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Cultivation and functional characterization of 79 planctomycetes uncovers their unique biology.

ABSTRACT: When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bacterial strains from all major taxonomic clades of the conspicuous bacterial phylum Planctomycetes. The samples were derived from different aquatic environments but close relatives could be isolated from geographically distinct regions and structurally diverse habitats, implying that 'everything is everywhere'. With the discovery of lateral budding in 'Kolteria novifilia' and the capability of the members of the Saltatorellus clade to divide by binary fission as well as budding, we identified previously unknown modes of bacterial cell division. Alongside unobserved aspects of cell signalling and small-molecule production, our findings demonstrate that exploration beyond the well-established model organisms has the potential to increase our knowledge of bacterial diversity. We illustrate how 'microbial dark matter' can be accessed by cultivation techniques, expanding the organismic background for small-molecule research and drug-target detection.

SUBMITTER: Wiegand S

PROVIDER: S-EPMC7286433 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Cultivation and functional characterization of 79 planctomycetes uncovers their unique biology.

Wiegand Sandra S Jogler Mareike M Boedeker Christian C Pinto Daniela D Vollmers John J Rivas-Marín Elena E Kohn Timo T Peeters Stijn H SH Heuer Anja A Rast Patrick P Oberbeckmann Sonja S Bunk Boyke B Jeske Olga O Meyerdierks Anke A Storesund Julia E JE Kallscheuer Nicolai N Lücker Sebastian S Lage Olga M OM Pohl Thomas T Merkel Broder J BJ Hornburger Peter P Müller Ralph-Walter RW Brümmer Franz F Labrenz Matthias M Spormann Alfred M AM Op den Camp Huub J M HJM Overmann Jörg J Amann Rudolf R Jetten Mike S M MSM Mascher Thorsten T Medema Marnix H MH Devos Damien P DP Kaster Anne-Kristin AK Øvreås Lise L Rohde Manfred M Galperin Michael Y MY Jogler Christian C

Nature microbiology 20191118 1

When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bac ...[more]

PMID: 31740763

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Project description:Background: Microbial gene expression is to a large extend determined by environmental growth conditions. Differential gene expression analysis between two conditions has been frequently used to reveal regulatory networks and to assign physiological function to unknown genes. In nature, microorganisms cohabit however these interactions have been rarely studied and reproduced in laboratory set-up. Thus to quantitatively explore the genome-wide responses of microbial interaction, we co-cultivated Penicillium chrysogenum and Bacillus subtilis in chemostat culture. Results: Time course expression analysis of P. chrysogenum to co-cultivation with B. subtilis was carried out to understand the natural responses of P. chrysogenum to prokaryotes. Steady state chemostats of P. chrysogenum in non-B-lactam producing conditions was pulsed with B. subtilis and co-cultivation was followed for 72 hours. The dynamic physiological and transcriptional responses of P. chrysogenum in mixed culture were monitored. B. subtilis outcompeted growth of P. chrysogenum resulting in an increased B. subtilis biomass by more than three fold of its original size and a reduction in P. chrysogenum biomass to half of its original size after 72 h of mixed culture. Genes of the penicillin pathway, synthesis of the side-chain and precursors were overall unresponsive to the presence of B. subtilis. Moreover Penicillium polyketide synthase and nonribosomal peptide synthetase genes either remained silent or down-regulated, whereas genes responsible for protein synthesis, metabolism, energy conservation, respiration and transport were upregulated in the presence of B. subtilis. Among highly responsive genes, two putative B-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production in P. chrysogenum. Mutanase activity was not observed in pure cultures of P. chrysogenum and B. subtilis or when P. chrysogenum was co-cultured with B. subtilis supernatant or heat inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum pulsed with filter sterilized supernatants from mixed cultures P. chrysogenum and B. subtilis. Heterologous expression of Pc12g07500 and Pc12g13330 genes in Saccharomyces cerevisiae confirmed that at least Pc12g07500 encoded an B-1,3 endoglucanase. Conclusion: Time course transcriptional profiling of P. chrysogenum revealed several differentially expressed genes during mixed culture, potentially reflecting interactions between the eukaryotic and the prokaryotic systems. M-oM-^AM-!-1,3 endoglucanase produced by P. chrysogenum against B. subtilis signals may have application in improving the efficacy of antibiotics by degrading exopolysacchride biofilms of pathogenic bacteria. The objective of the present study is to investigate the response of P. chrysogenum to co-cultivation with B. subtilis. To trigger an interaction specific behaviour, steady state chemostat of P. chrysogenum Wisconsin 54-1255 was pulsed with B. subtilis. The dynamic, transcriptional and physiological responses of P. chrysogenum in mixed culture were monitored and analyzed. Several differentially expressed genes potentially reflected interactions between the eukaryotic and the prokaryotic systems. To test whether any bacterial signaling molecules are responsible for differential expression of selected fungal genes, P. chrysogenum cultures were inoculated with supernatant of B. subtilis culture, supernatants from mixed culture and with heat-inactivated B. subtilis. The specific transcriptional responses identified using microarray was verified by analysis of fermentation broth and functional characterization by expression of selected genes in S. cerevisiae.

Project description:Background: Microbial gene expression is to a large extend determined by environmental growth conditions. Differential gene expression analysis between two conditions has been frequently used to reveal regulatory networks and to assign physiological function to unknown genes. In nature, microorganisms cohabit however these interactions have been rarely studied and reproduced in laboratory set-up. Thus to quantitatively explore the genome-wide responses of microbial interaction, we co-cultivated Penicillium chrysogenum and Bacillus subtilis in chemostat culture. Results: Time course expression analysis of P. chrysogenum to co-cultivation with B. subtilis was carried out to understand the natural responses of P. chrysogenum to prokaryotes. Steady state chemostats of P. chrysogenum in non-B-lactam producing conditions was pulsed with B. subtilis and co-cultivation was followed for 72 hours. The dynamic physiological and transcriptional responses of P. chrysogenum in mixed culture were monitored. B. subtilis outcompeted growth of P. chrysogenum resulting in an increased B. subtilis biomass by more than three fold of its original size and a reduction in P. chrysogenum biomass to half of its original size after 72 h of mixed culture. Genes of the penicillin pathway, synthesis of the side-chain and precursors were overall unresponsive to the presence of B. subtilis. Moreover Penicillium polyketide synthase and nonribosomal peptide synthetase genes either remained silent or down-regulated, whereas genes responsible for protein synthesis, metabolism, energy conservation, respiration and transport were upregulated in the presence of B. subtilis. Among highly responsive genes, two putative B-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production in P. chrysogenum. Mutanase activity was not observed in pure cultures of P. chrysogenum and B. subtilis or when P. chrysogenum was co-cultured with B. subtilis supernatant or heat inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum pulsed with filter sterilized supernatants from mixed cultures P. chrysogenum and B. subtilis. Heterologous expression of Pc12g07500 and Pc12g13330 genes in Saccharomyces cerevisiae confirmed that at least Pc12g07500 encoded an B-1,3 endoglucanase. Conclusion: Time course transcriptional profiling of P. chrysogenum revealed several differentially expressed genes during mixed culture, potentially reflecting interactions between the eukaryotic and the prokaryotic systems. -1,3 endoglucanase produced by P. chrysogenum against B. subtilis signals may have application in improving the efficacy of antibiotics by degrading exopolysacchride biofilms of pathogenic bacteria.

Project description:BACKGROUND: Microbial gene expression is strongly influenced by environmental growth conditions. Comparison of gene expression under different conditions is frequently used for functional analysis and to unravel regulatory networks, however, gene expression responses to co-cultivation with other microorganisms, a common occurrence in nature, is rarely studied under laboratory conditions. To explore cellular responses of the antibiotic-producing fungus Penicillium chrysogenum to prokaryotes, the present study investigates its transcriptional responses during co-cultivation with Bacillus subtilis. RESULTS: Steady-state glucose-limited chemostats of P. chrysogenum grown under penillicin-non-producing conditions were inoculated with B. subtilis. Physiological and transcriptional responses of P. chrysogenum in the resulting mixed culture were monitored over 72 h. Under these conditions, B. subtilis outcompeted P. chrysogenum, as reflected by a three-fold increase of the B. subtilis population size and a two-fold reduction of the P. chrysogenum biomass concentration. Genes involved in the penicillin pathway and in synthesis of the penicillin precursors and side-chain were unresponsive to the presence of B. subtilis. Moreover, Penicillium polyketide synthase and nonribosomal peptide synthase genes were either not expressed or down-regulated. Among the highly responsive genes, two putative ?-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold, respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production. Mutanase activity was neither observed in pure cultures of P. chrysogenum or B. subtilis, nor during exposure of P. chrysogenum to B. subtilis culture supernatants or heat-inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum exposed to filter-sterilized supernatants of mixed cultures of P. chrysogenum and B. subtilis. Heterologous expression of Pc12g07500 and Pc12g13330 genes in Saccharomyces cerevisiae confirmed that Pc12g07500 encoded an active ?-1,3 endoglucanase. CONCLUSION: Time-course transcriptional profiling of P. chrysogenum revealed differentially expressed genes during co-cultivation with B. subtilis. Penicillin production was not induced under these conditions. However, induction of a newly characterized P. chrysogenum gene encoding ?-1,3 endoglucanase may enhance the efficacy of fungal antibiotics by degrading bacterial exopolysaccharides.

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Cultivation and functional characterization of 79 planctomycetes uncovers their unique biology.

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Cultivation and functional characterization of 79 planctomycetes uncovers their unique biology.

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