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A normalization method to decipher quantitative proteome changes in microbial co-culture systems

ABSTRACT: The value of synthetic microbial communities in biotechnology is gaining traction due to their ability to undertake more complex metabolic tasks than monocultures. However, a thorough understanding of strain interactions, productivity and stability is often required to optimize growth and scale up cultivation. Quantitative proteomics can provide valuable insights into how microbial strains adapt to changing conditions in biomanufacturing or bioremediation scenarios. However, current workflows and methodologies are not suitable for simple artificial co-culture systems where strain ratios are dynamic. Here, we established a standard workflow for co-culture proteomics using an exemplar system containing two key members, Azotobacter vinelandii and Synechococcus elongatus. Factors affecting the quantitative accuracy of co-culture proteomics were investigated, including peptide physicochemical characteristics such as molecular weight, isoelectric point, hydrophobicity, and dynamic range, as well as factors relating to protein identification such as varying proteome size and shared peptides between species. Different quantification methods based on spectral counts and intensity were evaluated, demonstrating good correlations between protein amount and the six quantification methods at the protein level. We propose a new normalization method, named “LFQRatio”, to reflect the relative contributions of the two distinct cell types emerging from the cell ratio changes during co-cultivation. LFQRatio can be applied to real co-culture proteomics experiments, providing accurate insights into quantitative proteome changes in each strain.

ORGANISM(S): Synechococcus Elongatus Pcc 7942 = Fachb-805 Azotobacter Vinelandii Dj

SUBMITTER: Jagroop Pandhal

PROVIDER: PXD046627 | iProX | Thu Nov 02 00:00:00 GMT 2023

REPOSITORIES: iProX

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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:Determining how a bacterial pathogen responds to its host and other bacterial species by altering gene expression is key to understand its pathogenesis and environmental adaption. Here, we used RNA-Seq to comprehensively and quantitatively assess the transcriptional response of the rice bacterial pathogen Acidovorax avenae subsp. avenae strain RS-1 cultivated in vitro, in vivo and in co-culture with rice rhizobacterium Burkholderia seminalis R456. Results revealed a surprisingly large number of regulatory differences between these conditions indicating adaptation of A. avenae subsp. avenae to specific ecological conditions. In particular, a number of potential virulence factors such as type 3 secretion system proteins were specifically expressed under in vivo conditions, whereas genes whose protein products are involved in inter-bacterial interaction such as auxin efflux carrier, small mechanosensitive ion channel protein, and ureidoglycolate hydrolase were among those specifically up-regulated under co-culture conditions. In addition, global genomic analysis of strain RS-1 identified 406 putative non-coding (nc) RNA genes. Interestingly, 8 ncRNA genes that were uniquely expressed under in vivo may be linked to pathogenicity while 4 ncRNA genes that were uniquely expressed under coculture conditions may be involved in adaption to co-cultivation with B. seminalis. Expression data obtained by RNA-Seq were also confirmed for selected genes by quantitative real-time PCR and two-dimensional gel electrophoresis as well as knockout analysis. Aaa strain RS-1 and B. seminalis strain R456 was isolated from diseased rice plants (Li et al., 2011; Xie et al., 2011) and rice rhizosphere (Zhang et al., 2007; Li et al., 2011), respectively, in our previous studies, and were stored in 20-30% sterile glycerol at -80°C. The samples of Aaa strain RS-1 for in vitro and in vivo analysis were prepared as described before (Ibrahim et al., 2012). The co-culture analysis of Aaa strain RS-1 with B. seminalis strain R456 were conducted according to Ruiz et al. (2009) and Di Cagno et al. (2009). Briefly, Aaa strain RS-1 and B. seminalis strain R456 was inoculated and incubated in chambers of a double culture vessel apparatus separated by a 0.4-μm membrane filter (Millipore Isopore™). In order to avoid the possible contamination during in vivo and co-culture operation, all bacterial samples were further confirmed based on the sequence analysis of 16S-rRNA (Li et al., 2011). Then samples were processed for RNA harvesting, mRNA purification and cDNA synthesis.

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A normalization method to decipher quantitative proteome changes in microbial co-culture systems

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