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. 2021 Feb 26;11(1):4771.
doi: 10.1038/s41598-021-84057-4.

The σ54 system directly regulates bacterial natural product genes

Muqing Ma  1 , Roy D Welch  1 , Anthony G Garza  2
Affiliations

The σ54 system directly regulates bacterial natural product genes

Muqing Ma et al. Sci Rep. .

Abstract

Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutics and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery controlling their transcription. Recent findings suggest that the σ54 system, which includes σ54-loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explored this idea by analyzing a selected group of putative σ54 promoters identified in Myxococcus xanthus natural product gene clusters. We show that mutations in putative σ54-RNA polymerase binding regions and in putative Nla28 EBP binding sites dramatically reduce in vivo promoter activities in growing and developing cells. We also show in vivo promoter activities are reduced in a nla28 mutant, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the Nla28 binding sites are mutated. Together, our results indicate that M. xanthus uses σ54 promoters for transcription of at least some of its natural product genes. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product loci are located within genes and not in intergenic sequences.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The promoter regions of the MXAN1286, MXAN1579, MXAN1603 and MXAN3778 natural product loci. Nucleotides the match those in the consensus Nla28 binding site or the consensus σ54 RNA polymerase binding site are relatively large. The conserved GC dinucleotide in − 12-bp region and the conserved GG dinucleotide in − 24-bp region of the putative σ54 RNA polymerase binding sites are in bold. The underlined nucleotides represent the spacers between the two half Nla28 binding sites or the spacers between − 12 and − 24-bp promoter regions.
Figure 2
Figure 2
Locations of putative PK/NRP σ54 promoters identified in the M. xanthus genome. Of the 83 putative PK/NRP σ54 promoters identified in M. xanthus genome sequence, 74 (89%) are located in protein coding sequences (intragenic promoters) and 9 (11%) are located in non-coding sequences (intergenic promoters). Of the 74 intragenic promoters, 43 are located within a protein coding sequence in an operon or within the protein coding sequence of a single gene (internal promoters), and 31 are located in the protein coding sequence of an upstream gene (upstream promoters).
Figure 3
Figure 3
In vivo activities of wild-type MXAN1286, MXAN1579 and MXAN3778 promoters and derivatives of the promoters carrying a mutation in the putative − 12-bp region, − 24-bp region or spacer region. Wild-type and mutant fragments of the MXAN1286, MXAN1579 and MXAN3778 promoters were cloned into a lacZ expression vector and transferred to the wild-type M. xanthus strain DK1622. At various cell densities during growth (A–C) and time points during development (D–F), β-galactosidase-specific activities (defined as nanomoles of ONP produced per minute per milligram of protein) in cells carrying a wild-type or a mutant promoter fragment were determined. (N = 3 at each density or time point; error bars are standard deviations of the means; ***p < 0.001; **p < 0.01; *p < 0.05 for in vivo activities of mutant promoters versus wild-type promoters).
Figure 4
Figure 4
In vivo activities of MXAN1286, MXAN1579 and MXAN3778 promoters in nla28 mutant cells and when Nla28 binding sites are mutated. Wild-type fragments of the MXAN1286, MXAN1579 and MXAN3778 promoters were cloned into a lacZ expression vector and transferred to the wild-type M. xanthus strain DK1622 (Wild Type) or to a derivative of strain DK1622 with an inactivated nla28 gene (nla28 Mutant). In addition, fragments of the MXAN1286, MXAN1579 and MXAN3778 promoters containing one mutated Nla28 half binding site were cloned into a lacZ expression vector and transferred to strain DK1622 (Binding Site Mutant). At various cell densities during growth (A–C) and time points during development (D–F), β-galactosidase-specific activities in cells carrying a wild-type or a mutant promoter fragment were determined. (N = 3 at each density or time point; error bars are standard deviations of the means; ***p < 0.001; **p < 0.01; *p < 0.05 for in vivo activities of wild-type promoters in nla28 mutant versus in wild-type cells; ###p < 0.001; ##p < 0.01; #p < 0.05 for in vivo activities of binding site mutant promoters versus wild-type promoters in wild-type cells).
Figure 5
Figure 5
EMSAs performed with Nla28-DBD and a MXAN1286, MXAN1579 or MXAN3778 promoter fragment carrying a wild-type or mutated Nla28 binding site. Binding reactions were performed with (+) or without (−) 2 μM of purified Nla28-DBD and a Cy5 end-labeled promoter fragment containing a wild-type (WT) or mutated (Mut) Nla28 binding site.
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