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. 2023 Nov 16;19(11):e1011048.
doi: 10.1371/journal.pgen.1011048. eCollection 2023 Nov.

XRE transcription factors conserved in Caulobacter and φCbK modulate adhesin development and phage production

Maeve McLaughlin  1 , Aretha Fiebig  1 , Sean Crosson  1
Affiliations

XRE transcription factors conserved in Caulobacter and φCbK modulate adhesin development and phage production

Maeve McLaughlin et al. PLoS Genet. .

Abstract

The xenobiotic response element (XRE) family of transcription factors (TFs), which are commonly encoded by bacteria and bacteriophage, regulate diverse features of bacterial cell physiology and impact phage infection dynamics. Through a pangenome analysis of Caulobacter species isolated from soil and aquatic ecosystems, we uncovered an apparent radiation of a paralogous XRE TF gene cluster, several of which have established functions in the regulation of holdfast adhesin development and biofilm formation in C. crescentus. We further discovered related XRE TFs throughout the class Alphaproteobacteria and its phages, including the φCbK Caulophage, suggesting that members of this cluster impact host-phage interactions. Here we show that a closely related group of XRE transcription factors encoded by both C. crescentus and φCbK can physically interact and function to control the transcription of a common gene set, influencing processes including holdfast development and the production of φCbK virions. The φCbK-encoded XRE paralog, tgrL, is highly expressed at the earliest stages of infection and can directly inhibit transcription of host genes including hfiA, a potent holdfast inhibitor, and gafYZ, an activator of prophage-like gene transfer agents (GTAs). XRE proteins encoded from the C. crescentus chromosome also directly repress gafYZ transcription, revealing a functionally redundant set of host regulators that may protect against spurious production of GTA particles and inadvertent cell lysis. Deleting the C. crescentus XRE transcription factors reduced φCbK burst size, while overexpressing these host genes or φCbK tgrL rescued this burst defect. We conclude that this XRE TF gene cluster, shared by C. crescentus and φCbK, plays an important role in adhesion regulation under phage-free conditions, and influences host-phage dynamics during infection.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. A network of two-component regulators and XRE-family transcription factors control Caulobacter holdfast development.
A) Overlay of phase contrast and fluorescence micrographs of C. crescentus CB15 cultivated on peptone-yeast extract (PYE) medium, provides an example of unipolar polysaccharide adhesins in Alphaproteobacteria. The C. crescentus adhesin, known as the holdfast, is stained with wheat germ agglutinin conjugated to Alexa594 dye (yellow arrowheads). The holdfast is present at the tip of the stalk in a fraction of pre-divisional cells when cultivated in PYE. Scale bar = 1 μm. B) Schematic of a regulatory network that regulates transcription of the holdfast inhibitor, hfiA. Network includes the essential cell cycle regulators CtrA and GcrA [6,7,22]. A set of interacting sensor histidine kinases (LovK-SkaH-SpdS) activate the DNA-binding response regulator, SpdR. Dashed lines indicate post-transcriptional regulation and solid lines indicate transcriptional regulation. Black arrows indicate activation and red bar-ended lines indicate repression.
Fig 2
Fig 2. Conservation of XRE-family adhesion regulators in Caulobacter.
A) Pangenome constructed from whole-genome sequences of 19 Caulobacter species isolated from freshwater or soil. Internal dendrogram based on shared presence and absence of gene clusters generated using the Markov Cluster (MCL) algorithm (mcl-inflation = 3; min-occurrence = 2); gene clusters organized based on Euclidian distance metric and Ward linkage. Black bars in the internal 19 circles show presence of gene clusters; gray bars indicate absence of gene clusters. Number of genomes that contribute to a gene cluster (from 2–19), number of genes in a gene cluster (from 2–82), and maximum number of paralogs in any single species (from 1–7) are plotted on the outer 3 circles. Genomes are organized by average nucleotide identity (ANI). Dendrogram based on ANI shown above the red heatmap. Single-copy core genes (SCG), i.e. genes present in one copy in all species, are marked in dark green. Multi-copy core genes, i.e. genes present in all species and in more than one copy in at least one species, are marked with a dotted line outside the circle. Gene clusters containing previously reported holdfast regulators rtrA, rtrB, rtrC, and staR, and newly-discovered XRE-family transcriptional regulators, cdxA (CCNA_00049) and cdxB (CCNA_02755) are marked with black arrows.
Fig 3
Fig 3. Pangenome summary of holdfast regulators.
A) Number of paralogs in the gene clusters highlighted in Fig 2 (mcl-inflation = 3). C. crescentus members of GC_0003: RtrA, RtrB, CdxA (CCNA_00049); GC_0408: StaR; GC_2778: CdxB (CCNA_02755); GC_2397: RtrC. B) Percent pairwise amino acid identity between all of the XRE-family transcription factors (HTH_XRE; Conserved Domain Database accession—cd00093) encoded by C. crescentus NA1000. Numbers indicate the corresponding locus ID (CCNA_XXXXX).
Fig 4
Fig 4. C. crescentus and φCbK XRE regulators repress hfiA transcription and promote holdfast development.
A) hfiA expression evaluated using a PhfiA-mNeonGreen transcriptional reporter. Fluorescence was measured in a wild type background containing either the empty vector (EV), or rtrA, rtrB, cdxA, cdxB, or φCbK tgrL overexpression (++) vectors. Fluorescence was normalized to cell density. B) Percentage of cells with stained holdfast. Using the same strains as in (A), stained holdfasts were quantified by fluorescence microscopy. In A and B, cells were grown in M2-xylose defined medium. Bars represent the mean ± standard deviation of at least three biological replicates (dots). Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison (p-value ≤ 0.01,**).
Fig 5
Fig 5. C. crescentus and φCbK XRE regulators have redundant DNA binding repertoires.
A) XRE proteins bind the hfiA promoter in vivo. ChIP-seq profile from pulldowns of 3xFLAG-tagged XRE proteins are shown. Lines indicate the fold-enrichment from pulldowns compared to an input control. Genomic position and relative position of genes are indicated. Data are in 25 bp bins and are the mean of three biological replicates. B) XRE proteins bind overlapping sites on the C. crescentus chromosome. Venn diagram showing the number of ChIP-seq peaks (100 bp centered on summit) that occupy overlapping regions as identified by ChIPpeakAnno [83]. C) DNA sequence motif enriched in indicated XRE ChIP-seq peaks as identified by XSTREME [82].
Fig 6
Fig 6. Temporal expression profile of φCbK transcription during infection of C. crescentus.
A) Relative φCbK transcript levels over an infection time course as measured by RNA-seq. Percentage of reads from RNA-seq mapped to the φCbK genome compared to the C. crescentus host genome. B) φCbK tgrL is transcribed at the earliest stages infection. Normalized transcript levels (RPKM) for φCbK tgrL from RNA-seq were plotted over the course of an infection. C) Relative expression of φCbK genes during an infection. Transcript levels for each φCbK gene were plotted and relative values were calculated by normalizing transcript levels at a time point to the maximum transcript levels for that gene over the infection time course. Columns correspond to φCbK genes and genes are placed in the order that they appear on the phage chromosome. Genomic modules for structural, lysis, and DNA replication genes as indicated by [88] are marked below the heatmap. The position of φCbK tgrL is indicated by a vertical line and labeled. A-C) Wild type cells were infected during logarithmic growth at 10 multiplicity of infection (MOI). Samples for t = 0 min were harvested prior to phage addition. Data are the mean and error bars represent the standard deviation of three biological replicates.
Fig 7
Fig 7. TgrL regulates C. crescentus gene expression.
RNA-seq analysis of genes significantly regulated upon induction of φCbK tgrL expression. Volcano plot displays log2(fold-change) and -log10(FDR p-value) for all C. crescentus genes, comparing φCbK tgrL expression from a plasmid to that from an empty vector (EV). Experiment was conducted in a genetic background lacking the host XRE genes: ΔrtrA ΔrtrB ΔcdxA ΔcdxB background. Gray dots indicate genes for which expression does not change significantly, blue dots indicate genes without a TgrL ChIP-seq peak in an associated promoter, and pink dots indicate genes with a φCbK TgrL ChIP-seq in an associated promoter. Vertical red lines mark boundaries for ±1.5-fold-change; horizontal red line marks 0.0001 FDR p-value. Points represent the mean of three biological replicates.
Fig 8
Fig 8. C. crescentus and φCbK XRE regulators repress gafYZ and gene transfer agent (GTA) production.
A) XRE proteins bind the gafYZ promoter in vivo. ChIP-seq profile from pulldowns of 3xFLAG-tagged protein are shown. Lines show fold-enrichment from pulldowns compared to an input control. Genomic position and relative position of genes are indicated. Data are in 25 bp bins and are the mean of three biological replicates. B) gafYZ expression using a PgafYZ-mNeonGreen reporter. Fluorescence was measured and normalized to cell density. Data are the mean and error bars are the standard deviation of three biological replicates (black dots). Statistical significance was determined by one-way ANOVA followed by Šídák’s multiple comparisons test (p-value ≤ 0.0001,****). C) XRE proteins repress GTA production. Total DNA was purified, separated by gel electrophoresis, and imaged. GTA-associated DNA resolved at ~8 kb. Image is a representative gel from at least 3 biological replicates. B-C) Experiments were performed with wild type (WT) or strains harboring in-frame deletions (Δ) in rtrA, rtrB, cdxA, cdxB, gafYZ, and/or rogA. Δxre indicates the ΔrtrA ΔrtrB ΔcdxA ΔcdxB background. Strains contained either an empty vector (EV), rtrA, rtrB, cdxA, cdxB, or φCbK tgrL overexpression (++) vectors. Strains were grown to stationary phase in complex medium (PYE).
Fig 9
Fig 9. C. crescentus and φCbK XRE regulators support phage infection.
φCbK burst size in mutant backgrounds. A-B) Plaque forming units (PFU) per infected cell (i.e. burst size) was plotted for either wild type or mutants with in-frame deletions (Δ) of rtrA, rtrB, cdxA, and/or cdxB. Δxre indicates the ΔrtrA ΔrtrB ΔcdxA ΔcdxB quadruple deletion background. B) Strains contained either an empty vector (EV), or rtrA, rtrB, cdxA, cdxB, or φCbK tgrL overexpression (++) vectors. A-B) Strains were infected with φCbK at 0.01 multiplicity of infection (MOI) in logarithmic growth phase in complex medium (PYE). Data bars represent the mean and error bars are the standard deviation of at least three biological replicates (black dots). Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison (p-value ≤ 0.05,*; ≤ 0.01,**).
Fig 10
Fig 10. C. crescentus and φCbK XRE regulator interactions in a two-hybrid assay.
Heatmap summarizing interactions between RtrA, RtrB, CdxA, CdxB, StaR, and φCbK TgrL based on bacterial two-hybrid (BTH) assays. Proteins were fused to split adenylate cyclase fragments (T18c and T25) and co-expressed in E. coli. Interactions between the fused proteins reconstitutes adenylate cyclase, promoting expression of a lacZ reporter. Empty vector (EV) are the negative control and Zip is the positive control. β-galactosidase activity was measured for each pairing, and Miller units were calculated. Data are the mean of at least three biological replicates. See S6 Fig for statistical analysis.
Fig 11
Fig 11. XRE Transcription Factor Network in regulation of Caulobacter and φCbK gene expression.
Schematic of XRE transcription factor (TF) network (top) and φCbK infection schematic (bottom). The sensor histidine kinases LovK, SkaH, and SpdS physically interact and promotes SpdR activity [7]. SpdR activates rtrC expression [6], and RtrC and SpdR activate expression of XRE TF paralogs (rtrA, rtrB, cdxA, and cdxB) [6, 7]. XRE TFs repress holdfast and gene transfer agent (GTA) regulators, hfiA and gafYZ, respectively. φCbK tgrL is highly expressed during early infection of C. crescentus and regulates expression of C. crescentus genes. Dashed lines indicate post-transcriptional regulation and solid lines indicate transcriptional regulation. Black arrows indicate activation and red bar-ended lines indicate repression. Pink phage indicates φCbK phage, grey cells indicate C. crescentus, and cells outlined in dashed lines indicates lysed cells.
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