Transcription Inhibitors with XRE DNA-Binding and Cupin Signal-Sensing Domains Drive Metabolic Diversification in Pseudomonas

Abstract

Transcription factors (TFs) are instrumental in the bacterial response to new environmental conditions. They can act as direct signal sensors and subsequently induce changes in gene expression leading to physiological adaptation. Here, by combining transcriptome sequencing (RNA-seq) and cistrome determination (DAP-seq), we studied a family of eight TFs in Pseudomonas aeruginosa This family, encompassing TFs with XRE-like DNA-binding and cupin signal-sensing domains, includes the metabolic regulators ErfA, PsdR, and PauR and five so-far-unstudied TFs. The genome-wide delineation of their regulons identified 39 regulatory interactions with genes mostly involved in metabolism. We found that the XRE-cupin TFs are inhibitors of their neighboring genes, forming local, functional units encoding proteins with functions in condition-specific metabolic pathways. Growth phenotypes of isogenic mutants highlighted new roles for PauR and PA0535 in polyamines and arginine metabolism. The phylogenetic analysis of this family of regulators across the bacterial kingdom revealed a wide diversity of such metabolic regulatory modules and identified species with potentially higher metabolic versatility. Numerous genes encoding uncharacterized XRE-cupin TFs were found near metabolism-related genes, illustrating the need of further systematic characterization of transcriptional regulatory networks in order to better understand the mechanisms of bacterial adaptation to new environments.IMPORTANCE Bacteria of the Pseudomonas genus, including the major human pathogen Pseudomonas aeruginosa, are known for their complex regulatory networks and high number of transcription factors, which contribute to their impressive adaptive ability. However, even in the most studied species, most of the regulators are still uncharacterized. With the recent advances in high-throughput sequencing methods, it is now possible to fill this knowledge gap and help the understanding of how bacteria adapt and thrive in new environments. By leveraging these methods, we provide an example of a comprehensive analysis of an entire family of transcription factors and bring new insights into metabolic and regulatory adaptation in the Pseudomonas genus.

Keywords: DAP-seq; Pseudomonas aeruginosa; RNA-seq; XRE-cupin; global regulatory networks; transcription factors.

FIG 1

P. aeruginosa TFs belonging to the XRE family. (A) Maximum likelihood phylogenetic tree of the 19 proteins possessing an XRE DNA-binding domain in P. aeruginosa PAO1 (18). The branches corresponding to the eight XRE-cupin regulators are colored in red in the tree. (B) Stereo ribbon representation of the model of the XRE DNA-binding domain of ErfA. The five alpha-helices are annotated. (C) Stereo ribbon representation of the model of ErfA structure. The XRE domain is in red and the cupin domain is in blue. Model prediction was done using the SWISS-MODEL tool (34) using the structure with PDB ID 1Y9Q as the template.

FIG 2

Determination of the regulons of the XRE-cupin regulators. (A) Volcano plots displaying the RNA-seq results of the genes differentially expressed in the respective XRE-cupin mutants versus parental strain PAO1. Genes for which a DAP-seq binding peak was identified in the promoter are represented by red circles and annotated with their gene ID. The genes (yellow circles) found commonly downregulated in all mutants represent artifacts probably due to genetic manipulation. (B) Summary of number of regulatory targets per TF in P. aeruginosa PAO1.

FIG 3

The XRE-cupin regulators are inhibitors of transcription. (A) Repartition of binding sites identified by DAP-seq within target promoters. RNA polymerase binding sites were either inferred from experimentally determined transcription start sites (44) or predicted using BPROM (45) if no data were available. The transcription start site is shown as a black arrow, and the −10 and −35 boxes are shown as yellow rectangles. (B) RNA-seq expression fold changes of target genes in regulator mutants compared to the wild-type strain.

FIG 4

RT-qPCR and EMSA confirm the genome-wide results. Selected TFs and targets were PA0535 and pauB1 (PA0534) (A), PA1359 and PA1360 (B), PA1884 and PA1885 (C), PsdR and mdpA (PA4498) and dppA3 (PA4450) (D), PA4987 and PA4985-PA4986 (E), and PauR and davD (PA0265), PA1541 (by RT-qPCR), and PA2776 (by EMSA) (F). (Upper left panels) Local RNA-seq read abundance fold changes in the corresponding mutants compared to parental strain. Red lines show the deleted region in the mutant strain. (Lower left panels) Local fold enrichments in the corresponding regulator obtained by DAP-seq compared to negative controls. Target genes are shown as red arrows, genes encoding the studied TF as yellow arrows, and others as blue arrows. Suspected autoregulation is denoted with dashed yellow-red arrows. (Upper right panels) RT-qPCR showing the regulation of the target genes in the corresponding regulatory mutants and complemented strains. Experiments were performed in triplicates and normalized to the rpoD transcripts. Error bars indicate the SD. Statistical significance was assessed using two-tailed t test (P value < 0.05 [*], 0.01 [**], or 0.001 [***]) and is shown against wild-type (WT) strains for mutants and against mutants for complemented strains. (Lower right panels) EMSA on target binding sites. Recombinant XRE-cupin-His₁₀ proteins were incubated with 0.5 nM Cy5-labeled probes for 15 min before electrophoresis. For competition assays, excess of unlabeled probes (100 nM) is denoted by +.

FIG 5

The complete regulons of the XRE-cupin regulators. (A) Schematic views of the local targeted regions of the seven XRE-cupin TFs with determined regulons in P. aeruginosa PAO1. Genes in operons, as annotated in the Pseudomonas genome database (18), are connected with hyphens. Genes that both have a DAP-seq peak in their promoter region and are differentially expressed (P value < 0.05) are shown as regulated genes (red arrows). (B) Functional annotation of XRE-cupin regulatory targets. COG functional annotations were retrieved from the PAO1 genome on the Pseudomonas database (18) for all target genes.

FIG 6

Growth of the strains deficient in XRE-cupin regulators on putrescine and arginine. Bacterial growth in M9 minimal medium containing putrescine (A) or l-arginine (B) as carbon source. After overnight growth at 37°C in LB, bacteria were diluted to an OD₆₀₀ of 0.02 and grown in 96-well plates at 37°C with agitation for 24 h. Cultures were performed in biological duplicates.

FIG 7

Phylogenetic analysis of the conservation of the XRE-cupin regulatory family across the Pseudomonas genus. (A) Maximum-likelihood phylogenetic tree of 503 Pseudomonas complete genomes. The tree was generated from the multiple alignment of the concatenated sequences of 66 core genes for each strain with 100 bootstraps. The most represented species are delineated with a colored background. The number of XRE-cupin proteins detected through the hidden Markov model search is shown as the inner circle yellow-to-red heatmap. The outer circles show the results of homolog search by Reciprocal Best BLAST Hit search for the eight regulators studied here. (B) Dot plot showing the distribution of number of XRE-cupin regulators per strain for each species. Species represented by fewer than five strains are grouped in the “Others” column.

FIG 8

HMMER prediction of XRE-cupin regulators across the bacterial kingdom. Repartition of the number of predicted XRE-cupin regulators per genome. All 2,664 RefSeq representative complete bacterial genomes (>9.4 million unique protein sequences) were scanned using the XRE-cupin HMM and are shown in this figure across 40 different phylogenetic classes. Classes are sorted by increasing mean numbers of XRE-cupin regulators identified. A total of 4,515 XRE-cupin regulators were identified with HMM scores of >100 and were counted in this analysis.

FIG 9

Gene synteny and genetic environment of the XRE-cupin regulators. (A) Conservation of XRE-cupin neighboring target genes. Regulator-encoding genes are shown in yellow. Target genes are colored depending on how often they were found as conserved neighbors of their associated XRE-cupin TF. (B) Histogram showing the proportion of the most represented (>2%) GO functional annotations. Functional annotations were obtained from Pfam results of InterProScan search on 6,294 genes neighboring XRE-cupin regulators from 503 Pseudomonas genomes. Two categories are shown in gray as they correspond to the cycB gene, a conserved neighbor of pauR not regulated by PauR, and thus are not representing XRE-cupin regulatory targets.