BrrG encodes a putative antiterminator essential for bgtA-K expression and BaGTA-mediated gene transfer.

(A) Schematic representation of the BaGTA locus. Upper part: Structure and size of the bgtA-K and ror gene clusters. Lower part: Close-up of the bgtA-K locus encoding capsid and packaging functions and of the ror locus, encoding the run-off-replication origin oriROR and the associated brrA-G gene cluster. For the reporter strain bgtC-gfp and its derivatives used in D-G, the insertion site of the transcriptional gfp reporter within the bgtA-K locus is indicated. (B) Comparison of the structure of Q21 antiterminator (6p1b) of phage P21 and Alphafold2 model of BrrG (A0A0R4J952). Similar structural features are presented in identical colour. (C) Schematic presentation of (i) the flow cytometry assay to monitor expression of the bgtA-K locus using the bgtC-gfp transcriptional reporter strain (upper panel) and (ii) the assay to monitor GTA transfer via transfer of GmR from the GTA-positive donor strain to the ΔbgtCD SpR GTA-negative (ΔGTA) recipient strain resulting in SpRGmRbacteria and formula to calculate the frequency of double resistant bacteria (fDRB) as a proxy for GTA transfer. (D) BrrG overproduction increases the bacterial population expressing bgtA-K. BrrG expression was induced in strain bgtC-gfp (pPtet-brrG) with anhydrotetracycline (aTc). Strain bgtC-gfp H1GmR (bgtC-gfp BH01490::Himar1GmR) displaying wild-type properties served as a positive control. (E) BrrG overproduction does not enhance GTA transfer. Strain bgtC-gfp H1GmRdisplaying wild-type properties and the GTA-negative mutant ΔbgtCD H1GmR(ΔbgtCD BHRS00695::Himar1GmR) served as a positive and negative control, respectively. (F) Deletion of brrG (ΔbrrG bgtC-gfp H1GmR [ΔbrrG BH01490::Himar1GmR]) results in reduced expression of the bgtA-K locus. (G) Overproduction of BrrG in a ΔbrrG mutant background restores GTA transfer frequency to the wild-type level. BrrG expression was induced in strain ΔbrrG H1GmR(pPtet-brrG) with aTc. (D-G) The time-course experiments were started with the incubation of CBA agar-grown bacteria in M199 medium supplemented with 10% FCS. 25 ng/ml aTc was added where indicated to induce expression by the Ptet promoter. The experiments were performed in biological triplicates (D, F, G) or quadruplicates (E), error bars indicate SD.

BrrG binds in proximity to the PbgtA-K promoter located upstream of the putative bgtA-K operon.

(A) Location of the PbgtA-K promoter and of the BrrG binding site identified by ChIP-Seq (gray histogram). ΔbrrG + pPtet-brrG (no tag) is shown in dark grey; ΔbrrG + pPtet-3x-FLAG-brrG is shown in green. The fragments upstream of the bgtA-K locus were tested for promoter activity in promoter-probe plasmids as indicated. (B) The fusion of the 999 bp fragment spanning from the region between bgtK and clpB to dsRed (strain WT (PPf3-dsRed) displays a temporal transcriptional profile similar to the chromosomal transcription reporter for the bgtA-K locus (strain bgtC-gfp H1GmR [bgtC-gfp BH01490::Himar1GmR]). (C) The PbgtA-K promoter is located far upstream of the bgtA-K locus. Left: Temporal transcriptional profiles controlled by fragments spanning nucleotide 1-416 (plasmid psF1), nucleotides 271-782 (plasmid psF2), or nucleotides 594-999 (plasmid psF3) of the 999 bp fragment in Pf3. Right: Corresponding histograms of fluorescence distribution at the 6 h time-point. Strain bgtC-gfp displaying wild-type expression pattern of the bgtA-K locus was used as control. (D) DNA sequence alignment of the predicted promoter region and putative BrrG binding site within psF3 fragment with homologous sequences from various Bartonella species. The predicted -35 and -10 promoter elements and the inferred transcriptional start site (+1) are indicated. Numbers below indicate the location of the promoter elements relative to bgtK. (B, C) The time-course experiments were started with the incubation of CBA agar-grown bacteria in M199 medium supplemented with 10% FCS. The experiments were done in biological triplicates, error bars indicate SD.

A putative terminator is encoded in the long non-protein coding sequence downstream of the PbgtA-K promoter.

(A) Schematic representation of the 5’-part of the bgtA-K locus with the truncations tested with promoter-probe plasmids. Locations of the PbgtA-Kpromoter and the predicted terminator are shown. (B) Histograms of fluorescence distribution measured for the bacterial populations containing the promoter-probe plasmids with various truncations at 6 h. (C) Predicted secondary structure and free energy of the putative TbgtA-K terminator. Numbers in parentheses indicate the location of the terminator relative to bgtK. The time-course experiment was started with the incubation of CBA plate-grown bacteria in M199 medium supplemented with 10% FCS. Experiments were performed in biological triplicates.

The -10-like element downstream of the PbgtA-K promoter is crucial for the antitermination by BrrG.

(A) Alignment of Bartonella spp. consensus and lambdoid phage sequences (P21, phi80 and lambda) sequences containing PbgtA-K and the PR’ promoters followed by the -10-like elements, respectively. Promoter -35 and -10 elements, as well as the -10-like elements are marked. (B) Histograms of fluorescence distribution for the bacterial populations containing the promoter-probe plasmid pF6 (compare to Fig. 3A) with the PbgtA-Kpromoter and the adjacent native -10-like element or its derivative, plasmid pF6(-10mut), carrying mutations in two conserved positions of the -10-like element. Plasmid pPtet-brrG encodes an aTc-inducible brrG expression cassette. BrrG overproduction was induced by 25 ng/ml aTc. The time-course experiment was started with the incubation of CBA plate-grown bacteria in M199 medium supplemented with 10% FCS. Inducer aTc was added at time-point 0 h. Experiments were performed in biological triplicates.

Antiterminator BrrG allows RNA polymerase to bypass an engineered termination site.

(A) Schematic representation of the experimental setup. Plasmid pF2-TBBa_1006-gfp carries the insertion of the artificial strong terminator TBBa_1006 in between the terminator-less promoter fragment F2 and gfp of plasmid pF2-gfp. (B) Histograms of fluorescence distribution for the bacterial populations containing the fragment F2, the fragment F2 with the terminator and the sequence with the mutated -10-like element (pF6- 10mut). Time of the measurements is indicated. Induction was done with 25 ng/ml aTc. Bacteria were cultivated in M199 medium supplemented with 10% FCS and aTc inducer at indicated concentrations. Experiments were performed in biological triplicates.

Model of BrrG-antitermination.

(A) Schematic representation of terminator-dependent transcription inhibition. RNAP initiates transcription at the TSS (+1) and proceeds until the termination site (TbgtA-K), where it falls off. (B) Transcriptional antitermination of the BaGTA in presence of BrrG. BrrG acts as antiterminator enabling the transcription of the downstream bgtA-K cassette.

List of strains used in this study.

List of plasmids used in this study.

List of oligonucleotides used in this study.

BrrG, GafA and GafZ share structural features.

Structure of Q21 (6p1b) and AlphaFold2 models of BrrG (partial, A0A0R4J952), GafA (partial, D5AUH1) and GafZ (partial, A0A0H3C763). Similar features are shown in the same colour. Table below illustrates DALI Z-score and Rmsd score analysis of structural similarities. AlphaFold2 models were aligned to structure of Q21 (6p1b) using DALI Server (http://ekhidna2.biocenter.helsinki.fi/dali/). Higher Z-scores, respectively lower Rmsd values suggest better alignment and similarity to the reference structure.

BrrG binds in the promoter region PbgtA-K in front of the bgtA-K cluster.

Location of the PbgtA-K promoter and of the BrrG binding site of ΔbrrG + pPtet-3xFLAG-brrG- identified by ChIP-Seq. IP DNA is shown in green and input DNA in dark grey. ΔbrrG + pPtet-brrG was used as control. One replicate per strain is shown. Scale on the left indicates the coverage of the enriched DNA. Scale on the top indicates the genomic position in the Bartonella henselae genome.

The PbgtA-Kpromoter is located far upstream of the bgtA-K locus.

Fluorescence distribution at the 6 h time-point. Strain bgtC-gfp displaying wild-type expression pattern of the bgtA-K locus was used as control. The time-course experiments were started with the incubation of CBA agar-grown bacteria in M199 medium supplemented with 10% FCS. The experiments were done in biological triplicates.

Fluorescence distribution for the bacterial populations containing the promoter-probe plasmids with various truncations at 6 h.

The time-course experiments were started with the incubation of CBA agar-grown bacteria in M199 medium supplemented with 10% FCS. The experiments were done in biological triplicates.

The -10-like element downstream of the PbgtA-K promoter is crucial for the antitermination by BrrG.

Histograms of fluorescence distribution for the bacterial populations containing the promoter-probe plasmid pF6 with the PbgtA-K promoter and the adjacent native - 10-like element or its derivative, plasmid pF6(-10mut), carrying mutations in two conserved positions of the -10-like element. Plasmid pPtet-brrG encodes an aTc-inducible brrG expression cassette. BrrG overproduction was induced by 25 ng/ml aTc. The time-course experiment was started with the incubation of CBA plate-grown bacteria in M199 medium supplemented with 10% FCS. Inducer aTc was added at time-point 0 h. Experiments were performed in biological triplicates.

Fluorescence distribution for the bacterial populations containing the fragment F2 (Fig. 2A), the fragment F2 with the terminator and the sequence with the mutated -10-like element (pF6-10mut).

Time of the measurements is indicated. Induction was done with 25 ng/ml aTc. Bacteria were cultivated in M199 medium supplemented with 10% FCS and aTc inducer at indicated concentrations. Experiments were performed in biological triplicates.