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\begin{document}

\title{c-di-GMP regulates RNA chaperone activity of PlzA in the Lyme disease
spirochete}



\author[1]{Meghan C. Lybecker}%
\author[1]{Taylor Van Gundy}%
\author[2]{Dhara Patel}%
\author[3]{Bruce E. Bowler}%
\author[3]{Michael T. Rothfuss}%
\author[1]{Allie J. Hall}%
\author[4]{Christopher Davies}%
\author[5]{Laura Hall}%
\author[5]{Dan Drecktrah}%
\author[2]{Richard T. Marconi}%
\author[3]{D Scott Samuels}%
\affil[1]{National Center for Emerging and Zoonotic Infectious Diseases Division of Vector Borne Diseases}%
\affil[2]{Virginia Commonwealth University Department of Microbiology and Immunology}%
\affil[3]{University of Montana}%
\affil[4]{University of Southern Alabama}%
\affil[5]{University of Montana Division of Biological Sciences}%


\vspace{-1em}



  
  \date{June 18, 2024}


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\selectlanguage{english}
\begin{abstract}
PlzA is a c-di-GMP-binding protein crucial for adaptation of the Lyme
disease spirochete \emph{Borrelia} ( \emph{Borreliella})
\emph{burgdorferi} during its enzootic life cycle. Unliganded
\emph{apo}-PlzA is important for vertebrate infection, while liganded
\emph{holo}-PlzA is important for tick acquisition; however, the
biological function of PlzA has remained enigmatic. Here we report that
PlzA has RNA chaperone activity that is inhibited by c-di-GMP binding.
\emph{Holo}- and \emph{apo}-PlzA bind RNA and accelerate RNA annealing,
while only \emph{apo}-PlzA can strand displace and unwind
double-stranded RNA. Guided by the crystal structure of PlzA, we
identified several key aromatic amino acids protruding from the
\emph{N}- and \emph{C}-terminal domains that are required for RNA
binding and unwinding activity. Our findings illuminate c-di-GMP as a
switch controlling the RNA chaperone activity of PlzA and we propose
that complex RNA-mediated modulatory mechanisms allow PlzA to regulate
gene expression during both the vector and host phases of the \emph{B.
burgdorferi} life cycle.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

The Lyme disease agent \emph{Borrelia} (\emph{Borreliella}
)\emph{burgdorferi} is maintained in an enzootic cycle traversing
between\emph{Ixodes} ticks and vertebrates (1,2). The spirochete
regulates gene expression in a phase-specific fashion as it cycles
between its tick vector and its vertebrate hosts (3,4). \emph{Ixodes}
larvae feed on an infected host and acquire the spirochetes, which
persist in the midgut. Following the molt, nymphs feed on a vertebrate
and the spirochetes migrate from the midgut through the hemocoel to the
salivary glands and transmit to the na\selectlanguage{ngerman}ïve host (5,6). Gene expression is
globally regulated by the alternative sigma factors RpoS
(\selectlanguage{greek}σ\selectlanguage{english}\textsuperscript{S}) and RpoN (\selectlanguage{greek}σ\selectlanguage{english}\textsuperscript{N}) (7-9), the
stringent response via guanosine tetraphosphate and pentaphosphate
{[}(p)ppGpp{]} (10,11), and cyclic dimeric GMP (c-di-GMP) (12-15). While
transcriptomics studies of these signaling systems have revealed gene
products important for infectivity and persistence, our understanding of
post-transcriptional regulation remains sparse.

Many mechanisms of post-transcriptional gene regulation rely on RNA
chaperones, a heterogeneous group of proteins that modulate RNA
structures by disrupting RNA secondary and tertiary structure
(unwinding, unfolding, and strand-displacing) or accelerating base
pairing of RNAs (annealing) (16-18). Bacterial RNA chaperones include
Hfq, StpA, FinO/ProQ, CsrA, cold shock proteins (CSPs), and the
ribosomal proteins S1 and S12; mutants have pleiotropic phenotypes
including reduced virulence, impaired growth, and reduced ability to
respond to and survive environmental stresses (19,20). RNA chaperones
can display sequence-independent, transient annealing and strand
displacement activity (17,18,21). Hfq and, likely, ProQ are considered
``matchmaker'' chaperones that facilitate base pairing of\emph{trans}
-acting small regulatory RNAs and their RNA targets (19,22-24). \emph{B.
burgdorferi} harbors several RNA-binding proteins that regulate
post-transcriptional gene expression, but their RNA chaperone activity
and mechanisms of regulation have not been extensively studied (4). In
2010, we identified BB0268 as a unique RNA chaperone (25). BB0268
partially complements an \emph{Escherichia colihfq} mutant and the
\emph{hfq} gene of \emph{E. coli} partially complements several defects
of the pleiotropic \emph{bb0268} mutant of\emph{B. burgdorferi} ;
however, BB0268 has negligible homology with Hfq orthologs from
well-studied model bacteria (25).

Small RNAs (sRNAs) regulate gene expression, which depends on their
three-dimensional structure as well as RNA chaperones (26,27). Many
pathogenic bacteria utilize sRNAs to regulate virulence (20,28-30). Only
three sRNAs to date have been functionally characterized in \emph{B.
burgdorferi} : DsrA\textsubscript{Bb}, a \emph{trans} -acting sRNA,
regulates translation of an \emph{rpoS} mRNA species (31); Bb6S RNA, a
protein-interacting sRNA, globally regulates gene expression by
affecting RNA polymerase \selectlanguage{greek}σ \selectlanguage{english}factor selectivity (32); and \emph{ittA} ,
another sRNA, affects expression of several genes and is required for
dissemination in the vertebrate host by an unknown molecular mechanism
(33). However, the catalog of non-coding sRNAs in \emph{B.
burgdorferi}increased dramatically following high-throughput sequencing
studies (4,34-38).

c-di-GMP is a global secondary messenger responsible for numerous
physiological adaptations of bacteria, including biofilm formation,
virulence, motility, phage resistance, and cell morphology (39-43).
Diguanylate cyclases synthesize c-di-GMP from two GTP molecules and
phosphodiesterases degrade c-di-GMP into linear
5'-phosphoguanylyl-(3'-5')-guanosine (pGpG) and/or GMP. In \emph{B.
burgdorferi} , c-di-GMP is produced by a sole diguanylate cyclase, Rrp1,
and degraded by two phosphodiesterases, PdeA and PdeB (44-47). Rrp1
forms a prototypical two-component signal transduction system with
histidine kinase 1 (Hk1), although the activating ligand remains unknown
(12,48). \emph{rrp1} and \emph{hk1} mutants have wild-type transmission
to and colonization of vertebrates via needle inoculation; however, both
mutants are quickly destroyed in the tick midgut during larvae and nymph
blood meal feeding (12,13,49,50). In other bacteria, c-di-GMP regulates
gene expression at the transcriptional, post-transcriptional, and
post-translational levels by binding to different effectors
(riboswitches, transcriptional regulators, enzymes, and proteins) (51).
In most \emph{B. burgdorferi} , PlzA is the only c-di-GMP effector
identified to date (52-55). Mouse infectivity is markedly impaired in
a\emph{plzA} mutant (52,56,57), unlike the \emph{hk1} and
\emph{rrp1}mutants. Groshong et al. (14) recently demonstrated that a
\emph{plzA}point mutant unable to bind c-di-GMP (R145D) is fully
infectious in mice confirming that the function of PlzA in mammals is
c-di-GMP-independent. The c-di-GMP-dependent role of PlzA in the tick is
controversial. Kostick-Dunn et al. (56) found that PlzA is not required
to establish infection in ticks, while Pitzer et al. (55) report that
\emph{plzA}mutants do not survive during acquisition.

The binding of c-di-GMP induces a large conformational change in PlzA
(14,53). The crystal structure of \emph{holo} -PlzA revealed a
two-domain structure separated by a short linker (58). Both the \emph{N}
- and\emph{C} -terminal domains contain seven-stranded \selectlanguage{greek}β-\selectlanguage{english}barrels that
are nearly superimposable. Differences between the two domains are found
in the loops connecting the \selectlanguage{greek}β-\selectlanguage{english}strands. The \emph{N} -terminal domain has
\selectlanguage{greek}α \selectlanguage{english}helices joining the \selectlanguage{greek}β-\selectlanguage{english}strands, while the \emph{C} -terminal domain has
disordered linkers. Notably, \emph{apo} -PlzA did not yield crystals,
suggesting more structural flexibility than the liganded form.

In this study, we demonstrate that PlzA has RNA chaperone activity that
includes RNA annealing, strand displacement and unfolding. Our data show
that c-di-GMP inhibits PlzA strand displacement and RNA unfolding
activities \emph{in vitro} and in a heterologous system \emph{in vivo} .
We hypothesize that unliganded \emph{apo} -PlzA facilitates
conformational changes in RNA that alter their expression, remodeling
the RNA structure to stimulate or inhibit transcription or translation,
thus enabling spirochete transmission and survival in the vertebrate. In
contrast, c-di-GMP does not affect the RNA annealing activity of
PlzA\emph{in vitro} , as \emph{apo} - and \emph{holo} -PlzA have similar
RNA annealing properties. We hypothesize that \emph{holo} -\emph{}
and\emph{apo} -PlzA are ``matchmaker'' RNA chaperones that facilitate
sRNA and target RNA annealing resulting in the regulation of gene
expression throughout the \emph{B. burgdorferi} enzootic cycle. To our
knowledge, this is the first protein with RNA chaperone activity that is
modulated by a second messenger.

\textbf{MATERIALS AND METHODS}

\textbf{Bacterial strains and growth conditions}

\emph{B. burgdorferi} low-passage virulent B31-5A4 was cultivated in
Barbour-Stoenner-Kelly II (BSK-II) complete medium at 34\selectlanguage{ngerman}°C (59).
Cultures were passaged to 1 × 10\textsuperscript{5} cells/ml and grown
to 1-3 × 10\textsuperscript{7} cells/ml. Cell density was determined
using a Cellometer counting chamber (Nexcelom). \emph{E. coli} strains,
HI-control 10G or BL21(DE3) cells, were grown overnight in lysogeny
broth (LB) at 37°C to a density of \textasciitilde{}1
OD\textsubscript{600}. Cells were then passaged 1:100 unless stated
otherwise. Descriptions and genotypes of all strains used (\emph{E.
coli} and \emph{B. burgdorferi} ) can be found in Table S1.

\textbf{Construction and expression of recombinant proteins}

StpA and GrpE recombinant proteins were purified in \emph{E. coli} using
the pETite C-His vector utilizing the Expresso T7 system protocol
(Lucigen) as described in Brandt et al. (60). Genes were amplified by
PCR from \emph{B. burgdorferi} strain B31-5A4 genomic DNA using primers
listed in Table S2. pETite C-His plasmids were transformed into \emph{E.
coli} Top 10G Solo cells (Lucigen) and plated on LB plates supplemented
with 50 \selectlanguage{greek}μ\selectlanguage{english}g/ml kanamycin. Positive transformants were screened by PCR and
confirmed by sequencing. Correct plasmids were transformed into \emph{E.
coli} BL21(DE3) cells (Lucigen). Positive transformants were screened
and grown in LB-kanamycin (50 \selectlanguage{greek}μ\selectlanguage{english}g/ml) overnight. The overnight culture
was used as an inoculum at a 1:100 dilution in LB-kanamycin (50 \selectlanguage{greek}μ\selectlanguage{english}g/ml)
in 100 ml for protein expression. Protein expression was induced by the
addition of isopropyl-d-thiogalactopyranoside (IPTG; 1 mM) when cells
were at mid-logarithmic phase (OD\textsubscript{600} = 0.4-0.6). Cells
were harvested at late-log-phase growth (3-4 h), and recombinant protein
was purified under nondenaturing conditions using
a~nickel-nitrilotriacetic acid~(Ni-NTA) Fast Start~His tag~affinity
purification kit (Qiagen) per the manufacturer's protocol. Proteins were
dialyzed into PBS (pH 7.4) and quantified by bicinchoninic acid (BCA)
assay (Thermo-Fisher Scientific). Protein purification was examined by
immunoblot and Coomassie brilliant blue staining.

\emph{plzA} R145D and R145K mutant alleles were generated using splice
overlap exchange techniques (Table S2) (53,61). The amplicons were
digested with AgeI and XhoI, ligated into pET45b(+) using Instant
Sticky-end Ligase Master Mix (New England Biolabs), and purified using a
QIAquick PCR Purification Kit (Qiagen). \emph{plzA} R145D F92A
F95\emph{A} mutant and \emph{plzA} R145D F168A F218A mutant gene
sequences were synthesized with flanking BamHI and EagI restriction
sites and provided in pET45b(+) for expression with an \emph{N}
-terminal Hexa-His-tag (GenScript). To propagate the plasmids, \emph{E.
coli}NovaBlue(DE3) cells (Novagen) were transformed according to the
manufacturer's protocol with the following modification: 100 ng of the
plasmid was added to 20 \selectlanguage{greek}µ\selectlanguage{english}l of competent cells. Transformants were
selected with 100 \selectlanguage{greek}µ\selectlanguage{english}g/ml ampicillin. Positive transformants were screened
by PCR and confirmed by DNA sequencing. Isolated colonies were selected
and transferred into 10 ml of prewarmed LB-ampicillin (100 \selectlanguage{greek}μ\selectlanguage{english}g/ml) and
grown overnight (37\selectlanguage{ngerman}°C; 250 rpm). The overnight culture was used as an
inoculum at a 1:200 dilution in LB-ampicillin (100 \selectlanguage{greek}μ\selectlanguage{english}g/ml) in 500 ml for
protein expression. Protein expression was induced by the addition of 1
mM IPTG when cells were at mid-logarithmic phase (OD\textsubscript{600}=
0.4). Cells were harvested at late-log-phase growth (4 h) (5000
\selectlanguage{ngerman}×\emph{g} ; 15 min; 4°C). The cell pellet was resuspended and vortexed
in binding/washing (B/W) buffer (20 mM
NaH\textsubscript{2}PO\textsubscript{4}, 500 mM NaCl, 20 mM imidazole,
pH 7.4) containing protease inhibitor cocktail (1\% v/v; Sigma) and
Pierce Universal Nuclease for Cell Lysis (ThermoFisher; final
concentration 0.5 \selectlanguage{greek}μ\selectlanguage{english}g/ml). The samples were passed through an
EmulsiFlex-C3 high-pressure homogenizer (three times at 1000-1500 psi;
4\selectlanguage{ngerman}°C), and the resulting slurry was centrifuged (15,500 × \emph{g} ; 30
min; 4°C) to separate the soluble and insoluble fractions. To purify
proteins from the soluble fraction, nickel affinity chromatography was
performed using a Fast Protein Liquid Chromatography platform (ÄKTA Pure
25 M; Cytiva) with a 1-ml HisTrap FF column (Cytiva) at a flow rate of 1
ml/min (4°C). The column was equilibrated with five column volumes of
B/W buffer. Samples were loaded into a 10 ml Superloop (Cytiva),
followed by washing with 20 column volumes of B/W buffer. Proteins were
eluted with five column volumes of elution buffer (20 mM
NaH\textsubscript{2}PO\textsubscript{4}, 500 mM NaCl, 250 mM imidazole).
One-ml fractions were collected from under the 280 nm peak, combined,
and dialyzed into phosphate-buffered saline (PBS, pH 7.4) overnight at
4°C using Spectra/Por 1 (6--8 kDa cutoff) dialysis membranes (Spectrum
Laboratories). The concentrations of the recombinant proteins were
determined using a BCA assay (Pierce) as instructed by the supplier.

\textbf{Circular Dichroism Spectroscopy}

An Applied Photophysics Chirascan Circular Dichroism (CD)
spectrophotometer was used to acquire CD spectra of wild-type and mutant
forms of PlzA. All measurements were made in a temperature-regulated
sample holder at 25°C in 10 mM sodium phosphate buffer, pH 7.0, using a
1-mm pathlength quartz cuvette. Prior to measuring CD spectra, protein
samples were diluted to approximately 20-25 \selectlanguage{greek}µ\selectlanguage{english}M and their UV-Vis spectra
measured using a Beckman DU 800 UV-Vis spectrometer. An extinction
coefficient of 14,900 M\textsuperscript{-1} cm\textsuperscript{-1} at
280 nm, determined by the method of Pace and coworkers (62) using the
Expasy ProteinParam tool (63) was used to evaluate concentration from
the UV spectra. Samples were diluted fivefold further to concentrations
ranging from 4.2-5.5 \selectlanguage{greek}µ\selectlanguage{english}M so that high-quality spectra could be obtained
down to 180 nm without saturating the CD detector. Spectra were
collected from 260 nm to 180 nm using a 1-nm bandwidth, a 1-nm step size
and 5 s sampling time per point. Ellipticity was converted to Molar
Residue Ellipticity (MRE) using equation 1, where \selectlanguage{greek}θ \selectlanguage{english}is the ellipticity
in degrees, \emph{l} is the pathlength in cm, \emph{c} is concentration
in mol/L and \emph{n} is the

\(MRE=\left(\frac{100\times\theta}{l\times c\times n}\right)\)(Eq. 1)

number of residues in the protein (261 for PlzA). The STRIDE webserver
(64) was used to evaluate secondary structure in the PDB file for PlzA
(7MIE). The DichroWeb server (65) was used to analyze the CD data using
a truncated (190-240 nm) version of the SP175 reference set (66) and the
CDSTTR method (67). The normalized root mean squared deviation NRMSD
between the experimental and the spectra calculated by CDSTTR with the
SP175 reference set ranged from 0.019-0.024.

\textbf{Gel RNA annealing assay}

The gel annealing assay protocol was adapted from Doestch et al. (68),
except manganese was used instead of magnesium in the FRET buffer
(69,70). The assay utilizes two short unstructured complementary 21-mer
RNAs, termed J1 and M1, that are labeled with Cy5 and Cy3, respectively,
and are found in Table S3. All RNAs were synthesized by Dharmacon. RNA
annealing reactions were performed in 1\selectlanguage{ngerman}× MnCl\textsubscript{2} FRET
buffer (50 mM Tris-HCl, pH 7.5, 1 mM MnCl\textsubscript{2}, 10 mM NaCl,
1 mM DTT) at 37°C. Two controls were included with each annealing assay.
Control 1 (C1) is single stranded M1 RNA (10 nM M1, 1×
MnCl\textsubscript{2} FRET Buffer) and control 2 (C2) is preformed J1M1
dsRNA (10 nM M1, 10 nM J1, 1×MnCl\textsubscript{2} FRET Buffer). C2
dsRNA was formed by heating the two RNAs (J1 and M1) at 95°C for 2 min
and then slowly cooling to room temperature. A master mix for the
annealing reaction was assembled (10 nM J1 RNA in 1×
MnCl\textsubscript{2} FRET buffer) on ice and moved to 37°C to
equilibrate. A time point zero aliquot (10 \selectlanguage{greek}µ\selectlanguage{english}l) was taken from the master
mix, added to M1 RNA and 2.5 \selectlanguage{greek}µ\selectlanguage{english}l of 5\selectlanguage{ngerman}× stop/load buffer (150 mM EDTA, 2\%
SDS, 15\% (w/v) sucrose, 12.5\% (w/v) Ficoll 400, 0.2 mg/ml yeast tRNA
and 3 \selectlanguage{greek}µ\selectlanguage{english}M of unlabeled M1 (uM1) and immediately loaded into a running
(100V) 20\% native polyacrylamide gel (20\% TBE, 12 well, Novex precast
gel). M1 RNA was added to the RNA annealing master mix to start the
annealing reaction. Time points were withdrawn from the annealing
reaction, stopped with 5\selectlanguage{ngerman}× stop/load buffer and loaded on the running
gel. To track the progression of the running gel, a lane was loaded with
5× stop/load buffer with the addition of Gel loading buffer II
(Invitrogen). Proteins were added to reactions for a final concentration
of 1.2 \selectlanguage{greek}µ\selectlanguage{english}M, which is similar to protein concentrations used by others to
test for non-specific RNA chaperone activity (18,21,68,71). Gels were
run at 100 V at 4\selectlanguage{ngerman}°C for 3 h. RNA was visualized using Cy3 and Cy5
channels on an Azure Sapphire using the SmartScan feature and overlays
for each channel were used for gel images. Quantification of band
intensity was measured using the rectangle and gel plot function in
ImageJ. The rate of dsRNA production was quantified by plotting the
amount of dsRNA (J1M1) divided by the total RNA (J1M1, J1uM1) over time,
which was normalized to the zero time point and graphed using GraphPad
Prism with linear regression analysis. The amount of RNA was quantified
using the Cy5 channel. Differences between the assay endpoints (at 15
min) for each annealing reaction were determined using Welch's \emph{t}
-test (GraphPad Prism).

\textbf{dsRNA formation and purification}

dsRNA was formed and purified using the J1M1 RNAs described above. 40 \selectlanguage{greek}µ\selectlanguage{english}M
M1J1 dsRNA was formed in 1\selectlanguage{ngerman}× FRET buffer by heating at 95°C for 5 min and
then slow cooling to room temperature. The reaction was stopped in 5×
stop/load buffer and separated on a pre-poured native gel (Novex 20\%
TBE gel at 100 V for 1-3 h). The dsRNA was purified from the gel via
electrophoresis. Gel slices containing the dsRNA were placed in
preparative well of a 1\% agarose gel. Four to six 1-inch pieces of
Whatman paper were placed in the prep well (between the gel slices and
the gel) the dsRNA was electrophoresed (100 V for 3 min) from the gel
slices onto the Whatman paper. The Whatman paper was imaged on a BioRad
ChemiDoc Gel Imager to verify dsRNA migration. Each Whatman paper was
placed in a 1.7-ml light sensitive Eppendorf tube with 500 \selectlanguage{greek}µ\selectlanguage{english}l of 10\selectlanguage{ngerman}×
MgCl\textsubscript{2} FRET buffer (500 mM Tris-HCl, pH 7.5, 10 mM
MgCl\textsubscript{2}, 100 mM NaCl, 10 mM DTT). Tubes were placed in a
microshaker (1400 rpm, 4°C) overnight to extract dsRNA. The 10×
MgCl\textsubscript{2} FRET buffer (\textasciitilde{}500 \selectlanguage{greek}µ\selectlanguage{english}l) was removed
from the Eppendorf tubes and added to 3 volumes of ethanol (EtOH), 0.3 M
sodium acetate, and 15 \selectlanguage{greek}µ\selectlanguage{english}g of GlycoBlue? (Invitrogen). RNA was incubated
at -20\selectlanguage{ngerman}°C overnight. RNA was precipitated at 19000 × \emph{g} for 45 min
at 4°C. Pellets were washed with 200 \selectlanguage{greek}µ\selectlanguage{english}l of 70\% EtOH and spun again at
19000 \selectlanguage{ngerman}× \emph{g} for 20 min at 4°C. The supernatant was decanted and RNA
was resuspended in water. The purified dsRNA was quantified by nanodrop
and used for the strand displacement gel assay.

\textbf{Gel strand displacement assay}

Gel strand displacement assays were adapted from Doestch et al. (68),
with Mn in the buffer instead of Mg (69,70). Strand displacement assays
utilize M1, J1 and uM1 RNAs, as described above. RNA strand displacement
reactions were performed in 1× MnCl\textsubscript{2} FRET buffer (50 mM
Tris-HCl, pH 7.5, 1 mM MnCl\textsubscript{2}, 10 mM NaCl, 1 mM DTT) at
37°C. Two controls were included with each strand displacement assay.
Control 1 (C1) is purified J1M1 dsRNA (30 nM purified M1J1, 1×
MnCl\textsubscript{2} FRET Buffer) and control 2 (C2) is J1uM1 dsRNA (30
nM purified M1J1, 0.3 \selectlanguage{greek}µ\selectlanguage{english}M uM1, 1\selectlanguage{ngerman}× FRET MnCl\textsubscript{2} Buffer). C2
dsRNA J1uM1 was formed by heating purified dsRNA J1M1 and uM1 at 95°C
for 2 min and slowly cooling to room temperature. Strand displacement
master mix was prepared with 30 nM purified dsRNA in 1×
MnCl\textsubscript{2} FRET buffer. A zero time point was removed from
the master mix, added to 0.3 \selectlanguage{greek}µ\selectlanguage{english}M uM1 and 2.5 \selectlanguage{greek}µ\selectlanguage{english}l of 5\selectlanguage{ngerman}× stop/load buffer,
and immediately loaded into a running (100 V) 20\% native polyacrylamide
gel (20\% TBE, 12 well, Novex precast gel). Strand displacement
reactions were started by adding uM1 RNA to the strand displacement
master mix at 37°C. Time points were withdrawn from the reaction,
stopped with 2.5 \selectlanguage{greek}µ\selectlanguage{english}l of 5\selectlanguage{ngerman}× stop/load buffer. Gels were run at 100 V at
4°C for 3 h. Gels were visualized using Cy3 and Cy5 channels on an Azure
Sapphire using the SmartScan feature and overlayed for gel images.
Quantification of band intensity was measured using the Cy5 channel with
the rectangle and gel plot function in ImageJ. The amount of strand
displacement in each lane was quantified by plotting the amount of J1M1
(Cy5) dsRNA divided by the total RNA (J1M1, J1uM1 and M1; Cy5), which
was normalized to the amount of dsRNA (J1M1; Cy5) at time point zero and
graphed using GraphPad Prism with second order polynomial regression
analysis. Differences between the assay endpoints (at 15 min) for each
strand displacement reaction were determined using Welch's \emph{t}
-test (GraphPad Prism).

\textbf{RL211 antitermination assay}

\emph{E. coli} RL211 harbors the \emph{cat} gene preceded by a
Rho-independent \emph{trpL} terminator and is sensitive to
chloramphenicol (Cm). To test antitermination activity as described in
Ammerman et al. (72), Bae et al. (73), and Landick et al. (74), the
genes encoding StpA in the pET7C-HIS plasmid and PlzA, PlzA R145K and
PlzA R145D in pET-45b(+) plasmid were electroporated into \emph{E.
coli}RL211 strain provided by Dr. Robert Landick (74). Transformed RL211
strains were grown in either with 100 \selectlanguage{greek}µ\selectlanguage{english}g/ml ampicillin or 50 \selectlanguage{greek}µ\selectlanguage{english}g/ml
kanamycin overnight (OD\textsubscript{600} 0.9-1.0). Cultures were
spotted (5 \selectlanguage{greek}µ\selectlanguage{english}l) on LB plates with and without 34 \selectlanguage{greek}µ\selectlanguage{english}g/ml Cm. Cells were
grown for 1-3 d at 37\selectlanguage{ngerman}°C. Plates were imaged in an Azure Sapphire imager.

\textbf{Filter binding assay}

10 nM single-stranded Cy3 labeled M1, \emph{trpL} RNA or purified 10 nM
double-stranded J1uM1 RNA were titrated with PlzA proteins (50 nM, 100
nM, 200 nM, 300 nM, 500 nM, 800 nM, 1000 nM, and 2000 nM) in 1× binding
buffer (50 mM Tris-HCl pH 7, 2 mM DTT, 6\% glycerol) and incubated at
room temperature for 5 min. Nitrocellulose (Whatman PROTRAN BA 83 0.2
\selectlanguage{greek}µ\selectlanguage{english}m) and nylon membranes (GE Healthcare positively charged nylon transfer
membrane 0.45 \selectlanguage{greek}µ\selectlanguage{english}m) were pre-equilibrated in 1\selectlanguage{ngerman}× binding buffer and loaded
into a slot blot apparatus (Hoefer Scientific Instruments PR 600 slot
blot). Wells were washed with 1× binding buffer and pulled through the
membranes with a vacuum. 90 \selectlanguage{greek}µ\selectlanguage{english}l of a 100-\selectlanguage{greek}µ\selectlanguage{english}l reaction was applied to the
wells and vacuumed. PlzA-RNA complexes bound to the nitrocellulose
membrane and the free RNA flowed through and bound to the nylon
membrane. Wells were washed four times with 90 \selectlanguage{greek}µ\selectlanguage{english}l 1\selectlanguage{ngerman}× binding buffer. RNA
was detected using the Cy3 channel on both membranes with a Azure
Sapphire imager. RNA bound to each membrane was quantified using ImageJ
and the fraction of PlzA bound RNA was calculated as a function of PlzA
concentration. At least three replicates were performed for each PlzA
protein. Mean curves and standard error of the means were calculated and
the data were fit to the Hill equation for multiple binding sites, which
yielded dissociation constants.

\textbf{Immunoblotting}

Recombinant purified protein (10 \selectlanguage{greek}µ\selectlanguage{english}l) or eluates (10 \selectlanguage{greek}µ\selectlanguage{english}l) and 2\selectlanguage{ngerman}× Laemmli
buffer (Sigma) (10 \selectlanguage{greek}µ\selectlanguage{english}l) were run on a precast Criterion Any kD TGX
Stain-Free gel (BioRad) at 150 V for 1 h. Gels were blotted onto
prepackaged Iblot2 stacks (PVDF membrane, Invitrogen) using a custom
Iblot2 program (20 V 3 min, 23 V 1 min, 25 V 1 min). Membranes were
blocked for 15 min in SuperBlock, TBS (Thermo-Fisher Scientific).
Recombinant proteins were detected using mouse anti-6x-His antibody
(Clonetech) {[}1:20,000 1\selectlanguage{ngerman}×TBST (1.5 M NaCl, 10 mM Tris-HCl pH 7.5,
0.05\% tween 20), 1 h at room temperature{]} and goat anti-mouse HRP
(1:20,000 1×TBST, 1 h at room temperature) (Seracare). PlzA protein was
detected using rat anti-PlzA antibody (1:5,000 1×TBST, 1 h at room
temperature) and goat anti-rat antibody (BioRad) (1:40,000 1×TBST, 1 h
at room temperature). Membranes were washed with 1×TBST three times for
5 min between antibodies and before detection. Membranes were developed
using chemiluminescence (ECL select, Amersham) on an Azure Sapphire
imager using the chemiluminescence setting with High Dynamic Range
(HDR), auto exposure, and cumulative capture. Membranes were also
stained with Coomassie brilliant blue to visualize protein purity.

\textbf{PlzA RNA binding assay}

The Pierce~His protein interaction pulldown kit was used following the
manufacturer's instructions with some modifications. Briefly, spin
columns were prepared with 50 \selectlanguage{greek}µ\selectlanguage{english}l of slurry and equilibrated with a 1:1
TBS: Pierce Lysis Buffer with 10 mM imidazole. 150 \selectlanguage{greek}µ\selectlanguage{english}g of recombinant
His-tagged PlzA proteins were immobilized on the slurry by incubating in
the spin columns for 30 min at 4\selectlanguage{ngerman}°C on a rotating platform. Unbound
protein was removed and columns were washed. 50 ml of B31 5A4 \emph{B.
burgdorferi} were grown to a cell density of 1 ×
10\textsuperscript{8~}cells/ml in BSK-II at 34°C and 5\%
CO\textsubscript{2}. Cells were pelleted at 5,000 × \emph{g} and frozen
at -80°C. Pellets were thawed on ice and resuspended in wash solution
(1:1 ratio of Pierce lysis buffer and BupH Tris Buffered Saline; 500 \selectlanguage{greek}µ\selectlanguage{english}l
total volume). Cells were lysed in a 2-ml microfuge tube with 500 \selectlanguage{greek}µ\selectlanguage{english}l of
0.1 mm glass beads with a Retsch at 30 Hz for 5-10 min in a frozen
cassette. Lysates were cleared at 12000 \selectlanguage{ngerman}× \emph{g} for 10 min, added to
the spin column and rotated for 1 h at 4°C. Columns were centrifuged at
1250 × \emph{g} for 30 sec and flow throughs were saved for SDS-PAGE
analysis. The columns were washed five times with 400 \selectlanguage{greek}µ\selectlanguage{english}l of wash
solution and centrifuged at 1250 \selectlanguage{ngerman}× \emph{g} for 30 s. Proteins were
eluted with 250 \selectlanguage{greek}µ\selectlanguage{english}l of wash buffer containing 300 mM imidazole. The
columns were rotated at 4\selectlanguage{ngerman}°C for 5 min and then centrifuged at 1250
×\emph{g} for 30 s. Two eluates were acquired per sample. 10 \selectlanguage{greek}µ\selectlanguage{english}l of each
eluate was saved for SDS-PAGE analysis. The remaining sample was
prepared for hot phenol RNA isolation as previously described in
Lybecker et al\emph{.} (75). RNA concentration was measured using a
Nanodrop spectrophotometer and analyzed on an Agilent 2100 Bioanalyzer
picochip.

\textbf{RESULTS AND DISCUSSION}

\textbf{PlzA has c-di-GMP-independent RNA annealing activity \emph{in
vitro}}

The role of PlzA in the enzootic cycle of \emph{B. burgdorferi} has been
intensively studied (14,52,55-57), but the molecular basis for PlzA
function remains unknown. A comparative genomic analysis revealed PlzA
shares modest sequence similarity with ProQ (Fig. S1), an RNA chaperone
(22,76-79). The recently determined crystal structure revealed
that\emph{holo} -PlzA has bilobed \selectlanguage{greek}β-\selectlanguage{english}barrel domains connected by a short
linker (58). Several RNA-binding proteins, including cold shock proteins
A and E and eukaryotic Y-box proteins, utilize small \selectlanguage{greek}β-\selectlanguage{english}barrels to
interact with RNA (16). We hypothesized that PlzA has RNA chaperone
activity, which could be modulated by the binding of c-di-GMP. To test
this hypothesis, we assayed the RNA annealing activity of PlzA \emph{in
vitro} utilizing two artificial unstructured complementary 21-nucleotide
RNAs, termed J1 and M1, that were 5' end-labeled with Cy5 or Cy3,
respectively (68). \emph{In vitro} assays that employ artificial RNA
substrates are often used to test RNA chaperone activity. RNA chaperones
typically possess non-specific RNA chaperone activities even if they
have specific RNA targets. To assay ``pure annealing'' (without melting
internal base pairs from structured RNA), the J1 and M1 RNAs were
designed without internal secondary structure (68,71,80). The annealing
reactions were initiated by adding the M1 RNA to the J1 RNA, samples
were removed from the reaction over time, mixed with stop buffer and
resolved on a native polyacrylamide gel (Fig. 1A). The stop buffer
contains an excess of uM1 that binds to the remaining single-stranded
J1, effectively stopping the annealing reaction (Fig. 1A and S2A). RNA
annealing is visualized by a gel shift of the individual single-stranded
RNAs (ssRNAs) green and red bands to a yellow/orange band
double-stranded RNA (dsRNA) that migrates more slowly (Fig. S2B).
Annealing of complementary RNAs occurs unaided \emph{in vitro} ;
however, addition of StpA, a protein with known RNA annealing activity
(17,71), significantly accelerated the base pairing (Fig. S2B). To
measure the rate of annealing, dsRNA formation was quantified using the
ratio of J1M1 dsRNA (yellow band) to total RNA over time (Fig. 1B). We
found that PlzA accelerated RNA annealing over time compared to the RNA
only reaction and the GrpE (which lacks RNA chaperone activity) and
c-di-GMP negative controls (Fig. 1B). Furthermore, the PlzA-mediated RNA
annealing rate was independent of c-di-GMP (Fig. 1B, compare green and
blue triangles). The amount of dsRNA formed by the endpoint of the assay
with PlzA was significantly higher than the negative controls, albeit
lower than the positive control (Figs. 1C and S3A).

We performed filter-binding assays to determine the binding affinity of
PlzA for the M1 ssRNA substrate (Fig. 1D). Cy3-labeled M1 RNA and serial
dilutions of PlzA proteins were incubated together, applied to a slot
blot apparatus, and filtered through two membranes (nitrocellulose and
positively charged nylon). The protein and protein-bound RNA bind to
nitrocellulose membrane, while unbound RNA flows through the
nitrocellulose and binds to the nylon membrane. The fraction of bound
RNA was calculated as a function of protein concentration (Fig. 1E). The
disassociation constant (\emph{K} \textsubscript{D}) of StpA, the
non-specific positive control, was 838.5 \selectlanguage{ngerman}± 171.9 nM (Fig. S4), which
agrees with the \emph{K} \textsubscript{D} determined previously (81).
Generally, non-specific interactions of RNA chaperones with RNA result
in \emph{K} \textsubscript{D} in the low \selectlanguage{greek}µ\selectlanguage{english}M range, while specific
interactions are in the low nM range (18,21,68,80). The\emph{K}
\textsubscript{D} of PlzA with and without c-di-GMP were 68.17 \selectlanguage{ngerman}± 7.77 nM
and 93.37 ± 37.94 nM, respectively, which was not statistically
different (\emph{p} = 0.4338), demonstrating that c-di-GMP does not
affect PlzA binding to single-stranded M1 (Fig. 1E).

\textbf{c-di-GMP inhibits PlzA strand displacement activity \emph{in
vitro}}

Proteins other than RNA chaperones can accelerate RNA annealing \emph{in
vitro} via indiscriminate mechanisms including molecular crowding and
shielding repulsive charge interactions between RNA molecules (82).
However, a hallmark of RNA chaperones is their ability to destabilize
dsRNA helices and displace one of the strands in the helix with another
RNA, termed strand displacement. Unlike RNA annealing, the melting and
strand displacement of stable, completely complementary dsRNA does not
occur without an RNA chaperone \emph{in vitro} . We performed strand
displacement gel assays (68) to determine if PlzA has strand
displacement activity \emph{in vitro} . Stable dsRNA (yellow band) was
formed and purified using the two unstructured fluorescent-labeled
complementary 21-nucleotide RNAs, J1 and M1, described above in Fig. 1.
The strand displacement assay was initiated by adding an excess of M1
unlabeled competitor RNA (uM1) with or without an RNA chaperone to the
J1M1 dsRNA; strand displacement activity is detected as a decrease in
dsRNA J1M1 (yellow band) and increases in J1uM1 dsRNA (red band) and M1
ssRNA (green band), as the unlabeled competitor RNA, uM1, replaces the
M1 RNA in the dsRNA (Fig. 2A). As noted, strand displacement did not
occur without an RNA chaperone (Fig. S2C, RNA only), while the known RNA
chaperone StpA had strong strand displacement activity (Fig. S2C, StpA).
Strand displacement was quantified using the ratio of J1M1 dsRNA (yellow
band) to total RNA and values graphed over time (Fig. 2B). We found that
PlzA had increased strand displacement activity and the amount of the
initial dsRNA at the endpoint of the assay was significantly lower
compared to the three negative controls, RNA only, c-di-GMP alone and
GrpE (Figs. 2B, 2C and S3B). However, the ability of PlzA to strand
displace was not as efficient as the positive control StpA in
the\emph{in vitro} assay. Furthermore, PlzA strand displacement activity
was modulated by c-di-GMP: the addition of c-di-GMP abrogated PlzA
strand displacement activity (Fig. 2B and C, compare green triangles and
blue inverted triangles). Inhibition of PlzA strand displacement
activity was specific to c-di-GMP as c-di-AMP had no effect (Fig. 2B,
compare green and orange triangles).

RNA-binding proteins affinity for RNA can be modulated by the binding of
metabolites, sRNAs, and proteins as well as by phosphorylation. There
are several RNA-binding proteins in \emph{B. subtilis} , HutB, PyrR, and
TRAP, that are activated by the binding of histidine and
Mg\textsuperscript{2+}, UMP and UTP, and tryptophan, respectively (83),
but there are no examples of RNA chaperone activity being regulated by a
second messenger. We hypothesized that the \emph{holo} -PlzA may have a
lower affinity for dsRNA compared to \emph{apo} -PlzA, thus bound
c-di-GMP would decrease strand displacement activity. We tested PlzA
affinity for dsRNA in the presence and absence of c-di-GMP by filter
binding assays, similar to Fig. 1D, using M1uJ1 dsRNA. The data
demonstrate c-di-GMP does not affect PlzA binding to double-stranded RNA
and the disassociation constants are nearly
identical:\emph{K\textsubscript{D}} of 61.7 ± 15.18 nM and 60.87 ± 8.45
nM with and without c-di-GMP, respectively (Fig. 2D). In agreement with
previous reports, the \emph{K\textsubscript{D}} of StpA was in the low
\selectlanguage{greek}µ\selectlanguage{english}M range (Fig. S4). Taken together, these data indicate that the binding
of c-di-GMP affects PlzA strand displacement activity but does not
affect the affinity of PlzA for ssRNA or dsRNA \emph{in vitro} . In
contrast to other known metabolite-sensing RNA-binding proteins (83),
RNA binding by PlzA is not affected by c-di-GMP, but the RNA chaperone
activity (strand displacement) of PlzA is inhibited by c-di-GMP. To our
knowledge, we have identified the first RNA chaperone whose activity is
regulated by a second messenger.

\textbf{c-di-GMP inhibits the RNA unwinding activity of PlzA in \emph{E.
coli}}

RNA chaperone activity has been assayed \emph{in vivo} utilizing a
transcription antitermination assay (74,84-86). \emph{E. coli} strain
RL211 harbors the chloramphenicol (Cm) acyltransferase gene (\emph{cat}
) preceded by a Rho-independent \emph{trpL} transcriptional terminator.
The terminator is a stem-loop followed by a poly(U) stretch that
efficiently terminates transcription of the \emph{cat} gene and results
in susceptibility of the RL211 strain to Cm. However, proteins with RNA
chaperone unwinding activity melt or unfold the \emph{trpL} terminator
leading to antitermination, transcription of the \emph{cat} gene and
resistance to Cm (Fig. 3A). We sought to determine if PlzA had
antitermination activity \emph{in vivo} in \emph{E. coli} using the
RL211 strain. Plasmids carrying \emph{stpA} and \emph{plzA} under the
control of the \emph{lac} promoter were transformed separately into the
RL211 strain. Cultures were grown overnight in the absence of Cm and
then spotted onto plates without (LB) and with Cm (Fig. 3B). As
expected, all strains grew similarly on the LB plates (Fig. 3B). RL211
was unable to grow in the presence of Cm and growth was restored in the
presence of Cm in the strain producing StpA, which is known to have RNA
unwinding activity (71,87,88). The strain producing PlzA had minimal
growth in the presence of Cm (Fig. 3B). We postulated that c-di-GMP may
be inhibiting RNA unwinding in \emph{E. coli,} an effect similar to what
we observed in the strand displacement assay \emph{in vitro} (Fig.
2).\emph{E. coli} harbors 12 genes encoding diguanylate cyclases that
generate c-di-GMP (89). Therefore, we tested the ability of a PlzA
mutant that is unable to bind c-di-GMP (PlzA R145D) and a mutant with
increased affinity for c-di-GMP (PlzA R145K) to rescue growth in Cm
(53). Expression of the PlzA R145D mutant, which cannot bind c-di-GMP,
rescued growth in Cm, while expression of PlzA R145K, which binds
c-di-GMP with a high affinity, did not rescue growth in Cm (Fig. 3B).
Taken together, these data suggest that PlzA has antitermination
activity and this activity is inhibited by c-di-GMP \emph{in vivo} , at
least in a heterologous system. Therefore, both the RNA displacement and
unwinding activities of PlzA, but not its RNA annealing activity, are
modulated by c-di-GMP.

\textbf{Potential RNA-binding sites of PlzA}

The recently solved crystal structure of \emph{holo} -PlzA reveals a
bilobed protein of small \emph{N} - and \emph{C} -terminal \selectlanguage{greek}β-\selectlanguage{english}barrels
connected by a short linker region (58). The antiparallel seven-stranded
\selectlanguage{greek}β-\selectlanguage{english}barrels in the \emph{N} - and \emph{C} -terminal domains are under 100
amino acids each and have multiple aromatic and basic side chains (Fig.
S5). Small \selectlanguage{greek}β-\selectlanguage{english}barrel domains (SBB) are characterized by their
\selectlanguage{greek}β-\selectlanguage{english}strand-rich secondary structure and their small size, less than
approximately 100 amino acids. Proteins containing SBB domains are
functionally diverse and can bind to DNA, RNA and proteins (90).
The\emph{E. coli} major cold shock protein CspA is an SBB protein that
binds RNA along one surface and is proposed to destabilize mRNA
secondary structures at low temperatures (91). The RNA chaperone
activity of CspA is dependent on three aromatic side chains protruding
from the surface of the \selectlanguage{greek}β-\selectlanguage{english}barrel, which disrupt the base pairs in an RNA
hairpin via stacking interactions (92). The surrounding basic amino
acids are also thought to contribute to the RNA chaperone activity of
CspA by compensating for the negative charges on the RNA backbone (92).
Other RNA-binding proteins also utilize the combination of aromatic and
basic amino acid side chains to bind RNA (93). PlzA has numerous
aromatic and basic amino acid side chains (Fig. 4 and Fig. S5). We
propose that the protruding aromatic amino acids are necessary for
RNA-binding and chaperone activity. The \emph{N} -terminal F92 and F95
and \emph{C} -terminal F168 and F218 amino acids are positioned to
interact with RNA (Fig. 4A and Fig. S5) and were targeted for
mutagenesis to investigate RNA-binding and unwinding activity (Fig. 5).
Wild-type PlzA has modest RNA antitermination activity in \emph{E. coli}
, while the PlzA R145D mutant has strong RNA antitermination activity
(Fig. 3), consequently we mutated the aromatic amino acids in an R145D
background. F92 and F95 in the \emph{N} -terminal domain and F168 and
F218 in the \emph{C} -terminal \selectlanguage{greek}β-\selectlanguage{english}barrel were mutated to alanine,
resulting in two triple-mutant \emph{plzA} alleles (F92A/F95A/R145D and
F168A/F218A/R145D). We measured the circular dichroism (CD) spectra of
wild-type PlzA and the mutants to determine if the mutations affected
the structure of the protein. The CD spectra of wild type and all the
mutants are characteristic of well-folded proteins (Fig. 4B). There are
small deviations between the wild-type and mutant CD spectra, but these
differences have small effects on the secondary structure content of the
variants relative to wild-type PlzA determined from the CD spectra
(Table S4). The secondary structure content differs somewhat from that
observed for the X-ray structure of PlzA, which was obtained in the
presence of c-di-GMP. The increase in coil and the decrease in \selectlanguage{greek}β-\selectlanguage{english}sheet
observed with CD data for wild type and all mutants of PlzA in the
absence of c-di-GMP are consistent with the structure of \emph{apo}
-PlzA being more flexible than \emph{holo} -PlzA.

The \emph{in vivo} antitermination assay was performed in the RL211
strain with plasmids expressing the PlzA triple mutant proteins. We
found that \emph{E. coli} expressing PlzA containing the mutated
aromatic residues were unable to grow in Cm, suggesting that these amino
acids are important for unwinding RNA and the antitermination activity
of PlzA (Fig. 5). We hypothesized that the triple mutants were unable to
antiterminate \emph{in vivo} because of a lower affinity for the RNA. To
examine the role of the aromatic residues in RNA binding, we performed
filter binding assays with ssRNA, dsRNA and the \emph{trpL} terminator
RNA (Fig. 5B). The data demonstrate that the triple mutants have
diminished (PlzA F92A/F95A/R145D) or abolished (PlzA F168A/F218A/R145D)
RNA binding to all the RNA substrates. Apparent disassociation constants
could not be calculated for all RNAs tested for PlzA F168A/F218A/R145D
and ssRNA and dsRNA for PlzA F92A/F95A/R145D.
The\emph{K\textsubscript{D}} for PlzA F92A/F95A/R145D could be
calculated but was over sevenfold higher than wild-type PlzA for the
\emph{trpL}RNA (Fig. S5). Again, the \emph{K\textsubscript{D}} for StpA
was in the low \selectlanguage{greek}µ\selectlanguage{english}M range, as expected (Fig. S5). The R145D mutant PlzA
protein also had a lower affinity for all RNAs tested compared to the
wild type, but still efficiently bound RNA (Fig. 5B and Table S5).
Regardless, PlzA R145D efficiently antiterminates transcription \emph{in
vivo} in the RL211 strain. The data suggest that the reduced affinity
for the\emph{trpL} RNA \emph{in vitro} does not affect the
antitermination activity of the PlzA R145D mutant \emph{in vivo} .
Surprisingly, the data show that PlzA binding to RNA does not
necessarily result in RNA unwinding and antitermination. Wild-type PlzA
can bind RNA, but in the presence of c-di-GMP cannot antiterminate. PlzA
R145D binds RNA, albeit with a reduced affinity, but cannot bind
c-di-GMP and has antitermination activity. However, the mutation of
several key aromatic residues in the R145D background results in
abolished or diminished RNA binding and loss of antitermination
activity. Taken together, the data suggest that PlzA must be able to
bind RNA and be in its apo-form to unwind RNA and antiterminate
transcription \emph{in vivo} . To our knowledge this is the first
example of a small molecule regulating the activity of an RNA binding
protein without affecting its ability to bind RNA.

RNA chaperones use multiple RNA-binding domains or multiple copies of
the same protein for their various activities, including refolding RNA
and annealing two RNAs as a matchmaker (16). The multiple RNA-binding
sites of PlzA may facilitate binding to multiple RNAs, thus allowing
them to interact. In addition, multiple interactions with the same RNA
may allow PlzA to rearrange contacts with the RNA without releasing
it.\emph{Apo} -PlzA could not be crystalized and has more coil structure
when analyzed, which indicates the protein structure may be dynamic in
the absence of c-di-GMP and suggests that PlzA becomes less flexible
upon c-di-GMP binding, possibly serving as a mechanism to inhibit the
RNA unfolding activity.

RNA-binding proteins and DNA-binding proteins both rely on positively
charged and polar amino acids for interacting with RNA and DNA. However,
the chemical and structural differences of RNA and DNA result in
distinct types of interactions with these residues (94-99). Despite the
dissimilar mechanisms of RNA binding and DNA binding, numerous proteins
bind both DNA and RNA, including Hfq, StpA and H-NS (100-102). The
domains, or specific residues, required to bind RNA and DNA may be
different in a protein. For example, human ADAR1 binds both RNA and
Z-form DNA via two separate domains. The electrostatic surface of PlzA
(Fig. 4A) reveals several electropositive surfaces including a deep,
electropositive groove where c-di-GMP binds. The groove is larger than
the c-di-GMP-binding site.~Perhaps this surface interacts with DNA
and/or RNA. Further experimentation is required to examine the role of
these surfaces in nucleic acid binding. Notably, our data demonstrate
that the electropositive surfaces of PlzA are not sufficient to bind RNA
as mutating two aromatic amino acids diminished or abolished RNA
binding.

\textbf{PlzA binds \emph{B. burgdorferi} RNAs \emph{in vitro}}

To determine if PlzA can bind \emph{B. burgdorferi} RNA, we performed
an\emph{in vitro} RNA-binding assay using PlzA as bait. His-tagged
wild-type and mutant PlzA proteins were immobilized on a cobalt column
and \emph{B. burgdorferi} cleared lysates were passed over the column
(Fig. 6A). Columns were washed and PlzA was eluted with imidazole.
Eluates were immunoblotted to assay for PlzA protein (Fig. 6B), and RNA
bound to PlzA was isolated and run on a bioanalyzer (Fig. 6C).
Immunoblot analyses demonstrate that PlzA proteins were found in both
eluates but were more concentrated in the first fraction (Fig. 6B, E1).
We found that wild-type PlzA and PlzA R145D, which does not bind
c-di-GMP, both bind \emph{B. burgdorferi} RNA, while neither of the PlzA
triple mutant proteins nor the control without PlzA protein bind RNA
(Fig. 6C). These results support the hypothesis that the aromatic amino
acids in the \emph{N} - and \emph{C} -terminal domains are important for
binding RNA and corroborate the \emph{in vitro} filter binding data.
These results also demonstrate that the positive surface charges of PlzA
are not sufficient to bind \emph{B. burgdorferi} RNA. Notably, the RNA
detected on the bioanalyzer was relatively small (25-150 nucleotides),
which suggest PlzA binds predominantly sRNAs, but the specific RNAs
bound by PlzA awaits further investigation.

\textbf{Concluding remarks}

\emph{Apo} - and \emph{holo} -PlzA function differently in the
vertebrate and tick phases of the enzootic cycle of \emph{B.
burgdorferi}(14,52,55-57). We and others have suggested that the
effector functions of \emph{apo} -PlzA and \emph{holo} -PlzA reflect
distinct activities and interaction partners (14,47,53,54,57). Here, we
demonstrate that c-di-GMP modulates the RNA strand displacement and
unwinding activity of PlzA, suggesting these activities may be important
for the vertebrate phase of the life cycle, but not the tick phase.
Conversely, PlzA has c-di-GMP-independent RNA annealing activity, and we
postulate that PlzA may act as a matchmaker RNA chaperone aiding in sRNA
and target RNA interactions throughout the enzootic cycle of the
spirochete (Fig. 7).

\textbf{DATA AVAILABILITY}

All data are available in the main article and Supplementary Data.

\textbf{SUPPLEMENTARY DATA}

Supplementary Data are available online.

\textbf{ACKNOWLEDGEMENTS}

We thank Bob Gilmore, Kevin Brandt and Brittany Armstrong for thoughtful
and critical reading of the manuscript. We thank Jean-Marc Lanchy, James
Kovaks and Crystal Vander Zanden for useful discussions. We thank Robert
Landick for the RL211 strain. We thank Irina Goodrich for excellent
technical support. The findings and conclusions in this report are those
of the authors and do not represent the official position of the Centers
for Disease Control and Prevention.

\textbf{FUNDING}

This work was supported by the National Institutes of Health {[}R01
AI130247 D.S.S, M.C.L. and D.D.{]}

\textbf{CONFLICT OF INTEREST}

The authors declare no competing interests.



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