Unknown,Transcriptomics,Genomics,Proteomics

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RNA-Seq of Escherichia coli Nissle 1917 strains modified to produce different amounts of the bacteriocin microcin C

ABSTRACT: A popular strategy for enhancing the antibacterial properties of probiotic bacteria is to retrofit them with the ability to overproduce heterologous bacteriocins. This is often achieved from strong, non-native promoters. How the dysregulated overproduction of heterologous bacteriocins affects the fitness and antibacterial efficacy of the retrofitted probiotic bacteria is often overlooked. We conferred the prototypical probiotic Escherichia coli strain Nissle (EcN) the ability to produce different amounts of the bacteriocin microcin C (McC). Expression of the bacteriocin synthesis genes was driven from the native promoter (Pmcc-WT), or from promoters manipulated to be stronger (Pmcc-High) and weaker (Pmcc-Low) than the WT, in a plasmid-based system. Pmcc-Low and Pmcc-High retained their native regulation. A strain harbouring a non-functional promoter (Pmcc-Mut) produces no McC and was used as a control. Each strain was grown to early stationary phase, when production of McC starts, in Luria-Bertani broth at 37 degrees. The RNA was isolated and the effects of different levels of production of McC on the transcriptome of EcN was examined by RNA-Seq.

INSTRUMENT(S): NextSeq 550

ORGANISM(S): Escherichia coli

SUBMITTER: Emma Bartram

PROVIDER: E-MTAB-13078 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Biosynthetic gene clusters (BGCs) encoding the production of bacteriocins are widespread among bacterial isolates and are important genetic determinants of competitive fitness within a given habitat. Staphylococci produce a tremendous diversity of compounds, and the corresponding BGCs are frequently associated with mobile genetic elements, suggesting gain and loss of biosynthetic capacity. Pharmaceutical biology has shown that compound production in heterologous hosts is often challenging, and many BGC recipients initially produce small amounts of compound or show reduced growth rates. To assess whether transfer of BGCs between closely related Staphylococcus aureus strains can be instantly effective or requires elaborate metabolic adaptation, we investigated the intraspecies transfer of a BGC encoding the ribosomally synthesized and posttranslationally modified peptide (RiPP) micrococcin P1 (MP1). We found that acquisition of the BGC by S. aureus RN4220 enabled immediate MP1 production but also imposed a metabolic burden, which was relieved after prolonged cultivation by adaptive mutation. We used a multiomics approach to study this phenomenon and found adaptive evolution to select for strains with increased activity of the tricarboxylic acid cycle (TCA), which enhanced metabolic fitness and levels of compound production. Metabolome analysis revealed increases of central metabolites, including citrate and α-ketoglutarate in the adapted strain, suggesting metabolic adaptation to overcome the BGC-associated growth defects. Our results indicate that BGC acquisition requires genetic and metabolic predispositions, allowing the integration of bacteriocin production into the cellular metabolism. Inappropriate metabolic characteristics of recipients can entail physiological burdens, negatively impacting the competitive fitness of recipients within natural bacterial communities.IMPORTANCE: Human microbiomes are critically associated with human health and disease. Importantly, pathogenic bacteria can hide in human-associated communities and can cause disease when the composition of the community becomes unbalanced. Bacteriocin-producing commensals are able to displace pathogens from microbial communities, suggesting that their targeted introduction into human microbiomes might prevent pathogen colonization and infection. However, to develop probiotic approaches, strains are needed that produce high levels of bioactive compounds and retain cellular fitness within mixed bacterial communities. Our work offers insights into the metabolic burdens associated with the production of the bacteriocin micrococcin P1 and highlights evolutionary strategies that increase cellular fitness in the context of production. Metabolic adaptations are most likely broadly relevant for bacteriocin producers and need to be considered for the future development of effective microbiome editing strategies.

Project description:Subclass IIa bacteriocins have strong antilisterial activity and can control the growth of Listeria monocytogenes in food. However, L. monocytogenes may develop resistance towards such bacteriocins. In this follow-up study, the transcriptomes of a high level (L502-1) and a low level (L502-6) spontaneous sakacin P-resistant mutant strain of L. monocytogenes were compared to the wild-type (L502). The growth of the resistant strains was reduced on mannose but not affected on cellobiose and the transcriptomics was performed during growth on these sugars. The mannose phosphotransferase system (PTS) encoded by the mptACD operon (mpt) is known for transporting mannose and also act as a receptor to class IIa bacteriocins. The mpt was repressed in L502-1 and this is in accordance with abolition of the bacteriocin receptor with resistance to class IIa bacteriocins. In contrast, the mpt was induced in L502-6. Despite the induction of the mpt, L502-6 showed 1,000 times more resistance phenotype and reduced growth on mannose suggesting the mannose-PTS may not be functional in L502-6. The microarray data suggests the presence of other transcriptional responses that may be linked to the sakacin P resistance phenotype particularly in L502-6. Most of commonly regulated genes encode proteins involved in transport and energy metabolism. The resistant strains displayed shift in general carbon catabolite control possibly mediated by the mpt. Our data suggest that the resistant strains may have a reduced virulence potential. Growth sugar- and mutant-specific responses were also revealed. The two resistant strains also displayed difference in stability of the sakacin P resistance phenotype, growth in the presence of both the lytic bacteriophage P100 and activated charcoal. Taken together, the present study showed that a single time exposure to the class IIa bacteriocin sakacin P may elicit contrasting phenotypic and transcriptome responses in L. monocytogenes possibly through regulation of the mpt. Data is also available from <ahref=http://bugs.sgul.ac.uk/E-BUGS-110 target=_blank>BuG@Sbase</a>

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RNA-Seq of Escherichia coli Nissle 1917 strains modified to produce different amounts of the bacteriocin microcin C

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