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10260569","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
FEMS microbesPub Date : 2023-01-01DOI: 10.1093/femsmc/xtad007
Haris Antypas, Tianqi Zhang, Ferdinand X Choong, Keira Melican, Agneta Richter-Dahlfors
{"title":"Dynamic single cell analysis in a proximal-tubule-on-chip reveals heterogeneous epithelial colonization strategies of uropathogenic <i>Escherichia coli</i> under shear stress.","authors":"Haris Antypas, Tianqi Zhang, Ferdinand X Choong, Keira Melican, Agneta Richter-Dahlfors","doi":"10.1093/femsmc/xtad007","DOIUrl":"https://doi.org/10.1093/femsmc/xtad007","url":null,"abstract":"<p><p>The urinary tract is a hydrodynamically challenging microenvironment and uropathogenic <i>Escherichia coli</i> (UPEC) must overcome several physiological challenges in order to adhere and establish a urinary tract infection. Our previous work <i>in vivo</i> revealed a synergy between different UPEC adhesion organelles, which facilitated effective colonization of the renal proximal tubule. To allow high-resolution real-time analysis of this colonization behavior, we established a biomimetic proximal-tubule-on-chip (PToC). The PToC allowed for single-cell resolution analysis of the first stages of bacterial interaction with host epithelial cells, under physiological flow. Time-lapse microscopy and single-cell trajectory analysis in the PToC revealed that while the majority of UPEC moved directly through the system, a minority population initiated heterogeneous adhesion, identified as either rolling or bound. Adhesion was predominantly transient and mediated by P pili at the earliest time-points. These bound bacteria initiated a founder population which rapidly divided, leading to 3D microcolonies. Within the first hours, the microcolonies did not express extracellular curli matrix, but rather were dependent on Type 1 fimbriae as the key element in the microcolony structure. Collectively, our results show the application of Organ-on-chip technology to address bacterial adhesion behaviors, demonstrating a well-orchestrated interplay and redundancy between adhesion organelles that enables UPEC to form microcolonies and persist under physiological shear stress.</p>","PeriodicalId":73024,"journal":{"name":"FEMS microbes","volume":"4 ","pages":"xtad007"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10117878/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9662433","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
FEMS microbesPub Date : 2023-01-01DOI: 10.1093/femsmc/xtad008
Catarina Ferreira, Saria Otani, Frank Møller Aarestrup, Célia M Manaia
{"title":"Quantitative PCR versus metagenomics for monitoring antibiotic resistance genes: balancing high sensitivity and broad coverage.","authors":"Catarina Ferreira, Saria Otani, Frank Møller Aarestrup, Célia M Manaia","doi":"10.1093/femsmc/xtad008","DOIUrl":"https://doi.org/10.1093/femsmc/xtad008","url":null,"abstract":"<p><p>The widespread occurrence of clinically relevant antibiotic resistance within humans, animals, and environment motivates the development of sensitive and accurate detection and quantification methods. Metagenomics and quantitative PCR (qPCR) are amongst the most used approaches. In this study, we aimed to evaluate and compare the performance of these methods to screen antibiotic resistance genes in animal faecal, wastewater, and water samples. Water and wastewater samples were from hospital effluent, different treatment stages of two treatment plants, and of the receiving river at the discharge point. The animal samples were from pig and chicken faeces. Antibiotic resistance gene coverage, sensitivity, and usefulness of the quantitative information were analyzed and discussed. While both methods were able to distinguish the resistome profiles and detect gradient stepwise mixtures of pig and chicken faeces, qPCR presented higher sensitivity for the detection of a few antibiotic resistance genes in water/wastewater. In addition, the comparison of predicted and observed antibiotic resistance gene quantifications unveiled the higher accuracy of qPCR. Metagenomics analyses, while less sensitive, provided a markedly higher coverage of antibiotic resistance genes compared to qPCR. The complementarity of both methods and the importance of selecting the best method according to the study purpose are discussed.</p>","PeriodicalId":73024,"journal":{"name":"FEMS microbes","volume":"4 ","pages":"xtad008"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/62/d3/xtad008.PMC10117749.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10036436","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
FEMS microbesPub Date : 2023-01-01DOI: 10.1093/femsmc/xtac028
Abd Alaziz Abu Quba, Marc-Oliver Goebel, Mariam Karagulyan, Anja Miltner, Matthias Kästner, Jörg Bachmann, Gabriele E Schaumann, Doerte Diehl
{"title":"Changes in cell surface properties of <i>Pseudomonas fluorescens</i> by adaptation to NaCl induced hypertonic stress.","authors":"Abd Alaziz Abu Quba, Marc-Oliver Goebel, Mariam Karagulyan, Anja Miltner, Matthias Kästner, Jörg Bachmann, Gabriele E Schaumann, Doerte Diehl","doi":"10.1093/femsmc/xtac028","DOIUrl":"https://doi.org/10.1093/femsmc/xtac028","url":null,"abstract":"<p><p>Determination of the effect of water stress on the surface properties of bacteria is crucial to study bacterial induced soil water repellency. Changes in the environmental conditions may affect several properties of bacteria such as the cell hydrophobicity and morphology. Here, we study the influence of adaptation to hypertonic stress on cell wettability, shape, adhesion, and surface chemical composition of <i>Pseudomonas fluorescens</i>. From this we aim to discover possible relations between the changes in wettability of bacterial films studied by contact angle and single cells studied by atomic and chemical force microscopy (AFM, CFM), which is still lacking. We show that by stress the adhesion forces of the cell surfaces towards hydrophobic functionalized probes increase while they decrease towards hydrophilic functionalized tips. This is consistent with the contact angle results. Further, cell size shrunk and protein content increased upon stress. The results suggest two possible mechanisms: Cell shrinkage is accompanied by the release of outer membrane vesicles by which the protein to lipid ratio increases. The higher protein content increases the rigidity and the number of hydrophobic nano-domains per surface area.</p>","PeriodicalId":73024,"journal":{"name":"FEMS microbes","volume":"4 ","pages":"xtac028"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/38/5d/xtac028.PMC10169395.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10036437","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
FEMS microbesPub Date : 2023-01-01DOI: 10.1093/femsmc/xtad010
Alhassan Sa-Eed, Eric S Donkor, Reuben E Arhin, Patience B Tetteh-Quarcoo, Simon K Attah, Daniel E K Kabotso, Fleischer C N Kotey, Nicholas T K D Dayie
{"title":"<i>In vitro</i> antimicrobial activity of crude propolis extracts and fractions.","authors":"Alhassan Sa-Eed, Eric S Donkor, Reuben E Arhin, Patience B Tetteh-Quarcoo, Simon K Attah, Daniel E K Kabotso, Fleischer C N Kotey, Nicholas T K D Dayie","doi":"10.1093/femsmc/xtad010","DOIUrl":"https://doi.org/10.1093/femsmc/xtad010","url":null,"abstract":"<p><p>The search for antimicrobials in propolis presents a new dimension for addressing the problem of antimicrobial drug resistance. The aim of this study was to determine the antimicrobial activity of extracts of crude propolis collected from different regions in Ghana and their active fractions. The antimicrobial activity of the extracts, as well as that of the chloroform, ethyl acetate, and petroleum ether fractions of the active samples were determined using the agar well diffusion method. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the most active fractions were determined. The various crude propolis extracts frequently produced zones of inhibition against <i>Staphylococcus aureus</i> (17/20) than <i>Pseudomonas aeruginosa</i> (16/20), and <i>Escherichia coli</i> (1/20) test isolates. Chloroform and ethyl acetate solvents produced fractions possessing greater antimicrobial activity than the petroleum ether fraction. The mean MIC range of the most active fractions was greatest for <i>S. aureus</i> (76.0 ± 34.8-48.0 ± 33.0 mg/ml) than for <i>P. aeruginosa</i> (40.8 ± 33.3-30.4 ± 6.7 mg/ml) and <i>E. coli</i>, as was the mean MBC. Propolis has antimicrobial potential, and hence should be exploited as an alternative for the treatment of bacterial infections.</p>","PeriodicalId":73024,"journal":{"name":"FEMS microbes","volume":"4 ","pages":"xtad010"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10165684/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10036432","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
FEMS microbesPub Date : 2023-01-01DOI: 10.1093/femsmc/xtad011
Daniela Rosado, Paula Canada, Sofia Marques Silva, Nuno Ribeiro, Pedro Diniz, Raquel Xavier
{"title":"Disruption of the skin, gill, and gut mucosae microbiome of gilthead seabream fingerlings after bacterial infection and antibiotic treatment.","authors":"Daniela Rosado, Paula Canada, Sofia Marques Silva, Nuno Ribeiro, Pedro Diniz, Raquel Xavier","doi":"10.1093/femsmc/xtad011","DOIUrl":"https://doi.org/10.1093/femsmc/xtad011","url":null,"abstract":"<p><p>The activity of the microbiome of fish mucosae provides functions related to immune response, digestion, or metabolism. Several biotic and abiotic factors help maintaining microbial homeostasis, with disruptions leading to dysbiosis. Diseases and antibiotic administration are known to cause dysbiosis in farmed fish. Pathogen infections greatly affect the production of gilthead seabream, and antibiotic treatment is still frequently required. Here, we employed a 16S rRNA high-throughput metataxonomics approach to characterize changes in the gut, skin, and gill microbiomes occurring due to infection with <i>Photobacterium damselae</i> subsp. <i>piscicida</i> and subsequent antibiotic treatment with oxytetracycline (OTC), as well as during recovery. Although microbiota response differed between studied tissues, overall changes in composition, diversity, structure, and predicted function were observed in all mucosae. The skin and gill microbiomes of diseased fish became largely dominated by taxa that have been frequently linked to secondary infections, whereas in the gut the genus <i>Vibrio</i>, known to include pathogenic bacteria, increased with OTC treatment. The study highlights the negative impacts of disease and antibiotic treatment on the microbiome of farmed fish. Our results also suggest that fish transportation operations may have profound effects on the fish microbiome, but further studies are needed to accurately evaluate their impact.</p>","PeriodicalId":73024,"journal":{"name":"FEMS microbes","volume":"4 ","pages":"xtad011"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10306326/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9741996","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
FEMS microbesPub Date : 2022-12-19eCollection Date: 2023-01-01DOI: 10.1093/femsmc/xtac030
Robin Weihmann, Sonja Kubicki, Nora Lisa Bitzenhofer, Andreas Domröse, Isabel Bator, Lisa-Marie Kirschen, Franziska Kofler, Aileen Funk, Till Tiso, Lars M Blank, Karl-Erich Jaeger, Thomas Drepper, Stephan Thies, Anita Loeschcke
{"title":"The modular pYT vector series employed for chromosomal gene integration and expression to produce carbazoles and glycolipids in <i>P. putida</i>.","authors":"Robin Weihmann, Sonja Kubicki, Nora Lisa Bitzenhofer, Andreas Domröse, Isabel Bator, Lisa-Marie Kirschen, Franziska Kofler, Aileen Funk, Till Tiso, Lars M Blank, Karl-Erich Jaeger, Thomas Drepper, Stephan Thies, Anita Loeschcke","doi":"10.1093/femsmc/xtac030","DOIUrl":"10.1093/femsmc/xtac030","url":null,"abstract":"<p><p>The expression of biosynthetic genes in bacterial hosts can enable access to high-value compounds, for which appropriate molecular genetic tools are essential. Therefore, we developed a toolbox of modular vectors, which facilitate chromosomal gene integration and expression in <i>Pseudomonas putida</i> KT2440. To this end, we designed an integrative sequence, allowing customisation regarding the modes of integration (random, at <i>att</i>Tn7, or into the 16S rRNA gene), promoters, antibiotic resistance markers as well as fluorescent proteins and enzymes as transcription reporters. We thus established a toolbox of vectors carrying integrative sequences, designated as pYT series, of which we present 27 ready-to-use variants along with a set of strains equipped with unique 'landing pads' for directing a pYT interposon into one specific copy of the 16S rRNA gene. We used genes of the well-described violacein biosynthesis as reporter to showcase random Tn5-based chromosomal integration leading to constitutive expression and production of violacein and deoxyviolacein. Deoxyviolacein was likewise produced after gene integration into the 16S rRNA gene of <i>rrn</i> operons. Integration in the <i>att</i>Tn7 site was used to characterise the suitability of different inducible promoters and successive strain development for the metabolically challenging production of mono-rhamnolipids. Finally, to establish arcyriaflavin A production in <i>P. putida</i> for the first time, we compared different integration and expression modes, revealing integration at <i>att</i>Tn7 and expression with NagR/P<i><sub>nagAa</sub></i> to be most suitable. In summary, the new toolbox can be utilised for the rapid generation of various types of <i>P. putida</i> expression and production strains.</p>","PeriodicalId":73024,"journal":{"name":"FEMS microbes","volume":"4 ","pages":"xtac030"},"PeriodicalIF":0.0,"publicationDate":"2022-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/9c/90/xtac030.PMC10117823.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10018452","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}