Evidence for Rho-dependent control of a virulence switch in Acinetobacter baumannii

ABSTRACT Acinetobacter baumannii strain AB5075 is able to interconvert at high frequency between virulent cells that form opaque colonies (VIR-O) and avirulent cells that form translucent colonies (AV-T). Cells switch from the VIR-O to the AV-T state by the combinatorial activation of at least three TetR-type transcriptional regulators (TTTRs). A genetic screen identified the transcription termination factor Rho as a major contributor that controls expression differences of these TTTRs between VIR-O and AV-T cells. Each TTTR has a long mRNA leader region where transcripts are terminated in VIR-O cells. However, in AV-T cells, the degree of termination in each TTTR leader was greatly reduced, allowing for higher levels of TTTR expression. In a strain with decreased Rho expression, or in wild-type VIR-O cells treated with the Rho inhibitor bicyclomycin, the degree of termination in each TTTR mRNA leader region was reduced. Mutations in the leader region of one TTTR, ABUW_1645, were identified that reduced the degree of termination. Purified Rho protein bound the leader region of the most frequently activated TTTR ABUW_1645 and the least frequently activated TTTR ABUW_1959 with similar affinities. This together with the observation that the levels of Rho protein were unchanged between VIR-O and AV-T cells, suggested that additional factors differentially modulate Rho activity between each variant. Finally, we demonstrate in AV-T cells that nutrient depletion is a condition that increases the levels of Rho-dependent transcription termination in the TTTR leader regions to regulate this phenotypic switch. IMPORTANCE Acinetobacter baumannii is a significant cause of infections in the healthcare setting. More recently, A. baumannii has been a leading cause of secondary bacterial pneumonia in patients infected with SARS-CoV-2 and the overall frequency of A. baumannii infection increased 78% during the COVID-19 pandemic. A. baumannii can exist in virulent or avirulent subpopulations and this interconversion is mediated by the expression of a family of TetR-type transcriptional regulators. In this study, we demonstrate that Rho is a key regulatory component in the expression of these TetR regulators. Overall, this study is the first to address a role for Rho in A. baumannii and provides additional evidence for the role of Rho in regulating diversity in bacterial subpopulations.

the skin and soft tissues, bloodstream infections, and urinary tract infections (1,6,7).A. baumannii is responsible for approximately 2 million infections and 450,000 deaths annually worldwide (8).A greater understanding of its mechanisms of antibiotic resistance and virulence are fundamental for the development of novel approaches to overcome A. baumannii infections.
Genetically identical populations of bacteria contain subpopulations that exhibit pronounced phenotypic differences due to distinct gene expression profiles relative to the general population (9)(10)(11).This phenotypic heterogeneity can regulate virulence, and examples of this can be found in both prokaryotes and eukaryotes (12)(13)(14)(15)(16). Phenotypic heterogeneity can be generated by using a bistable regulatory system, where the expression of a global transcriptional regulator is maintained in an "ON" state by positive autoregulation or by a pair of mutually repressing repressors (9,10).In a stochastic manner, a rare cell expresses more of the regulator typically due to random fluctuations in gene expression "noise, " which sets off an autoactivation cascade (9,10,17).This results in a single cell expressing the global regulatory protein (ON state) and this cell now exhibits a new set of phenotypes.Importantly, once a cell has activated a bistable switch, it can be propagated during cell division by a positive feedback loop and thus be inherited by daughter cells during division (13,(17)(18)(19)(20). Phenotypic heterogeneity provides an isogenic population of cells the flexibility to adapt to changing environ ments, both inside and outside the host (11,21).
In bacteria, one mechanism for transcriptional termination is mediated by the Rho protein, a hexameric ATP-dependent helicase that based on one model binds to sites on mRNA termed rut sites (Rho utilization) and displaces RNA polymerase (22,23).A typical rut site is an unstructured region of approximately 80 bp with regularly spaced C residues (24,25).In an alternative model, Rho directly associates with RNA polymerase during transcription and then mediates termination at the appropriate sites (26).In Escherichia coli, Rho-mediated termination can be modulated by both the NusG and NusA proteins and accounts for approximately 20% of termination events (27)(28)(29)(30).While Rho mediates termination at the 3′-end of genes, it is also responsible for termination within untransla ted 5′ upstream regions (UTR) of mRNAs, hereafter described as a leader region and in this capacity, it regulates gene expression (31,32).Rho-mediated termination within a mRNA leader region can be influenced by additional factors, such as riboswitches, sRNAs, translation of small ORFs and DNA inversions (23,(31)(32)(33)(34)(35).Rho has been shown to have a role in phenotypic heterogeneity in Clostridioides difficile and C. botulinum (16,36,37).In A. baumannii, Rho is essential for viability (38,39).
A phenotypic switch has been described in A. baumannii that generates virulent and avirulent subpopulations (40)(41)(42).Each cell variant can be distinguished by its colony morphology on agar plates using oblique lighting, with virulent variants forming opaque colonies (VIR-O) and avirulent variants forming translucent colonies (AV-T) (41,43).In addition to differences in virulence, the VIR-O variant possesses a thicker capsule than the AV-T variant; it is considerably more motile and secretes greater amounts of the quorum-sensing signal 3-hydroxy-dodecanoyl-l-homoserine lactone (3-OH-C 12 -HSL) (40,43).In contrast, the AV-T variant forms more robust biofilms and is metabolically more versatile.Despite our increasing knowledge of the underlying mechanisms that lead the interconversion between the VIR-O and the AV-T variants, a number of questions remain to be addressed.
We recently demonstrated that the stochastic activation of a family of TetR-type transcriptional regulators (TTTRs) is able to drive the cells from the VIR-O state to many of the phenotypes associated with the AV-T state (43).Activation of the TTTR ABUW_1645 (ABUW_RS08030) constitutes the preferred pathway to switch and it gives rise to avirulent AV-T variants.However, two additional TTTRs, ABUW_1959 (ABUW_RS09530), and ABUW_2818 (ABUW_RS13690) can also be activated alone or in different combina tions with ABUW_1645 to drive the cells to variants that form translucent colonies and are predominantly avirulent (AV-T) (43).This mechanism has an important biolog ical relevance, as it gives rise to diverse AV-T subpopulations that present variable patterns of TTTR expression, thus conferring unique phenotypic traits to each subvariant (43).Therefore, characterization of this phenotypic switch as well as the mechanism responsible for its regulation is important for a full understanding of A. baumannii virulence and how cells adapt to the environment.
In this study, we report a role for the transcription termination factor Rho in the VIR-O to AV-T switch.A transposon insertion in the rho upstream region converted the VIR-O cells to an AV-T-like state and increased the expression of ABUW_1645.Subsequent genetic analysis of this mutant and the use of the Rho inhibitor bicyclomycin (BCM) demonstrated that transcriptional termination within a long leader region of each TTTR mRNA was responsible for TTTR expression differences between VIR-O and AV-T variants.Moreover, we demonstrate that Rho can interact with the mRNA leader regions of ABUW_1645 and ABUW_1959 which represent the most frequently and least frequently activated TTTRs, respectively, during the VIR-O to AV-T switch (43).Finally, in AV-T cells, starvation increases Rho-dependent transcription termination and the resulting decrease in TTTR expression results in an increased rate of switching to the VIR-O state.Our findings provide new insight into the role of the transcription termination factor Rho in modulating phenotypic heterogeneity in A. baumannii.

Identification of Rho as a regulator of ABUW_1645 expression
Previously, the TTTR encoded by the ABUW_1645 gene was identified as an impor tant regulator of the switch between the VIR-O and the AV-T states (41).ABUW_1645 expression was upregulated 150-fold in the AV-T variant (AV-T.LS) (43), relative to VIR-O, and its overexpression in VIR-O cells drove them to the AV-T state, where cells became avirulent and exhibited all known phenotypes associated with the AV-T state (41,43).The AV-T.LS variant with the TTTRs ABUW_1645, ABUW_1959, and ABUW_2818 all in the ON state was used for experiments in this study.To search for regulators that controlled expression of ABUW_1645, transposon mutagenesis with a mariner derivative (MarTc) was used in A. baumannii ATCC 17978 containing a chromosomal 1645-lacZ fusion.After repeated screens of over 100,000 colonies for altered blue color on X-gal plates, a single colony with increased 1645-lacZ expression was identified as a darker blue colony.In addition, this insertion resulted in a small colony phenotype, indicating that the insertion had a detrimental effect on cell growth.The transposon insertion site was localized to the region immediately upstream of the ABUW_3277 (ABUW_RS15915) gene, encoding a 422 amino acid protein that exhibited 78% amino acid identity and 89% similarity to the E. coli Rho protein.Rho mediates transcriptional termination at the 3′-end of genes, and also within mRNA leader regions that precede coding regions (44,45).
To determine the phenotype of this insertion in the AB5075 background, a strain with a high rate of switching and where the role of ABUW_1645 was previously studied, the rho::MarTc insertion, hereafter designated rho::Tc, was moved into this strain by natural transformation of chromosomal DNA from the ATCC17978 rho::Tc mutant.We initially moved the insertion into the VIR-O background, where the ABUW_1645 gene is normally in the OFF state and expressed at 150-fold lower levels than the AV-T.LS variant (43).Since the MarTc insertion was between the promoter and the rho coding region, it was unclear if the insertion increased or decreased Rho expression.The use of qRT-PCR analysis demonstrated the rho::Tc insertion in the VIR-O background reduced rho expression 4.9-fold (Fig. 1A).Concurrently, this insertion increased ABUW_1645 expression 13.2-fold compared to the wild-type VIR-O strain (Fig. 1A).In addition, a VIR-O strain containing the rho::Tc insertion now exhibited a translucent colony phenotype, typical of cells overexpressing ABUW_1645 (not shown) (41).When the rho::Tc insertion was moved into the AV-T background, the mutant colonies were identical in appearance to the VIR-O rho::Tc mutant.Since both the VIR-O rho::Tc and AV-T rho::Tc mutants were locked into this translucent state, the frequency of switching between variants could not be determined.Because the rho::Tc insertion resulted in the conversion of VIR-O cells to an AV-T like-state based on colony opacity, we tested for an additional phenotype associated with the AV-T state, the inability to secrete the quorum sensing signal 3-OH C 12 -HSL (43).Using an Agrobacterium tumefaciens traG-lacZ fusion in a soft agar lawn as a biosensor to detect 3-OH C 12 -HSL secretion, the VIR-O rho::Tc mutant failed to secrete 3-OH C 12 -HSL, providing further evidence that the rho::Tc insertion in the VIR-O background converted cells to the AV-T state (Fig. 1B).

Complementation of the VIR-O rho::Tc mutant
To complement the rho::Tc mutation, a wild-type copy of the rho gene was introduced into the chromosome using a Tn7 derivative, where rho transcription from the tac promoter could be induced by IPTG (46).In the presence of IPTG, the VIR-O rho::Tc/ Tn7-rho cells recovered the wild-type VIR-O colony size (Fig. 1C).In addition, the ability to secrete 3-OH C 12 -HSL was restored to VIR-O rho::Tc cells containing Tn7-rho in the presence of IPTG (Fig. 1B).Expression of rho and ABUW_1645 in the VIR-O rho::Tc complemented strain was also assessed by qRT-PCR.In the VIR-O rho::Tc/Tn7-rho cells grown in the presence of IPTG, rho expression was increased 4.1-fold compared to the uninduced mutant.In the case of ABUW_1645, its expression was decreased 68.6-fold in the presence of IPTG, relative to uninduced cells (Fig. 1D).

Termination within a long mRNA leader region accounts for ABUW_1645 expression differences between VIR-O and AV-T cells
To determine the transcriptional start site (TSS) for the ABUW_1645 gene, reads from previously reported RNA-sequencing (41) were mapped to this region, allowing for the identification of a predicted 5′-end of the 1645 mRNA.This revealed a long leader region of 515 bp between the transcriptional start site and the 1645 coding region (Fig. S1).Interestingly, within this region, there was an abrupt decrease in reads mapping to this region, consistent with transcriptional termination.Based on this information and above data indicating a role for Rho in regulating ABUW_1645 expression, we hypothesized that the mRNA leader region of ABUW_1645 contained a Rho-dependent terminator and that the increased ABUW_1645 expression in AV-T cells, relative to VIR-O cells, could be due to differences in Rho-dependent termination within this 5′ leader region.This was first tested by using qRT-PCR to measure transcript levels using a primer set that amplified immediately downstream of the transcriptional start site (primer set A) or after the termination site predicted by the RNA-seq analysis, but before the 1645 coding region (primer set B) (Fig. 2A).When ABUW_1645 transcript levels were compared before and after the termination site between VIR-O and AV-T cells, primer set A revealed a 21-fold increase in AV-T cells (Fig. 2B).However, primer set B, which detects differences in termination within the ABUW_1645 leader revealed a 587-fold increase in AV-T cells, relative to VIR-O cells (Fig. 2B).Next, we tested the effects of the rho::Tc mutation in the VIR-O background on transcriptional readthrough.Compared to wild-type VIR-O cells, the rho::Tc mutation resulted in a 4.8-fold increase with primer set A, and a 19.5-fold increase after the site of termination with primer set B (Fig. 2B).Overall, this suggested that termination occurred within this region.
Although the above experiments demonstrated increased mRNA levels in the AV-T variant after the putative Rho-dependent terminator, the data also showed an apparent increase in promoter activity (primer set A in Fig. 2A).We hypothesized this was an indirect result of increased mRNA stability of this region in AV-T or rho::Tc cells, due to the longer length of the read-through transcripts.To address this possibility in an independent manner, transcriptional lacZ fusions were made to various derivatives containing the ABUW_1645 promoter and leader region.A DNA fragment comprised of the region immediately upstream of the +1 transcriptional start site and containing only the promoter without the downstream leader region exhibited a similar high level of β-galactosidase activity in VIR-O and AV-T cells (Fig. 2C-1).A DNA fragment containing only the terminator region had no promoter activity (Fig. 2C-2).However, the full-length fragment with the promoter and terminator region had minimal activity in VIR-O cells, but exhibited a 17-fold increase in β-galactosidase activity in AV-T cells, a level of expression similar to the promoter only in both VIR-O and AV-T cells (Fig. 2C-3).Based on these data, we conclude that ABUW_1645 expression differences between VIR-O and AV-T cells are largely due to differences in transcriptional termination in the untranslated leader region, and not from changes in promoter activity.

ABUW_1959 and ABUW_2818 also have long mRNA leader regions containing Rho-dependent terminators
We previously reported that in addition to ABUW_1645, the TTTRs ABUW_1959 and ABUW_2818 were also stochastically activated alone or in different combinations to drive VIR-O cells to the translucent state (43).To determine if the increased expression of ABUW_1645, ABUW_1959 and ABUW_2818 in AV-T cells was also due to reduced Rho-dependent termination, we first determined the predicted transcriptional start site for each gene by mapping reads from the RNA-seq analysis.This indicated that like ABUW_1645, both ABUW_1959 and ABUW_2818 had long leader regions of 383 and 457 bp, respectively (Fig. S1).In addition, the frequency of reads mapping to their leader regions fell dramatically midway through the leader, suggesting that a strong terminator was present.In fact, the degree of termination in all three leaders resulted in these termination products being recognized in a previous study as small RNAs (sRNAs) (47).To verify this observed termination, primer sets that monitor mRNA levels immediately after the transcriptional start site (Set A), and after the site of termination, but before the coding region (Set B) were used in qRT-PCR analysis.For both ABUW_1959 and ABUW_2818, the increased expression in AV-T cells, relative to VIR-O cells, was far more pronounced after the termination site, indicating reduced termination in AV-T cells drove the higher levels of TTTR expression (Fig. S2).

The Rho inhibitor bicyclomycin decreases termination in the ABUW_1645, ABUW_1959, and ABUW_2818 leader regions
BCM is an antibiotic that inhibits Rho activity (48).To further confirm that Rho had a role in regulating ABUW_1645, ABUW_1959, and ABUW_2818 expression, wild-type VIR-O cells, where these TTTRs are expressed at low levels were grown in the presence or absence of sub-inhibitory levels of BCM (25 µg/mL) and qRT-PCR analysis was carried out as described above to assess the levels of mRNA, either before, or after the Rho-depend ent termination site.In VIR-O cells treated with BCM, the levels of ABUW_1645 mRNA immediately after the start site of transcription were similar in untreated and BCM-trea ted cells; however, there was a 4-fold increase after the termination site in BCM-treated cells (Fig. 3A).For the ABUW_1959 and ABUW_2818 genes, BCM treatment decreased the degree of termination, resulting in increased expression of 9.2-and 6.6-fold, respectively, after the termination site for each gene (Fig. 3B and C).

Mutations within the mRNA leader region increase ABUW_1645 expression
To obtain additional evidence that termination within the leader region in VIR-O cells decreased ABUW_1645 expression, a transcriptional fusion between the ABUW_1645 promoter/leader region and the lacZ gene was constructed on plasmid pQF1645.Then, a genetic screen was used to identify cis-acting mutations that increased the expres sion of this fusion (see Materials and Methods).In the VIR-O variant, two mutants with increased β-galactosidase expression were isolated as colonies with increased blue color on X-gal plates, and in each case, single nucleotide changes were found in the leader region between the transcriptional start site and the predicted site of Rho-dependent termination (Fig. 4A).However, each mutation was outside the predicted rut site determined using the algorithm RhoTermPredict (25).These mutations resulted in 9.9-and 6.1-fold increases in β-galactosidase expression from the 1645-lacZ fusion, relative to the wild-type promoter/terminator (Fig. 4B).These mutations did not result in a further increase in expression in cells treated with the Rho inhibitor BCM.In contrast, BCM treatment of cells with the wild-type ABUW_1645 promoter/terminator resulted in a 7.2-fold increase (Fig. 4B).Interestingly, these mutations did not result in significant changes to the RNA secondary structure of the leader region as predicted by Mfold (49) (Fig. S3), and Rho binding to each leader region was similar (Fig. S4C).

Rho binds to the TTTR mRNA leader regions
To further demonstrate Rho-dependent regulation of the above TTTRs, the ability of purified Rho protein to interact with the mRNA leader regions was investigated by electrophoretic mobility shift assays (EMSAs).Previous work demonstrated that ABUW_1645 was the most frequently activated TTTR to mediate the VIR-O to AV-T switch, activated in ~94% of cells that switched from VIR-O and AV-T states (43).In contrast, ABUW_1959 was only activated in ~20% of AV-T cells.To determine if differences in Rho binding affinities influenced how these TTTR were activated during the VIR-O to AV-T switch, we focused on these two TTTR leader regions.Purified Rho bound both the ABUW_1645 and ABUW_1959 leader regions, forming a high-molecular weight complex that migrated a short distance into the gel (Fig. 5).The affinity of Rho for the ABUW_1645 and ABUW_1959 mRNA leader regions was similar, with K D values of 20 and 29 nM, respectively (Fig. 5).The binding of Rho to each leader was specific based on the were determined by qRT-PCR using oligonucleotides that amplify the region immediately after the transcriptional start site (Set A in Fig. 2A) and oligonucleotides that amplify the region after the termination site (Set B in Fig. 2A).Reported values represent the average of three biological replicates and standard error is shown.A * represents a P value < 0.02 and ** represents a P value < 0.004.following: (i) Rho did not bind a labeled control RNA composed of an antisense region of the ABUW_0939 gene (Fig. S4A), and (ii) the binding of Rho to labeled ABUW_1645 or ABUW_1959 leader regions was reduced by an excess of unlabeled competitor mRNA, but not by an excess of non-specific competitor mRNA (ABUW_0939 antisense) (Fig. S4B).

Rho levels are unchanged between VIR-O and AV-T cells
The changes in Rho-dependent termination between VIR-O and AV-T cells could be explained by differences in the levels of Rho between each variant.However, since Rho is essential for viability in A. baumannii (38,39), it was unlikely that significant differences in cellular Rho levels could account for the reduced termination in AV-T cells without having a substantial impact on cell growth.To examine the cellular levels of Rho, a His 6 -tagged version of Rho at its native chromosomal locus was constructed and Western blot analysis demonstrated the levels of Rho were unchanged between VIR-O and AV-T cells (Fig. S5).In addition, it has been shown that in Clostridium botulinum, Rho can exist in two forms, free or aggregated, the latter of which is mediated by a prion-like domain (37).The A. baumannii Rho protein is missing the prion-like domain and the use of semidenaturing detergent agarose gel electrophoresis did not reveal any evidence of Rho aggregation into prion-like forms (50).Therefore, differences in Rho-dependent termina tion between VIR-O and AV-T cells are likely due to other factors, whose expression or activity is differentially regulated to modulate Rho binding or activity.

Starvation increases the switching frequency of AV-T.LS cells to the VIR-O variant by modulating Rho-mediated termination
In AV-T.LS cells, where the ABUW_1645, 2818, and 1959 genes were in the ON state (43), we observed that the rate of switching to the VIR-O variant was higher when colonies were at high density (>200 colonies/plate) versus at low density (<10 colonies/plate).When this was quantitated, the AV-T to VIR-O switching frequency was 4.2 ± 1.9% in colonies at low density, but increased to 32.8 + 7.6% in colonies at high density, a 7.8-fold increase.Two mechanisms that could account for this density-dependent increase in switching were quorum sensing and/or nutrient depletion.As reported previously, the AbaI-dependent signal 3-OH C 12 -HSL did not influence switching (51).To determine if nutrient depletion was involved, the rate of switching in colonies at low density was compared on LB agar with normal (1×) concentrations of tryptone and yeast extract, and on agar with a reduced nutrient concentration, containing 0.125× concentrations of tryptone and yeast extract, but retaining normal levels of sodium chloride.These switching assays were done using 18-h-old colonies instead of the typical 24-h colonies to minimize the effects of nutrient depletion on 1X agar.The rate of AV-T to VIR-O switching in colonies was 9.6-fold higher at lower nutrient concentrations (10.6 + 1.4%) versus on normal nutrient levels (1.1 ± 0.5%).
We previously reported that the expression of the TTTRs ABUW_1645, ABUW_1959, and ABUW_2818 were required to keep cells in the AV-T state (43).To determine if the increase in AV-T to VIR-O switching during nutrient depletion was due to the downregulation of ABUW_1645, 2818, or 1959 expression, qRT-PCR was used to measure their expression in cells grown in rich (normal LB) and nutrient poor conditions (LB with 0.125× tryptone and yeast extract, but normal sodium chloride levels).Expression of ABUW_1645, 1959, and 2818 was downregulated 14.6-, 12.1-, and 32.6-fold, respec tively, under nutrient-poor conditions, relative to nutrient-rich conditions (Fig. 6A).To determine if these changes in expression were due to differences in Rho-mediated transcriptional termination, qRT-PCR analysis was used to measure mRNA levels before and after the site of termination for each TTTR leader region in AV-T.LS cells grown in 0.125× LB compared to cells grown in 1× LB.For all three TTTRs, the levels of expression were similar immediately after the start site of transcription, but there was a striking downregulation of expression after the termination site (Fig. 6B).Analysis of cellular Rho levels in cells grown in 1× LB and 0.125× LB showed no significant changes (Fig. S5B).

DISCUSSION
Previous work demonstrated a novel regulatory mechanism to control phenotypic switching in A. baumannii, where the stochastic activation of TTTRs alone or in different combinations was responsible for driving VIR-O cells to cells that formed translucent colonies, the majority of which were avirulent (AV-T) (43).In this study, we demonstrate that differences in Rho-dependent transcriptional termination in the leader regions of three of these TTTRs were largely responsible for the expression differences between VIR-O and AV-T variants.In Fig. 7, our model for the Rho-dependent control of switching is outlined.In VIR-O cells, Rho binds a rut site in one or more TTTR leader regions to terminate transcription.However, in a subpopulation of VIR-O cells, Rho-mediated termination is inhibited by a yet to be determined mechanism, and one or more TTTRs are expressed.This then results in the cell switching to the AV-T state.
Rho levels were similar in VIR-O and AV-T variants; therefore, this could not explain the differential Rho-dependent termination in the TTTR leader regions between VIR-O and AV-T cells (Fig. S5).However, we hypothesized that differential Rho binding might influence the frequency by which each TTTR was activated, i.e., Rho affinity would be lower for the most frequently activated TTTR ABUW_1645 and higher for the ABUW_1959 mRNA leader that is the least frequently activated.Our data did not support this hypothesis, as the affinity of Rho was similar for both ABUW_1645 and ABUW_1959 mRNA leader regions (Fig. 5).This suggested another factor may bind the TTTR leader regions to modulate Rho-dependent termination.Interestingly, mutations in the leader regions of ABUW_1645 that decreased Rho-dependent termination fell outside the predicted rut site determined using RhoTermPredict (25) (Fig. 4).In addition, Rho bound each mutant leader in-vitro with similar affinities as the wild-type leader (Fig. S4C).This could indicate either: (i) the mutant leader regions adopt different conformations in-vitro and in-vivo where Rho is unable to bind the latter condition, (ii) changes in transcriptional pausing, or (iii) the ability of Rho to bind the TTTR leader regions is regulated by one or more additional factors and the mutations alter the ability of these factors to bind the leader.One candidate for such a factor is a sRNA designated SrvS that increases the rate of VIR-O to AV-T switching, and influences which TTTR was activated during the VIR-O to AV-T switch (43,52).Examples of sRNAs altering the binding of Rho to modulate transcrip tional termination have been described (32,34,53).A second possible factor is CsrA, an RNA-binding protein that has been shown to modulate Rho-dependent termination (54), although in the cited example, Rho-dependent termination was stimulated by CsrA.Experiments are in progress to test the role of these factors in Rho-dependent termina tion in the TTTR leader regions.
In this study, a strong increase in AV-T to VIR-O switching was observed during nutrient depletion, which prompted us to analyze the effect of nutrient depletion on TTTR expression.In AV-T cells where TTTR expression is high, nutrient depletion resulted in a significant decrease in expression (Fig. 6A).Moreover, this TTTR downregulation resulted from increased levels of Rho-dependent termination (Fig. 6B).In E. coli, it has been shown that during reduced translation under nutrient starvation, Rho has the opportunity to bind to the available rut sites on the nascent mRNA, thus increasing transcriptional termination (45).A second possibility is that the structure of the TTTR mRNA exhibits a conformational change under starvation and Rho is better able to terminate transcription.During starvation, the concentration of ions such as Mg 2+ may be altered, resulting in different rates of transcription termination, either by altering Rho activity (55), or mRNA leader structure (31).A third possibility is that a specific protein senses starvation and then directly or indirectly alters Rho-dependent termination.
Overall, our study provides new insights in uncovering the molecular mechanism of VIR-O to AV-T phenotypic opacity switch in A. baumannii.Our results bring to the forefront the importance of Rho-dependent transcription termination in the activation of TTTRs that drive cells from a virulent state to an avirulent state.There are additional examples of Rho-mediated control of phenotypic heterogeneity in bacteria.In C. difficile, differential Rho binding to the leader region of the flgB flagellar gene influences motility.Interestingly, the flgB upstream region contains an invertible DNA segment within a leader region that precedes the flgB coding region and the orientation of this invertible segment in a cell subpopulation determines whether Rho can bind the mRNA to mediate termination (16).In C. botulinum, Rho can exist in two forms to mediate heterogeneity, free or aggregated, the latter of which is mediated by a prion-like domain (37).We found no evidence of Rho aggregation in A. baumannii.In summary, our findings provide the foundation for future work aimed at discovering other uncharacterized elements involved in Rho-dependent termination, as well as additional environmental conditions that affect this mechanism.Understanding this Rho-dependent phenotypic virulence switch represents a step in the development of new therapeutic options aimed at combating A. baumannii infections.

Strains and growth conditions
A. baumannii strain AB5075 was used in all the experiments conducted in this study except for the mariner transposon mutagenesis screen to look for ABUW_1645 regulators where strain A. baumannii ATCC 17978 containing a 1645-lacZ reporter fusion was used.Strains were grown in full-strength LB (1× LB) containing 10 g tryptone, 5 g yeast extract, and 5 g NaCl per liter and 1.5% agar or half-strength LB (0.5× LB) containing 5 g tryptone, 2.5 g yeast extract, and 2.5 g NaCl per liter and 0.8% agar.For the starvation experiments, 1× LB (media prepared as described above) or 0.125× LB containing 2.5 g tryptone, 1.25 g yeast extract, and 5 g NaCl per liter were used.When required, the media were supplemented with tetracycline (5 µg/mL), hygromycin (150 µg/mL), or apramycin (30 µg/mL).

Construction of transposon mutant library
A. baumannii ATCC 17978 containing a 1645-lacZ transcriptional fusion was mutagen ized with a derivative of the mariner transposon MAR2XT7 (56), where a tetracycline resistance gene was inserted into the gentamicin resistance gene, resulting in MarTc.Overnight cultures of SM10 pMarTc E. coli and A. baumannii ATCC 17978 containing a chromosomal ABUW_1645-lacZ transcriptional fusion were grown at 37°C to mid-log phase, 300 µL of each culture was spun down and the cells washed two times with fresh LB to remove the antibiotic present in the cultures.Cells were resuspended in 300 µL of fresh LB and 100 µL of each strain spotted on a dried LB plate.The conjugation was carried out for 6 h at 37°C and cells were recovered and plated on LB plates containing tetracycline 5 µg/mL and X-gal.Plates were incubated overnight at 37°C and mutants with a more intense blue color were rescreened to confirm the phenotype.The site of the mariner transposon insertion was determined by cloning the chromosomal region containing the insertion as a partial Sau3A fragment and using DNA sequencing to identify the insertion site.

Complementation of the rho::Tc mutant
The wild-type rho gene with its native ribosome binding site was amplified by PCR using the primers listed in Table S1.This PCR product was cloned into the SmaI site of pUC18mini-Tn7 LAC Apra (46) and transformed into competent E. coli Transformax EC100D cells (Epicentre Biotechnologies) by electroporation.A clone containing the plasmid with the insert in the correct orientation where expression is driven by the IPTG-induci ble tac promoter was identified.The pUC18-mini-Tn7 LAC Apra rho plasmid was then transformed along with the pTNS2 plasmid (containing the Tn7 transposase) into the VIR-O rho::Tc mutant.Transformation was plated on LB plates containing apramycin at 30 µg/mL and transformants were screened by PCR for insertion of the Tn7-rho into the glmS site.Isopropyl-β-d-thiogalactopyranoside (IPTG) was used at a concentration of 2 mM to induce rho expression for the complementation experiments.

Construction of transcriptional lacZ fusions
Oligonucleotides were designed to amplify by PCR various fragments containing the ABUW_1645 promoter only (1645 P1 and 1645 TSS.rev, Table S1), the mRNA leader region only (1645 TSS and 1645 P2), or the promoter/leader region (1645 P1 and 1645 P2) as shown in Fig. 2C.Fragments were cloned into SmaI digested pQF1266W using the Fast-Link Ligation Kit (Lucigen).pQF1266W is a plasmid vector and is a derivative of the pQF50 (57) plasmid containing a hygromycin resistance gene and the origin of replication of the pWH1266 vector (58).Ligation was transformed into E. coli Transformax EC100D competent cells (Lucigen) and plated on regular LB plates containing hygromy cin at 150 µg/mL and 20 µL/mL X-gal.Plasmids with inserts were confirmed by DNA sequencing (Genewiz) and transformed into the A. baumannii VIR-O and AV-T.LS variants for subsequent β-galactosidase assays as previously described.

Isolation of mutations in the ABUW_1645 leader region that decrease termination
Plasmid pQF1645 (43) containing the ABUW_1645 promoter and entire leader region fused to lacZ was mutagenized by passage through the E. coli mutator strain XL1-Red.Three independent XL1-red transformants were grown and plasmid DNA was isolated and transformed in the AB5075 VIR-O variant.Colonies with increased ABUW_1645 expression were identified by a blue colony phenotype on LB agar plates containing X-gal.One mutant from each pool was analyzed further and revealed a single mutation in the leader region downstream of the transcriptional start site.The promoter/leader region from each mutant was amplified by PCR and recloned into pQF1266W.DNA sequence analysis confirmed the single original mutation was present.Plasmids were then transformed into the VIR-O variant and β-galactosidase activity was determined as described below.

β-galactosidase assays
Strains with various plasmid-based ABUW_1645-lacZ fusions were grown with hygromy cin 150 µg/mL or bicyclomycin (25 µg/mL) as necessary to an OD 600 of 0.5 and cells were pelleted and resuspended in 0.9 mL of Z Buffer containing 27 µL of beta-mercap toethanol.Then, 10 µL of 0.1% SDS and 20 µL of chloroform were added to the tubes and vortexed vigorously for 20 s.About 200 µL of O-nitrophenyl β-D-galactopyranoside (ONPG) was added to each tube and reactions were stopped by adding 500 µL of 1M Na 2 CO 3 .Samples were centrifuged for 8 min at maximum speed and optical density of the supernatant was measured at 420 nm.β-galactosidase activity was determined by the method of Miller (59).

Total RNA extraction and quantitative reverse transcription PCR
Cultures of the desired strains were grown from glycerol stocks until OD 600 of 0.5 and cells were pelleted and flash-frozen in an ethanol-dry ice bath.All cultures were checked

FIG 1
FIG 1 Effects of the rho::Tc insertion in the VIR-O background and complementation analysis.Panel (A): qRT-PCR analysis of ABUW_1645 and rho expression in the VIR-O rho::Tc mutant strain is shown compared to the VIR-O wild-type parental strain using the 16S gene as an internal control.Values represent the average of three biological replicates with standard error shown.A * designates a P value < 0.05.Panel (B): Qualitative assay to measure 3-OH C 12 -HSL secretion in the VIR-O rho::Tc mutant and the VIR-O rho::Tc/Tn7-rho complemented mutant.Cultures of the VIR-O wild-type strain, the VIR-O rho::Tc and the VIR-O rho::Tc/Tn7-rho complemented mutant were grown in broth to OD 600 of 0.2 and then spotted onto a soft agar lawn containing X-Gal and an Agrobacterium tumefaciens traG::lacZ biosensor strain.Plates were incubated for 24 h at 28°C.Panel (C): A line of 1M IPTG was applied to an LB agar plate and allowed to be absorbed before the indicated cells perpendicular to the IPTG.Plates were incubated for 24 h at 37°C.Panel (D): Expression levels of ABUW_1645 and rho in the VIR-O rho::Tc/Tn7-rho mutant strain without IPTG and in the presence of 2 mM IPTG compared to the VIR-O rho::Tc mutant.Values represent the average of three biological replicates with standard error shown.A *** designates a P value < 0.001.

FIG 2
FIG 2 Expression analysis of ABUW_1645 upstream region.Panel (A): The ABUW_1645 promoter, putative Rho-dependent terminator (red) in the mRNA leader region, and ABUW_1645 coding region (green) are depicted.Panel (B): Expression levels of the ABUW_1645 leader region in the AV-T.LS variant and VIR-O rho::Tc mutant compared to the VIR-O variant.Primer set A was used to amplify the region immediately after the transcriptional start site and primer set B amplified the region after the termination site.Values represent the fold-activation relative to the VIR-O variant and are the average of three biological replicates using 16S as control.Error bars indicate standard error.Panel (C): β-galactosidase activity of lacZ fusions to the ABUW_1645 promoter and downstream regions in the VIR-O and AV-T.LS variants (1) corresponds to a lacZ fusion to a fragment containing ABUW_1645 promoter only with no DNA downstream of the +1 site (2), corresponds to a lacZ fusion of a fragment of ABUW_1645 containing the mRNA leader only region, and (3) corresponds to the lacZ fusion of a fragment of ABUW_1645 containing the promoter and mRNA leader region.Values represent the average of duplicate samples from two biological replicates.

FIG 4
FIG 4 Mutations in the ABUW_1645 leader region increase expression.In panel (A), single nucleotide substitutions in the ABUW_1645 leader region that decreased Rho-dependent termination are shown and a predicted rut site is underlined.In panel (B), the expression of β-galactosidase was measured from wild-type or mutant ABUW_1645 promoter/leader regions fused to lacZ.Red bars denote cells treated with bicyclomycin (25 µg/mL).Values represent the mean of duplicate samples from two to four biological replicates with standard deviations.

FIG 5
FIG 5 Interaction of Rho with the ABUW_1645 and ABUW_1959 mRNA leader regions.Biotinylated RNAs immediately after the transcriptional start site to the predicted site of termination were incubated with the indicated amounts of Rho for 60 min, separated on a 5% acrylamide gel and developed as described in the Materials and Methods.Red arrows represent bound mRNAs relative to unbound RNA designated by a black arrow.Bands denoted with an asterisk were not always present between experiments and their nature is unknown.The apparent dissociation constant (K D ) values are shown and were determined from the averages of three separate EMSA experiments for each mRNA leader.

FIG 6
FIG 6 Rho-dependent termination in the TTTR leaders is influenced by nutrient depletion.Panel (A): AV-T.LS cultures were grown in either regular LB or 0.125× LB media and the expression profiles of the ABUW_1645, ABUW_1959, and ABUW_2818 genes were determined by qRT-PCR analysis.The reported values represent the relative expression in 0.125× LB relative to regular LB.Values were calculated using the 2 −ΔΔCT method and 16S as an internal control.Values represent the average of three biological replicates with standard error shown.** represents a P value < 0.001 and *** represents a P value < 0.00002.Panel (B): Expression levels of the ABUW_1645, ABUW_1959, and ABUW_2818 upstream regions in AV-T.LS cultures grown in 0.125× LB were determined as described in panel (A).Oligonucleotide set A amplifies the region immediately after the transcriptional start site and oligonucleotide set B amplifies the region after the termination site.Reported values represent the average of three biological replicates with standard error shown.A * represents a P value < 0.008, ** represents a P value < 0.001, and *** represents a P value < 0.00004.

FIG 7
FIG 7 Proposed model for the role of Rho in the VIR-O to AV-T switch.The regulatory region of a representative TTTR is shown in a VIR-O cell with Rho bound to the mRNA leader and terminating TTTR transcription to keep cells in the VIR-O state.In subpopulation of VIR-O cells, a rare cell switches to the AV-T state by loss of Rho-dependent termination, which leads to TTTR expression and a switch to the AV-T state.