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\begin{document}

\title{In Salmonella enterica, the pathogenicity island 2 (SPI-2) regulator
PagR regulates its own expression and the expression of a five-gene
operon that encodes transketolase C}



\author[1]{Jorge Escalante-Semerena}%
\author[1]{Anastacia R. Parks}%
\author[1]{Regan D. McCormick}%
\author[1]{Jordan A. Byrne}%
\affil[1]{University of Georgia Department of Microbiology}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The enteropathogen \emph{Salmonella enterica} subsp. \emph{enterica} sv.
Typhimurium str. LT2 (hereafter \emph{S.} Typhimurium) utilizes a
cluster of genes encoded within the pathogenicity island 2 (SPI-2) of
its genome to proliferate inside macrophages. The expression of SPI-2 is
controlled by a complex network of transcriptional regulators and
environmental cues, which now include a recently characterized
DNA-binding protein named PagR. Growth of \emph{S.} Typhimurium in low
phosphate low magnesium medium mimics conditions inside macrophages.
Under such conditions, PagR ensures SPI-2 induction by upregulating the
transcription of \emph{slyA}, a known activator of SPI-2. Here we report
that PagR represses the expression of a divergently transcribed
polycistronic operon that encodes the two subunits of transketolase TktC
( \emph{i.e., tktD, tktE}) of this bacterium. Transketolases contribute
to the non-redox rearrangements of phosphorylated sugars of the pentose
phosphate pathway, which provide building blocks for amino acids,
nucleotides, cofactors, etc. We also demonstrate that PagR represses the
expression of its own gene and define two PagR binding sites between
\emph{stm2344} and \emph{pagR}.%
\end{abstract}%



\sloppy


\textbf{In \emph{Salmonella enterica,} the pathogenicity island 2
(SPI-2) regulator PagR regulates its own expression and the expression
of a five-gene operon that encodes transketolase C}

\textbf{Anastacia R. Parks\textsuperscript{1+\#}, Regan D.
McCormick\textsuperscript{1\#}, Jordan A. Byrne\textsuperscript{1§}, and
Jorge C. Escalante-Semerena*\textsuperscript{1}}\selectlanguage{ngerman}

\textsuperscript{1}Department of Microbiology, University of Georgia,
Athens, GA 30602, USA

*Corresponding author: Department of Microbiology, University of
Georgia, 212C Biological Sciences Building, 120 Cedar Street, Athens, GA
30602, USA, T: 1 (706)-542-2651, F: 1 (706)-542-2815; Email:
jcescala@uga.edu, T: +1 (706)-542-2651

\selectlanguage{english}\textsuperscript{+}Current address: Department of Microbiology and
Immunobiology, Harvard Medical School, Boston, MA 02115

\textsuperscript{SS}Current address: Molecular, Cellular, and
Developmental Biology, University of Michigan, Ann Arbor, MI 48109

\textsuperscript{\#}These authors contributed equally to this work

Running title: PagR regulates the synthesis of transketolase C

\textbf{AUTHOR CONTRIBUTIONS}

A.R.P., R.D.M., and J.C.E.-S. conceptualized the project, designed the
research, analyzed data, wrote the first draft of the paper; A.R.P.
performed most of the experiments. J.A.B. assisted in the performance of
experiments and edited the paper. J.C.E.-S obtained funding for the
performance of the work.

\textbf{Summary}

The enteropathogen \emph{Salmonella enterica} subsp. \emph{enterica} sv.
Typhimurium str. LT2 (hereafter \emph{S.} Typhimurium) utilizes a
cluster of genes encoded within the pathogenicity island 2 (SPI-2) of
its genome to proliferate inside macrophages. The expression of SPI-2 is
controlled by a complex network of transcriptional regulators and
environmental cues, which now include a recently characterized
DNA-binding protein named PagR. Growth of \emph{S.} Typhimurium in low
phosphate low magnesium medium mimics conditions inside macrophages.
Under such conditions, PagR ensures SPI-2 induction by upregulating the
transcription of \emph{slyA} , a known activator of SPI-2. Here we
report that PagR represses the expression of a divergently transcribed
polycistronic operon that encodes the two subunits of transketolase TktC
(\emph{i.e., tktD, tktE} ) of this bacterium. Transketolases contribute
to the non-redox rearrangements of phosphorylated sugars of the pentose
phosphate pathway, which provide building blocks for amino acids,
nucleotides, cofactors, etc. We also demonstrate that PagR represses the
expression of its own gene and define two PagR binding sites
between\emph{stm2344} and \emph{pagR} .

\textbf{Keywords} . PagR protein, pentose phosphate pathway,
\emph{tktDE}expression, transketolase, transcriptional regulation,
\emph{pagR} gene expression, \emph{Salmonella} pathogenesis

\textbf{Introduction}

Transketolases are well-characterized enzymes that catalyze the
non-oxidative transfer of a C2 unit from a ketose to an aldose,
shortening the ketose by two carbons and lengthening the aldose by two
carbons (Schneider and Lindqvist, 1998). Transketolases use thiamin
pyrophosphate (TPP) as cofactor, whose ylid carbanion triggers a
nucleophilic attack on the carbonyl group of the ketose. The addition
product cleaves in a retro-aldol like reaction and the resulting enamine
adds to the carbonyl group of the aldose. Elimination of the TPP ylid
carbanion yields the final ketose product. Transketolase-catalyzed
reactions are critical in processes such as the non-oxidative branch of
the pentose phosphate pathway (non-oxPPP) (Stincone et al., 2015) and
the Calvin-Benson-Bassham (CBB) cycle (Raines, 2003). Up until recently,
the genome of \emph{S.} Typhimurium was thought to encode only two
transketolases.

TktC was recently characterized as an isozyme of transketolase A and
transketolase B (Shaw et al., 2018). The TktA and TktB transketolases
are housekeeping enzymes involved in the non-oxPPP of this bacterium.
The reversibility of reactions catalyzed by transketolase enzymes
involved in the non-oxPPP allows the production of intermediate
compounds to meet metabolic demands of the cell through the diversion of
metabolites to other pathways such as replenishment of glycolysis, or to
provide building blocks for the biosynthesis of aromatic amino acids,
nucleotides, or lipopolysaccharides (Stincone et al., 2015). PagR is a
transcriptional regulator that indirectly induces the expression of
SPI-2 through \emph{slyA} , which is important for \emph{S.} Typhimurium
replication in macrophages and pathogenicity in mice (Jiang et al.,
2020). During the analysis of the genomic context of the \emph{pagR}gene
(locus tag \emph{stm2345} ), we noticed that \emph{pagR} was transcribed
divergently from a set of genes encoding a putative sugar transporter
(\emph{stm2342} , \emph{stm2343} , \emph{stm2344} ) and two genes
(\emph{tktD = stm2341} and \emph{tktE} = \emph{stm2340} ) encoding the
subunits of transketolase C (TktC, Fig. 1). In this paper we show that
i) PagR regulates the expression of the divergently
transcribed\emph{tktDE} genes that encode the subunits of the TktC
transketolase of\emph{S.} yphimurium; ii); PagR represses the expression
of its own gene; and iii); PagR directly binds the promoter region
between\emph{stm2344} and \emph{pagR} , protecting two binding sites.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. Genomic context of the \emph{pagR} and \emph{tktD,
tktE} genes} . PagR activates the transcription of the five-gene operon
shown in the figure. The genes encoding the TktC transketolase are the
most promoter-distal genes of the operon. \emph{tktD} = 953
nt,\emph{tktE} = 830 nt, \emph{stm2342} = 1391 nt, \emph{stm2343} = 272
nt,\emph{stm2344} = 443 nt, \emph{pagR} = 1019 nt. At present, the
location of the promoters is hypothetical. The operon spans the region
from 2,257,129 to 2,454, 101 nt (\emph{i.e.,} 3,028 nt) of the
\emph{S.}Typhimurium chromosome. \emph{pagR} spans the region from
2,457,382 to 2,458401 nt. All genes are at scale.



\textbf{Results and Discussion}

\textbf{A \emph{S.} Typhimurium [?]\emph{pagR} strain grows poorly with
succinate as a sole carbon and energy source} . We probed for areas
of\emph{S.} Typhimurium metabolism that might be regulated by PagR. To
this end, we tested the ability of a \emph{S.}
Typhimurium\emph{pagR1::kan} \textsuperscript{+} strain to catabolize
various carbon sources. During this screen, we observed that
a\emph{pagR1::kan} \textsuperscript{+} strain (JE21566) struggled to
grow when succinate was the sole carbon and energy source (Fig. 2A, gray
triangles \emph{vs} red circles). The observed phenotype was solely due
to the lack of PagR, since we restored growth to the rate and cell
density of a culture of the \emph{pagR} \textsuperscript{+} strain by
ectopically expressing the \emph{pagR} \textsuperscript{+} allele (Fig.
2A, dark green triangles). Surprisingly, we also corrected the growth
phenotype by deleting the \emph{tktD} and \emph{tktE} genes encoding the
two subunits of the TktC transketolase (\emph{tktDE1}
::\emph{cat\textsuperscript{+}} , Fig. 2A, blue squares). The latter was
a notable result because the correction of the phenotype was as
efficient as the ectopic expression of\emph{pagR} \textsuperscript{+}.
Conversely, the ectopic expression of the \emph{tktDE}
\textsuperscript{+} genes repressed growth (Fig. 2B, magenta squares),
mimicking the growth behavior of a\emph{pagR1}
::\emph{kan\textsuperscript{+}} strain (Fig. 2B, gray triangles). We
wanted to learn whether the phenotype observed when the\emph{tktDE}
\textsuperscript{+} genes were overexpressed was a result of having
excess transketolase activity or whether TktC, was somehow different to
TktA and TktB. For this purpose we expressed the other two\emph{S.}
Typhimurium transketolases in the\emph{pagR1}
::\emph{kan\textsuperscript{+}tktDE1} ::\emph{cat\textsuperscript{+}}
strain and monitored cell growth. Neither the ectopic expression of
\emph{tktA\textsuperscript{+}} nor\emph{tktB\textsuperscript{+}}
repressed the growth of the\emph{pagR1}
::\emph{kan\textsuperscript{+}tktDE1} ::\emph{cat\textsuperscript{+}}
strain (Fig. 2B, light green squares and yellow squares \emph{vs}
magenta squares). Rather, expression of \emph{tktA\textsuperscript{+}}
and\emph{tktB\textsuperscript{+}} improved growth relative to the vector
control. From these results we learned that the cell could not overcome
whatever the deleterious effect of having TktC in excess was when
growing on succinate.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} \textbf{Growth of \emph{S.} Typhimurium on succinate
as a function of \emph{pagR} and \emph{tktDE}.} \emph{S.} Typhimurium
was grown on NCE (no-carbon essential) minimal medium (Berkowitz et al.,
1968) supplemented with sodium succinate (30 mM) as the sole source of
carbon and energy. \textbf{A.} The\emph{pagR1}
::\emph{kan\textsuperscript{+}} strain failed to grow after 36 h of
incubation at 37\selectlanguage{ngerman}ºC on succinate as the sole carbon source, and ectopic
expression of \emph{pagR} restores growth to wild-type. Removal of
\emph{tktDE} in the \emph{pagR1}
::\emph{kan\textsuperscript{+}}background restores growth as well.
Growth of the\emph{pagR\textsuperscript{+}tktDE}
::\emph{cat\textsuperscript{+}} strain doesn't significantly differed
from the parent strain. Ectopic gene expression was induced with
L-(+)-arabinose (100 \selectlanguage{greek}µ\selectlanguage{english}M). ``pPagR'' refers to plasmid
pPagR-7.\textbf{B.} Expression of TktC, but not TktA or TktB, repressed
growth of the \emph{pagR1} ::\emph{kan\textsuperscript{+}tktDE}
::\emph{cat\textsuperscript{+}} strain. Ectopic gene expression was
induced with L-(+)-arabinose (1 mM). The strains used were\emph{pagR}
\textsuperscript{+} \emph{tktDE\textsuperscript{+}} / vector (JE22070,
red circles), \emph{pagR1} ::\emph{kan}
\textsuperscript{+}\emph{tktDE\textsuperscript{+}} / vector (JE21566,
grey triangles),\emph{pagR1} ::\emph{kan}
\textsuperscript{+}\emph{tktDE\textsuperscript{+}} / pPagR (JE21577,
dark green triangles),\emph{pagR} \textsuperscript{+}\emph{tktDE1}
::\emph{cat\textsuperscript{+}} / vector (JE25521, orange diamonds),
\emph{pagR1} ::\emph{kan} \textsuperscript{+}\emph{tktDE1}
::\emph{cat\textsuperscript{+}} / vector (JE25523, blue squares),
\emph{pagR1} ::\emph{kan} \textsuperscript{+}\emph{tktDE1}
::\emph{cat\textsuperscript{+}} / pTktC (JE25524, magenta squares),
\emph{pagR1} ::\emph{kan} \textsuperscript{+}\emph{tktDE1}
::\emph{cat\textsuperscript{+}} / pTktA (JE257201, light green squares),
\emph{pagR1} ::\emph{kan} \textsuperscript{+}\emph{tktDE1}
::\emph{cat\textsuperscript{+}} / pTktB (JE27202, yellow squares);
`vector' stands for the empty cloning vector pCV1 that contains an
arabinose-inducible promoter (VanDrisse and Escalante-Semerena, 2016).
This experiment was conducted in technical triplicate of biological
duplicates three independent times. Error bars represent one standard
deviation from the mean. Error bars that are not visible are smaller
than the symbol. The statistical analysis was performed with Prism v9
(GraphPad)

\textbf{The \emph{tktD and tktE} genes are part of a five-gene operon} .
Bioinformatics analysis suggested that genes \emph{stm2340
(tktE),stm2341 (tktD)} , \emph{stm2342} , \emph{stm2343} ,
and\emph{stm2344} comprised a polycistronic operon (Biocyc.org). To test
this possibility, we performed operon PCR with RNA isolated from
a\emph{pagR1} ::\emph{kan\textsuperscript{+}} strain. The RNA was
treated with DNase, and cDNA was generated from purified total RNA.
Genomic DNA, cDNA, and RNA were PCR amplified with four primer sets to
generate products of four potential regions that overlap in neighboring
putative genes (Fig. 3A). As shown in figure 3B, the use of such primers
generated amplicons for the cDNA and the gDNA, but none amplified for
the RNA, which served as the negative control. PCR amplification of all
overlapping regions between genes \emph{stm2340} and
\emph{stm2344}suggested these genes were part of a polycistronic unit.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3. Genes encoding TktD and TktE are promoter-distal in a
five-gene operon. A.} The \emph{pagR} gene is divergently transcribed
from the operon containing the genes encoding the two subunits of TktC
(\emph{tktD} and \emph{tktE} ). The first three genes of the operon are
annotated as putative phosphotransferase system (PTS)-type
transporters.\textbf{B.} Operon PCR of the putative polycistronic operon
containing the transketolase genes \emph{tktD} and \emph{tktE} encoding
the subunits of TktC. RNA, cDNA and gDNA were PCR amplified using
primers specific to overlapping regions between genes of the putative
operon designated 1-4 in panel A. Positive controls are amplification of
gDNA; negative controls are no amplification of RNA samples;
experimental samples were the cDNA amplifications. Ladder on left shows
corresponding amplification size (2-Log DNA Ladder, NEB).

\textbf{PagR binds directly to the promoter region for its own gene and
the polycistronic mRNA encoding \emph{tktD} and \emph{tktE}.} To
determine whether PagR directly bound to the promoter driving the
expression of the \emph{tktD and tktE} genes or was acting indirectly to
repress the expression of the operon, PagR protein was purified to
\textgreater{}95\% homogeneity (Fig. S1) and tested for its ability to
bind to the putative promoter region between \emph{stm2344}
and\emph{pagR} (Fig. 3A). The putative promoter fragment of \emph{pagR}
and the polycistronic operon comprised of the \emph{stm2340}
-\emph{stm2344}genes was incubated with increasing concentrations of
PagR protein then separated by native polyacrylamide electrophoresis
(Fig. 4). The mobility of the DNA fragment (2,457,048 to 2,457,454 nt of
the chromosome; 406 nt) that included the putative promoter region
decreased as a function of increasing concentrations of PagR protein as
compared to the no-PagR-protein-added control. This suggested that the
oligomerization state of PagR changed as its concentration increased or
that multiple PagR-binding sites were present in the DNA fragment.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4. PagR binds the region between \emph{pagR} and the
five-gene operon that includes \emph{tktD and tktE.}} Representative
results of an experiment designed to determine whether PagR could bind
to the intergenic region between \emph{pagR} and the five-gene operon
containing genes encoding TktC. In this experiment we used 0.5 pmol of
probe. The experiment was repeated three times and the amount of free
probe (406 nt) decreased as a function of the amount of PagR added to
the reaction mixture. More details about how the experiment was
performed can be found under \emph{Experimental procedures.}

\textbf{PagR represses the expression of the operon
containing\emph{tktDE,} and its own gene} . Given the proximity of
\emph{pagR} to the operon containing the \emph{tktD and tktE} genes
(Fig. 3A) and the data presented in figure 2, we investigated the
possibility that PagR repressed the expression of the operon containing
\emph{tktD} and\emph{tktE} . We isolated RNA from three strains,
namely\emph{pagR} \textsuperscript{+} / vector,\emph{pagR1}
::\emph{kan\textsuperscript{+}} / vector, and\emph{pagR1}
::\emph{kan\textsuperscript{+}} / pPagR, and quantified the amount of
\emph{stm2343} , \emph{tktD,} and \emph{tktE} mRNA using RT-qPCR. For
this purpose, cells were grown on lysogeny broth (LB) plus ampicillin
and L-(+)-arabinose (100 \selectlanguage{greek}µ\selectlanguage{english}M), and RNA was isolated when cultures reached
an OD\textsubscript{600} of 0.5. As shown in figure 5A, expression of
\emph{stm2343} , \emph{tktD} and \emph{tktE} increased
\textasciitilde{}10 fold in the\emph{pagR1}
::\emph{kan\textsuperscript{+}} / vector strain (red bars) when compared
to the \emph{pagR\textsuperscript{+}} / vector and the\emph{pagR1}
::\emph{kan\textsuperscript{+}} / pPagR strains (black and blue bars,
respectively). To assess whether PagR regulated its own expression, we
constructed a \emph{lacZY} transcriptional fusion with the chromosomal
\emph{pagR} gene and assayed \selectlanguage{greek}β-\selectlanguage{english}galactosidase activity with and without
ectopic expression of \emph{pagR} . Cells were grown to an
OD\textsubscript{600} of \textasciitilde{}0.5 in LB plus ampicillin and
L-(+)-arabinose (100 \selectlanguage{greek}µ\selectlanguage{english}M), then quantified \selectlanguage{greek}β-\selectlanguage{english}galactosidase activity
following a described protocol (Miller and Hershberger, 1984). The
\selectlanguage{greek}β-\selectlanguage{english}galactosidase activity of the\emph{pagR3}
::\emph{lacZY\textsuperscript{+}} strain was significantly lower in the
strain expressing \emph{pagR} than in the vector control (Fig. 5B, blue
vs red bars). Together, these data suggested that PagR repressed
expression of the operon containing \emph{tktDE,} and its own gene. This
conclusion was consistent with its annotated homology to the LacI family
of repressors.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5. Expression analysis of the operon
containing\emph{tktDE} and of \emph{pagR,} as a function of PagR.}
\textbf{A.}Effect of PagR on the expression of \emph{stm2343} ,
\emph{tktD} , and\emph{tktE} as shown by RT-qPCR. The experiment was
performed in biological and technical triplicates. \textbf{B.} Activity
of the\emph{pagR} promoter (in Miller Units) without and with\emph{pagR}
\textsuperscript{+} expressed \emph{in trans} (red vs blue bars,
respectively). * = \emph{p} [?] 0.05; ** = \emph{p} \textless{} 0.01, ***,
\emph{p} [?] 0.001, **** = \emph{p} [?] 0.0001. Cultures were grown in LB,
and details of the RT-qPCR and \selectlanguage{greek}β-\selectlanguage{english}galactosidase assay conditions used can
be found in the \emph{Experimental procedures}section. Vector = pCV1
(VanDrisse and Escalante-Semerena, 2016).

\textbf{PagR protects two binding sites in the \emph{stm2344}
--\emph{pagR} promoter region.} DNase I footprinting was performed using
a 291-nt DNA fragment spanning from 2,457,048 to 2,457,339 nt of the
chromosome to define the DNA sequence bound by PagR. Results of an EMSA
comparing PagR binding to this fragment with the one used in figure 4
suggested that the shorter probe used here contained all the relevant
PagR binding sites (Fig. S1). Figure 6 shows the digestion results for
the coding strand of the operon containing \emph{tktD} and
\emph{tktE}(left to right going towards \emph{stm2344} ). The presence
of PagR (Fig. 6, black trace) clearly protected two 21-bp regions of
the\emph{stm2344} - \emph{pagR} promoter region that were not protected
without PagR (Fig. 6, orange trace). A nearly identical sequence was
observed in both (Fig. 6, red underlines). The underlined sequence in
the protected region on the left is a perfect 14 base pair palindrome,
5'-TGATAGCGCTATCA-3', and the protected region on the right differs at
only 2 bases (5'-TG\emph{G} TAGCGCTATC\emph{T} -3', differences
underlined). These data supported our conclusion that PagR was a LacI
family regulator, since LacI family repressors typically bind
palindromic or inverted repeat DNA (Weickert and Adhya, 1992).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6. PagR binds two regions of the \emph{stm2344}promoter.}
DNase 1 footprinting electropherograms from the coding strand of
P\emph{\textsubscript{stm2344}} . Digestions were performed with 75 pmol
PagR (black trace) and with no PagR (orange trace). This image is
representative of two independent experiments.

\textbf{PagR is likely a member of the LacI/GalR family of
transcriptional regulators.} Herein, we have presented data showing that
PagR regulates gene expression in a manner like other proteins that
belong to the LacI/GalR family of regulators. That is, we hypothesize
PagR binds an as-yet-unidentified co-effector, dimerizes and then binds
to the identified sequences within the region between \emph{pagR} and
the \emph{tktDE} operon with the concomitant repression of its own
expression, and the expression of adjacent genes encoding a putative
transport system and subunits of a metabolic enzyme (Fig. 7).

\par\null

\textbf{Figure 7. Working model of PagR regulation of the
\emph{tktDE}operon.} We hypothesize PagR requires a co-effector to
dimerize, bind to the identified sites within the intergenic region
between \emph{pagR}and the 5-gene operon whose distal genes encode the
subunits of transketolase C (TktC). Roman numerals represent: I,
\emph{stm2344}(putative PTS ascorbate transporter subunit IIA), II,
\emph{stm2343}(putative PTS mannitol subunit IIB), III, \emph{stm2342}
(putative PTS ascorbate transporter subunit IIC). No experimental
evidence has been reported for the putative transporter. The red polygon
represents the as-yet-unidentified PagR co-effector.

Moreover, PagR binding to the promoter region between its gene
and\emph{stm2344} protects a palindromic DNA sequence (Fig. 6). The
repressor activity of PagR presented here contrasts with previous data
reported by others, who showed that PagR is an activator of
the\emph{slyA} gene (Jiang et al., 2020). This dual activity, however,
is not unprecedented for LacI/GalR family proteins (Swint-Kruse and
Matthews, 2009). It is currently unknown what causes the switch between
these functions for PagR. It is possible that the target of the
uncharacterized, putative phosphotransferase system (PTS)
(\emph{stm2342-4} ) adjacent to \emph{pagR} may provide the signal, like
galactose derepressing GalR (Swint-Kruse and Matthews, 2009). Expression
of \emph{pagR} increases under low phosphate and low magnesium
conditions (Jiang et al., 2020), and it could be that PagR is regulated
by phosphorylation or as a function of magnesium ions. All these
interesting questions are currently being investigated.

\textbf{Conclusions}

Previous work demonstrated that PagR is necessary for full induction of
the SPI-2 type-3 secretion system (Jiang et al., 2020). Our data show
that PagR also regulates expression of the genes encoding the subunits
of the TktC transketolase (\emph{i.e., tktD, tktE} ), the third
transketolase enzyme identified in \emph{S.} Typhimurium (Shaw et al.,
2018). Transketolases are essential for \emph{S.} Typhimurium virulence
(Shaw et al., 2018), and PagR appears to be a link between genetic
regulation of the pathogenic machinery and a metabolic function
essential for the pathogenic lifestyle of this bacterium. Moreover, the
data presented here indicate that TktC has a function in the cell that
differs from those of TktA and TktB. Future research into the identity
of the co-efector of PagR and into the function of TktC, will illuminate
the connections between central metabolism and the pathogenic machinery
in \emph{S.} Typhimurium.

\textbf{Experimental procedures}

\textbf{Bacterial strains.} All strains were derivatives
of\emph{Salmonella enterica} subsp. \emph{enterica} serovar Typhimurium
str. LT2 (hereafter \emph{S} . Typhimurium) (Table 1). Gene deletions
were constructed using the protocol that employs \selectlanguage{greek}λ \selectlanguage{english}Red recombinase to
engineer in-frame deletions as described elsewhere (Datsenko and Wanner,
2000).

\textbf{Table 1. Strains and plasmids used in this study}.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Strain}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Relevant genotype\textsuperscript{1}}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Reference/source\textsuperscript{2}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{S.} \textbf{Typhimurium\textsuperscript{3} strains}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE6583\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{metE205} [?]\emph{araB9}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
K. Sanderson\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Derivatives of JE6583}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE22070\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
/ pCV1 (cloning vector) \emph{bla\textsuperscript{+}}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
(VanDrisse and Escalante-Semerena, 2016)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE21107\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pagR1}::\emph{kan}\textsuperscript{+}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE24887\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{[?]pagR2} resolved\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE27072\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pagR3}::\emph{lacZY\textsuperscript{+}
kan\textsuperscript{+}}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE25373\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{tktDE1}::c\emph{at}\textsuperscript{+}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE25443\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pagR1}::\emph{kan}\textsuperscript{+}
\selectlanguage{greek}Δ\selectlanguage{english}\emph{tktDE1}::c\emph{at}\textsuperscript{+}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE21566\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pagR1}::\emph{kan}\textsuperscript{+} / pCV1
\emph{bla\textsuperscript{+}}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE21577\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pagR1}::\emph{kan}\textsuperscript{+} / pPagR-7
\emph{bla\textsuperscript{+}}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE25521\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pagR\textsuperscript{+} tktDE1}::\emph{cat\textsuperscript{+}} /
pCV1 \emph{bla\textsuperscript{+}}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE25523\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pagR1}::\emph{kan}\textsuperscript{+}
\emph{tktDE1}::c\emph{at}\textsuperscript{+} /pCV1
\emph{bla\textsuperscript{+}}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE25524\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pagR1}::\emph{kan}\textsuperscript{+}
\emph{tktDE1}::c\emph{at}\textsuperscript{+ /} pTktC-1
\emph{bla\textsuperscript{+}}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE27201\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pagR1}::\emph{kan}\textsuperscript{+}
\emph{tktDE1}::c\emph{at}\textsuperscript{+ /} pTktA-1
\emph{bla\textsuperscript{+}}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE27202\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pagR1}::\emph{kan}\textsuperscript{+}
\emph{tktDE1}::c\emph{at}\textsuperscript{+ /} pTktB-1
\emph{bla\textsuperscript{+}}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE27091\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pagR3}::\emph{lacZY\textsuperscript{+} kan\textsuperscript{+}} /
pCV1 \emph{bla\textsuperscript{+}}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE27092\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pagR3}::\emph{lacZY\textsuperscript{+} kan\textsuperscript{+}} /
pPagR-7 \emph{bla\textsuperscript{+}}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
JE6692\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
/ pKD46 \emph{bla}\textsuperscript{+}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Laboratory collection\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{E. coli} \textbf{strains}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{E. coli} C41 (\selectlanguage{greek}λ\selectlanguage{english}DE3)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{pka12}::\emph{kan+ ompT hsdS}
(r\textsubscript{B}m\textsubscript{B}) \emph{gal} (\selectlanguage{greek}λ\selectlanguage{english}DE3)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Laboratory collection\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
\emph{E. coli} DH5\selectlanguage{greek}α\selectlanguage{english}\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
[?]\emph{(argF-lacZ)169 [?]phoA8 l\textsuperscript{-}}f80d\emph{lacZ58}(M15)
\emph{thiE1} \emph{glnX44}(AS) \emph{deoR481 hsdR17}
\emph{gyrA96}(Nal\textsuperscript{R}) \emph{recA1 endA1}\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Laboratory collection\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{1}In strains JE22070 and JE6692 the genotype preceding
the forward slash is: \emph{metE205} [?]\emph{araB9.}

\textsuperscript{2}Unless otherwise indicated, all strains used in this
study were generated during the work.

\textsuperscript{3}\emph{S.} Typhimurium = \emph{Salmonella
enterica}subsp. \emph{enterica} sv. Typhimurium strain LT2.

\textbf{Deletion strain construction.} Primers used in this study were
synthesized by Integrated DNA Technologies, Inc. (IDT, Coralville, IA),
and are listed in Table 2. The\emph{pagR1} ::\emph{kan}
\textsuperscript{+} and\emph{tktDE1} ::\emph{cat} \textsuperscript{+}
markers were engineered as follows: PFU Ultra II Fusion DNA polymerase
(Stratagene) was used to amplify flanking regions of plasmids pKD3
(\emph{cat} \textsuperscript{+}marker) or pKD4 (\emph{kan}
\textsuperscript{+} marker) using primers designed with 36 bp of
overlapping region to the beginning or the end of\emph{pagR} or
\emph{tktD-tktE} genes. Polymerase chain reaction (PCR) amplicons were
visualized by post-staining with ethidium bromide (0.5 mg /mL) for 10
min. Products were cleaned with the Wizard? SV gel and PCR clean up kit
(Promega), and \textasciitilde{}200 ng of PCR product was electroporated
into \emph{S.} Typhimurium strain JE6692 (\emph{metE205}[?]\emph{araB9} /
pKD46 \emph{bla} \textsuperscript{+}) using a 0.2-cm electroporation
cuvette (MidiSci) and a microPulser electroporator (Bio-Rad
Laboratories) on Ec2 setting. Cells were recovered by incubation at 37
\selectlanguage{ngerman}ºC with shaking and plated on lysogeny broth (LB, Difco) agar
supplemented with either 12.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL of chloramphenicol for the
\emph{cat} \textsuperscript{+} marker or 25 \selectlanguage{greek}µ\selectlanguage{english}g/mL of kanamycin for the
\emph{kan} \textsuperscript{+} marker. Correct antibiotic insertions
were PCR verified then moved by P22-mediated transduction into strain
JE6583 as described elsewhere (Davis et al., 1980). Colonies were
streaked to isolation and the genomes of isolated colonies were
sequenced using Sanger sequencing technology to verify correct insertion
location and sequence using primers flanking genes of interest.

\textbf{Construction of \emph{pagR3::lacZY} chromosomal fusion
strain.}The kanamycin resistance cassette was resolved from strain
JE21107 (\emph{pagR1} ::\emph{kan} \textsuperscript{+}, yielding
\selectlanguage{greek}Δ\selectlanguage{english}\emph{pagR2} ) using pCP20 and a method described elsewhere (Datsenko
and Wanner, 2000). The \emph{lacZY\textsuperscript{+}} fusion was
created by FLP mediated integration of pKG136 (Ellermeier et al., 2002).
Briefly, pCP20 was transformed into the resolved \selectlanguage{greek}Δ\selectlanguage{english}\emph{pagR} strain
(JE24887). Plasmid pKG136 was transformed into the resulting strain and
cells were recovered overnight in LB at 37 \selectlanguage{ngerman}ºC. The next morning cells
were plated on LB agar with 50 \selectlanguage{greek}µ\selectlanguage{english}g/mL kanamycin to select for
integration. Correct\emph{lacZY\textsuperscript{+}}
\emph{kan\textsuperscript{+}} insertions were PCR verified then moved by
P22-mediated transduction into strain JE6583 using a described protocol
(Davis et al., 1980).

\textbf{Table 2. Primers and DNA probes used in this
study\textsuperscript{1}.}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{Primer Name}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{Primer Sequence 5' 3'}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{EMSA primers}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{EMSA primers}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_\emph{pagR}\_EMSA (5' 6-FAM label)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
CGCAAATTTCCAGCGCCTGCG\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_\emph{pagR}\_EMSA (5' HEX label)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
TACACCGGCAAGTTGGGCTAC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{Deletion, Cloning, and RT-qPCR primers}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
5'\_\emph{pagR}\_wanner\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
ATGTCGATACCCCGTAAACGGCGCAGTA CCGGTAAAGTGTAGGCTGGAGCTGCTTC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
3'\_\emph{pagR}\_wanner\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
TTAGCAGCTTTCCCGCATACATAAACTG GAGGCGATCATATGAATATCCTCCTTAG\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_\emph{pagR}\_BspQI\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NNGCTCTTCNTTCATGTCGATACCCCGTAAACG\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_\emph{pagR}\_BspQI\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NNGCTCTTCNTTATTAGCAGCTTTCCCGCATAC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_\emph{tktD}\_BspQI\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NNGCTCTTCNTTCATGAACGTAACCGAAA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_\emph{tktE}\_BspQI\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NNGCTCTTCNTTATTAAAGCAGTGATTTCG\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_\emph{tktA}\_BspQI\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NNGCTCTTCNTTCATGTCCTCACGTAAAGAGCTTG\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_\emph{tktA}\_BspQI\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NNGCTCTTCNTTATTACAGCAGTGCTTTCGCTT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_\emph{tktB}\_BspQI\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NNGCTCTTCNTTCATGTCCCGTAAAGACCTTGC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_\emph{tktB}\_BspQI\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NNGCTCTTCNTTATTAGCCTTTGATGTTTAGCACC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
5'\_\emph{tktD}\_wanner\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
TCACCCAACTGGCGCGCGATATTCGCGT CGCCACGCGTGTAGGCTGGAGCTGCTTC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
3'\_\emph{tktE}\_wanner\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
TTTCGCGGCTTCTACAATCGCTTCGGCGG TGAGTCCCATATGAATATCCTCCTTAG\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_\emph{stm2343}\_qPCR\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
GTTCCGGCCTGGGTAGTAGTT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_\emph{stm2343}\_qPCR\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
CCGATGAAAGATCGGAGTGT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_\emph{tktD}\_qPCR\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
CGATGATATCGCCGGACTAC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_\emph{tktD}\_qPCR\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
CCTCATTGAGCGTCTGCTTC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_\emph{tktE}\_qPCR\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
CTATCGATCGCATGTTGGTG\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_\emph{tktE}\_qPCR\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
CTGCCCATAACGCTCTTTGA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{Operon PCR Primers}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_operon PCR\_1\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
AGCGCCATAAAGAGCTGA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_operon PCR\_1\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
CGATGATATCGCCGGACTAC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_operon PCR\_2\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
TATTCAGCTCTTCGCGCG\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_operon PCR\_2\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
TCCTGCCAGTGCTGTTAC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_operon PCR\_3\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
ACATACCCCCTAGCGG\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_operon PCR\_3\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
GTTCCGGCCTGGGTAGTAGTT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_operon PCR\_4\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
CCGATGAAAGATCGGAGTGT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_operon PCR\_4\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
TTGGGCCATATTATGTATTAGCG\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{DNase 1 Footprinting Primers}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5'\_sequencing template\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
GGTACTGCGCCGTTTAC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_sequencing template\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
GAACAATCGCCGTAAGGTATTC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
5'\_digestion/sequencing (5' 6-FAM label)\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
CGCAAATTTCCAGCGCCTGCG (same as 5'\_\emph{pagR}\_EMSA)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
3'\_digestion/sequencing (5' HEX label)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
TGTCGTTCTTTGCTTCGCATGAC\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Plasmid construction for complementation of function and gene
overexpression.} All plasmids used here are listed in Table 3.

\textbf{Table 3. Plasmids used in this study}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Plasmid} & \textbf{Genotype} & \textbf{Description} &
\textbf{Reference/source\textsuperscript{*}}\tabularnewline
pCV1 & \emph{araC\textsuperscript{+} bla\textsuperscript{+}} &
P\emph{\textsubscript{araBAD}} expression vector & (VanDrisse and
Escalante-Semerena, 2016)\tabularnewline
pTEV19 & \emph{bla\textsuperscript{+}} & Overexpression vector for
MBP-H\textsubscript{6}- \emph{N}-terminally tagged rTEV-cleavable
protease & (VanDrisse and Escalante-Semerena, 2016)\tabularnewline
pPagR7 & \emph{pagR\textsuperscript{+} bla\textsuperscript{+}} &
\emph{pagR}\textsuperscript{+} (\emph{stm2345}\textsuperscript{+})
cloned into pCV1 &\tabularnewline
pTktC1 & \emph{tktD\textsuperscript{+} tktE\textsuperscript{+}
bla\textsuperscript{+}} & \emph{tktD}\textsuperscript{+} and
\emph{tktE}\textsuperscript{+} cloned into pCV1 &\tabularnewline
pTktA1 & \emph{tktA\textsuperscript{+} bla\textsuperscript{+}} &
\emph{tktA}\textsuperscript{+} cloned into pCV1 &\tabularnewline
pTktB1 & \emph{tktB\textsuperscript{+} bla\textsuperscript{+}} &
\emph{tktB}\textsuperscript{+} cloned into pCV1 &\tabularnewline
pPagR-8 & \emph{pagR\textsuperscript{+} bla\textsuperscript{+}} &
\emph{pagR} \textsuperscript{+} \emph{(stm2345\textsuperscript{+})}
cloned into pTEV19 &\tabularnewline
pCP20 & \emph{flp\textsuperscript{+} bla\textsuperscript{+}
cat\textsuperscript{+}} & FLP expression plasmid, temperature sensitive
above 37 \selectlanguage{ngerman}°C & (Cherepanov and Wackernagel, 1995)\tabularnewline
pKG136 & \emph{lacZY\textsuperscript{+} kan\textsuperscript{+}} & For
FLP integration of \emph{lacZY\textsuperscript{+}
kan\textsuperscript{+}} cassette at FLP-resolved deletion sites & J. M.
Slauch\tabularnewline
\bottomrule
\end{longtable}

The high-efficiency cloning method of BspQI digestion described
elsewhere (Galloway et al., 2013, VanDrisse and Escalante-Semerena,
2016) was used to clone \emph{pagR} into plasmids pCV1 and pTEV19,
and\emph{tktD-tktE} , \emph{tktA} and \emph{tktB} into pCV1. All genes
cloned were PCR amplified from \emph{S.} Typhimurium strain JE6583
genomic DNA using PFU Ultra II Fusion DNA polymerase and PCR products
were visualized as described above. PCR products were cleaned using the
Promega Wizard? SV gel and PCR clean up kit, and cloning using the
method cited above. After transformation into \emph{E. coli} DH5\selectlanguage{greek}α \selectlanguage{english}cells
and colony PCR screening of correctly ligated plasmids, plasmids were
isolated using the Wizard? Plus SV miniprep kit (Promega). Sanger DNA
sequencing was performed by Eton Bioscience to rule out mutations and
confirm insertions.

\textbf{Culture media, chemicals, and \emph{in vivo} growth
analyses.}Growth behavior studies were conducted as follows: i) starter
bacterial cultures were grown from a single colony in nutrient broth
(NB, Difco) with overnight shaking at 180 rpm at 37 \selectlanguage{ngerman}ºC; ii) after
\textasciitilde{}16 h of growth, cells
(\textasciitilde{}10\textsuperscript{8} cfu) were sub-cultured into
no-carbon essential (NCE) minimal medium (Berkowitz et al., 1968)
containing MgSO\textsubscript{4} (1 mM), Wolfe's trace minerals (Balch
and Wolfe, 1976), ampicillin (100 \selectlanguage{greek}µ\selectlanguage{english}g/mL), L-(+)-arabinose (concentration
stated in figure legends), with sodium succinate (30 mM) as the sole
carbon and energy source. Inoculum used was routinely 1\% (v/v) of the
final volume of the culture. Growth studies were performed in 96-well
polystyrene (Falcon) microtiter plates with each well containing 198 \selectlanguage{greek}µ\selectlanguage{english}L
of fresh medium and a 2-\selectlanguage{greek}µ\selectlanguage{english}L inoculum. Microtiter plates were incubated at
37 \selectlanguage{ngerman}ºC shaking continuously inside a PowerWave microtiter plate reader
(Bio-Tek Instruments). Density of cells was monitored at 630 nm and data
were analyzed using Prism 9 (GraphPad).

\textbf{RNA isolation.} Strains JE22070 (\emph{pagR}
\textsuperscript{+}/ vector), JE21566 (\emph{pagR1}
::\emph{kan\textsuperscript{+}} / vector), JE21577 (\emph{pagR1}
::\emph{kan\textsuperscript{+}} / pPagR) were grown overnight in
triplicate in nutrient broth (2 mL; NB, Difco) with shaking at 37 ºC.
After incubation, cultures were diluted 1:100 into 5 mL of fresh
lysogeny broth rich medium supplemented with L-(+)-arabinose (100 \selectlanguage{greek}µ\selectlanguage{english}M).
Cultures were grown shaking at 37 \selectlanguage{ngerman}ºC to an optical density of 0.5 at 600
nm, then 5 mL of each sample were quickly centrifuged in 15-mL Falcon
tubes at 4000 x \emph{g} , supernatant was removed, and pellets were
flash-frozen in liquid nitrogen and kept on dry ice. RNA was isolated
following the RNAsnap\textsuperscript{TM}protocol (Stead et al., 2012).
Pellets were re-suspended in 150 \selectlanguage{greek}µ\selectlanguage{english}L of boil solution
(ethylenediaminetetraacetic acid (EDTA, 18 mM), SDS (0.025\%, w/v)
formamide (95\%, v/v; RNA grade), 2-mercaptoethanol (1\% v/v) in
RNase-free water) and were vortexed vigorously to break up the cell
pellet. Pellets were incubated at 95 \selectlanguage{ngerman}ºC for 7 min and centrifuged at
16,000 x \emph{g} for 5 min at room temperature; 100 \selectlanguage{greek}µ\selectlanguage{english}L of supernatant
was transferred to a fresh tube. A sodium acetate/ethanol RNA
precipitation was then conducted by the addition of 400 \selectlanguage{greek}µ\selectlanguage{english}L of RNase-free
water, 50 \selectlanguage{greek}µ\selectlanguage{english}L of sodium acetate (3 M, pH 5.2: final concentration of 0.3
M), and finally 1.65 mL of ice-cold absolute ethanol (100\%), with
mixing briefly before the addition of the next reagent. The mixture was
incubated at -80 \selectlanguage{ngerman}ºC for one hour, centrifuged at 16,000 x \emph{g} for
30 min at 4 ºC, and ethanol was decanted. Ethanol (300 \selectlanguage{greek}µ\selectlanguage{english}L of cold 70\%
v/v) was added, and pellets were centrifuged at 8,000 x \emph{g} for 5
min at 4 \selectlanguage{ngerman}ºC in an Eppendorf 5415D centrifuge. Ethanol was removed and
pellets were allowed to dry. RNA pellets were re-suspended in RNase-free
water. Subsequent RNase-free DNase I treatment was conducted using the
Ambion Turbo DNA-free kit according to manufacturer's instructions
(ThermoFisher Scientific). After DNA cleavage, a final sodium
acetate/ethanol precipitation was performed as described above, except
using 360 \selectlanguage{greek}µ\selectlanguage{english}L of water, 50 \selectlanguage{greek}µ\selectlanguage{english}L of 3 M sodium acetate, and 1.5 mL of cold
100\% ethanol. After overnight incubation at -80 \selectlanguage{ngerman}ºC, RNA was centrifuged
at 16,000 x \emph{g} for 30 min, then washed with 300 \selectlanguage{greek}µ\selectlanguage{english}L of cold 70\%
ethanol (v/v). Ethanol was decanted, and RNA pellets were dried for 20
min at room temperature. RNA was resuspended in 100 \selectlanguage{greek}µ\selectlanguage{english}L of water. A small
sample of each preparation was used for quantification with the RNA
Broad Range (BR) Assay kit by Qubit on a Qubit 4 fluorometer. A small
amount of each preparation was also tested for quality and integrity
using the Qubit RNA IQ Assay. Primers for qPCR were designed using
Primer3 (Untergasser et al., 2012, Koressaar and Remm, 2007, Koressaar
et al., 2018)

\textbf{cDNA synthesis and real-time quantitative polymerase chain
reaction (RT-qPCR).} Total RNA (972 ng) from each sample was used for
the synthesis of cDNA using the iScript\textsuperscript{TM} cDNA
synthesis Kit from Bio-Rad Laboratories according to manufacturer's
protocol. Each cDNA reaction was then diluted to 7.5 ng/\selectlanguage{greek}µ\selectlanguage{english}L and used as
template for PCR. For real-time PCR, 20 \selectlanguage{greek}µ\selectlanguage{english}L reactions were prepared with
10 \selectlanguage{greek}µ\selectlanguage{english}L of 2X FastSYBR Green master mix (Applied Biosystems), 500 nM of
each gene-specific primer (1 \selectlanguage{greek}µ\selectlanguage{english}L of 10 \selectlanguage{greek}µ\selectlanguage{english}M primer stock), and 15 ng of
cDNA (2 \selectlanguage{greek}µ\selectlanguage{english}L of 7.5 ng/\selectlanguage{greek}µ\selectlanguage{english}L cDNA). The real-time PCR reaction was performed
using a 7500 Fast real-time PCR system (Applied Biosystems). The
threshold cycle value of \emph{gyrB} were checked first to ensure it was
optimal for use as reference genes for these strains under the
conditions chosen for RT-qPCR. Cycle threshold (C\textsubscript{T}) data
were normalized to the \emph{gyrB} gene. These normalized values
([?]C\textsubscript{T}) were transformed using the
2(e-[?]C\textsubscript{T})/10\textsuperscript{-6} method (Livak and
Schmittgen, 2001), and were reported as the gene expression ratio
(2\^{}[?][?]C\textsubscript{T}) of the mutant strains/the parent strain
(JE22070 \emph{pagR} \textsuperscript{+}). Mean
2\^{}[?][?]C\textsubscript{T}values were used to calculate the standard
error of the mean (SEM) using Prism9 (GraphPad) from three biological
replicates that were each tested in technical triplicate. Differences in
2\^{}[?][?]C\textsubscript{T}between strains were compared using Welch's
\emph{t} -test with Prism9 software.

\selectlanguage{greek}\textbf{β-Galactosidase assays.}\selectlanguage{english} Plasmids pCV1 and pPagR7 were
independently transformed into JE27072 (\emph{pagR}
::\emph{lacZY\textsuperscript{+} kan\textsuperscript{+}} ). Three
independent colonies of each strain were grown overnight in 2 mL of NB
plus ampicillin (100 \selectlanguage{greek}µ\selectlanguage{english}g/mL), then sub-cultured 1:100 into 5 mL of LB
plus ampicillin (100 \selectlanguage{greek}µ\selectlanguage{english}g/mL) and arabinose (100 \selectlanguage{greek}µ\selectlanguage{english}M). Cells were grown
shaking at 180 rpm at 37C until an OD\textsubscript{600} nm of 0.4-0.6,
and \selectlanguage{greek}β-\selectlanguage{english}galactosidase units were measured as described (Miller and
Hershberger, 1984).

\textbf{Operon PCR.} As described above, total RNA was isolated from
strain JE21107 (\emph{pagR1} ::\emph{kan\textsuperscript{+}} ) and was
used to generate cDNA. cDNA and genomic DNA isolated from strain JE21107
were used in PCR reactions containing Green GoTaq (Promega) master mix
to amplify overlapping genes within the \emph{stm2340}
-\emph{stm2344}operon. Primer pairs are listed in Table S2.

\textbf{Purification of PagR protein.} PagR protein was purified to
homogeneity from plasmid pPagR-8 encoding a PagR protein with a maltose
binding protein-hexahistidine (MBP-H6) tag fused to its N terminus. The
tag was removed after incubation with recombinant tobacco etch virus
(rTEV) protease since the plasmid used to produce
MBP-H\textsubscript{6}-PagR (pTEV19) contained an rTEV protease cleavage
site (VanDrisse and Escalante-Semerena, 2016). A sample (10 mL) of an
overnight culture of \emph{E. coli} C41 (\selectlanguage{greek}λ\selectlanguage{english}DE3) / pPagR-8 strain was used
to inoculate one liter of lysogeny broth containing 100 \selectlanguage{greek}µ\selectlanguage{english}g/mL of
ampicillin. Cells were grown shaking at 125 rpm at 37 \selectlanguage{ngerman}°C until the
culture reached an optical density at 600 nm of 0.7. Expression of genes
of interest encoded by the plasmids was induced by the addition of
isopropyl \selectlanguage{greek}β-\selectlanguage{english}D-1-thiogalactopyranoside (IPTG, 0.25 mM) followed by
\textasciitilde{}12 h of overnight incubation at 37 \selectlanguage{ngerman}°C. The next morning
cells were harvested by centrifugation at 6,000 x \emph{g} for 15 min
using a refrigerated Beckman-Coulter Avanti J-20-XPI centrifuge equipped
with a JLA 8.1 rotor. Cell pellets were resuspended in 20 mL of buffer A
{[}(4-(2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES) buffer (50
mM, pH 7.5 at 4 ºC) containing NaCl (0.5 M), glycerol (20\% v/v), and
imidazole (20 mM){]} and were sonicated thrice for 30-s intervals, and
during each interval, sonication was on for 2 s and off for 2 s, at 60\%
amplitude. The resulting whole-cell lysates were centrifuged for 30 min
at 40,000 x \emph{g} and the supernatants were filtered with a 0.45-\selectlanguage{greek}µ\selectlanguage{english}m
filter (VWR) to remove large particulates. Each filtered lysate was
applied onto a 1-mL nitrilotriacetic acid (NTA) affinity chromatography
column pre-equilibrated with buffer A. Fractions were collected by
gravity at 4 \selectlanguage{ngerman}°C. The purification was performed as follows: After all
the lysate was loaded onto the column, the column was washed with 10
column volumes (CV, \emph{i.e.,} 10 mL) of buffer A, seven CV
(\emph{i.e.,} 7 mL) of buffer A containing 4\% elution buffer B {[}HEPES
buffer (50 mM, pH 7.5 at 4 ºC), NaCl (0.5 M), glycerol (20\% v/v), and
imidazole (0.5M){]}, and finally, MBP-H\textsubscript{6}-PagR was eluted
in two fractions, first with one CV (\emph{i.e.,} 1 mL) of 100\% elution
buffer B, and the second fraction being four CV (\emph{i.e.,} 4 mL) of
100\% elution buffer B. Both elution fractions were pooled and
MBP-H\textsubscript{6}-PagR was cleaved with rTEV protease at a 1 mg
1:100 rTEV:PagR protein ratio while dialyzing at room temperature in
HEPES buffer (50 mM, pH 7.5 at 4 ºC) containing NaCl (0.5 M), glycerol
(10\% v/v), and dithiothreitol (DTT, 1 mM). Cleaved protein was dialyzed
twice more at 4 °C in buffer A. Cleaved PagR protein was loaded again
onto a 2-mL NTA column to remove MBP-His and rTEV protease, both of
which were fused to a hexahistidine tag. Tag-less PagR protein did not
interact with the NTA resin and was collected in the flow-through
fraction. To further remove MBP from PagR, flow-through fractions from
the second NTA purification were run over a 1-mL amylose resin to remove
contaminating MBP. Pure, tag-less PagR protein was dialyzed overnight
against HEPES buffer (50 mM, pH 7.5 at 4ºC) containing NaCl (150 mM) and
glycerol (20\% v/v) at 4\selectlanguage{english}@C. Fifteen mL of dialyzed PagR solution was
dispensed into Eppendorf microcentrifuge tubes, flash frozen in liquid
nitrogen and stored at -80@\selectlanguage{english}C until used. Protein concentration was
determined using the Qubit\textsuperscript{TM} Protein Assay Kit
(ThermoFisher) and the Qubit\textsuperscript{TM} 4 fluorometer.

\textbf{Electrophoretic mobility shift assays (EMSAs).} EMSAs were
performed to quantify PagR binding to DNA. EMSAs were performed as
follows: purified PagR protein was incubated at 0, 0.5, 1.0, 2.5, 5.0
and 10.0 pmol of protein with 0.5 pmol of a 5(6)-carboxyfluorescein
(5(6)-FAM) and hexachlorofluorescein (HEX)-labeled DNA probe (1: from
2,457,048 to 2,457,454 nt of the chromosome; 406 nt, 2: from 2,457,048
to 2,457,339 nt of the chromosome; 291 nt). EMSA buffer {[}HEPES buffer
(50 mM, pH 7.5 at 4\selectlanguage{ngerman}ºC) containing NaCl (150 mM), and glycerol (10\%
v/v){]} was added to the reaction mixture (total volume = 25 \selectlanguage{greek}µ\selectlanguage{english}L) and DNA
and protein were incubated at room temperature for 40 min. During
incubation, a 7.5\% Tris-Boric acid-EDTA (TBE) polyacrylamide gel was
pre-developed at 100 V for 40 min in 0.5X TBE buffer at 4@\selectlanguage{english}C. After
incubation, 5 \selectlanguage{greek}µ\selectlanguage{english}L of glycerol (50\% v/v) was added to the reaction
mixtures, and 20 \selectlanguage{greek}µ\selectlanguage{english}L of each reaction mixture was resolved by the
polyacrylamide gel. A lane of xylene cyanol and bromophenol blue dye was
added as a tracking indicator, and the gel was run until bromophenol
blue reached the bottom of the gel. The gel was imaged using a Typhoon
Trio Imager (GE Healthcare) at 525 nm with the 488 (Blue) filter.

\textbf{DNase I footprinting.} The promoter region of \emph{stm2344}
and\emph{pagR} (from 2,457,048 to 2,457,339 nt of the chromosome; 291
nt) was PCR amplified using a 5(6)-FAM-labeled primer and a HEX-labeled
primer from JE6583 genomic DNA. The product was purified with the
Wizard? SV Gel and PCR Cleanup System (Promega). 7.5 pmol of
5(6)-FAM/HEX-labeled probe was incubated with either no PagR protein or
75 pmol PagR for 40 min at room temperature in 250 \selectlanguage{greek}µ\selectlanguage{english}L EMSA Buffer.
Twenty-five ng of DNase I (Sigma) was added to each reaction and
incubated for 5 min at room temperature. DNase was heat inactivated at
80\selectlanguage{ngerman}°C for 10 min. Digested fragments were purified with the Wizard\selectlanguage{english}? SV
Gel and PCR Cleanup System (Promega), eluted from the column with 30 \selectlanguage{greek}µ\selectlanguage{english}L
diH\textsubscript{2}O, and diluted 1:2 in diH\textsubscript{2}O for
analysis. Fragment analysis by capillary electrophoresis was performed
at the University of Illinois DNA Core Sequencing facility using the
Applied Biosystems 3730xl DNA Analyzer. The results were processed with
GeneMapper6 and aligned to the sequencing results to determine the
protected region(s).

\textbf{Dideoxy Sanger sequencing.} The promoter region of\emph{stm2344}
/ \emph{pagR} (from 2,456,991 to 2,457,412 nt of the chromosome; 421 nt)
was PCR amplified from JE6583 genomic DNA and purified with the Wizard?
SV Gel and PCR Cleanup System (Promega) to create a template for dideoxy
termination sequencing. This template was sequenced using the USB\selectlanguage{ngerman}®
ThermoSequenase Cycle Sequencing Kit (Affeymetrix) with the HEX and
5(6)-FAM labeled primers used to generate the digested fragment.
Sequencing was performed following the manufacturer's instructions for
dideoxy termination sequencing using 2pmol of primer, 200ng template,
and 60 cycles for each reaction. Samples were diluted 1:2 in deionized
water and analyzed at the University of Illinois DNA Core Sequencing
facility using the Applied Biosystems 3730xl DNA Analyzer. The resulting
electropherograms were analyzed with GeneMapper\selectlanguage{english} Software 6 (Thermo
Fisher Scientific).

\textbf{Acknowledgements.} This work was supported by NIH grant
R35-GM130399 to J.C.E.-S. The authors thank the DNA Services Lab of the
Roy J. Carver Biotechnology Center of The University of Illinois at
Urbana-Champaign for the performance of the DNAse I Footprinting
Analysis.

\textbf{Conflict of interest statement.} The authors have no conflict of
interest to declare.

\textbf{Data availability.} All the data generated by these studies is
reported in this paper and its Supplementary Material file.

\textbf{ORCID}

\emph{Anastacia R. Parks} : 0000-0003-0425-0106

\emph{Regan D. McCormick} : 0000-0001-6342-0051

\emph{Jordan A. Byrne} : 0000-0002-9480-6587

\emph{Jorge C. Escalante-Semerena} : 0000-0001-7428-2811

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\textbf{Supporting Information}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{File Name} & \textbf{Description}\tabularnewline
\midrule
\endhead
Figure S1, 44.2MB tif file & \textbf{PagR purified to \textgreater{}95\%
homogeneity.} PagR was purified as described in \emph{Experimental
procedures} for Electrophoretic Mobility Shift Assays and DNase 1
footprinting. Serial 1:2 dilutions of the final preparation were
electrophoresed through a 12\% acrylamide SDS-PAGE gel, and percent
purity of the 44.8 pmol lane was calculated using the TotalLab TL 100
program. Valley to valley background correction was employed, and the
final percent purity of PagR was calculated to be 96.9\%. MM, molecular
markers.\tabularnewline
Figure S2, 53.6 MB tif file & \textbf{PagR generates the same banding
pattern when assayed against two probes of the \emph{stm2344 -- pagR}
promoter region.} Electrophoretic Mobility Shift Assays against the
probe spanning 2,457,048 nt to 2,457,454 nt of the chromosome (left box,
shown in Fig. 4) and the probe spanning from 2,457,048 nt to 2,457,339
nt of the chromosome (right box, used for DNase footprinting in Fig. 6).
0.5 pmol of fluorescent DNA probe was used for each assay. There are two
clear mobility shifts for both probes, even with 20:1 PagR:probe
ratio.\tabularnewline
Figure S3, 113.8 MB tif file & \textbf{Identification of PagR binding
sites in the promoter region of the \emph{stm2344-stm2340} operon.}
\textbf{A.} Combined electropherograms from dideoxy termination Sanger
sequencing of the \emph{stm2344} promoter. Each trace is colored based
on traditional presentation of BigDye termination: adenine is green,
thymine is red, cytosine is blue, and guanine is black. \textbf{B.}
DNase I digestion of P\emph{\textsubscript{stm2344}} with (black trace)
or without (orange trace) PagR present. Protected bases were identified
by aligning the sequencing traces (\textbf{A}) with the digestion traces
(\textbf{B}).\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{english}
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