The effect of cell growth phase on the regulatory cross-talk between flagellar and Spi1 virulence gene expression

pre-printThe flagellar regulon controls Salmonella biofilm formation, virulence gene expression and the production of the major surface antigen present on the cell surface: flagellin. At the top of a flagellar regulatory hierarchy is the master operon, flhDC, which encodes the FlhD4C2 transcriptional complex required for the expression of flagellar, chemotaxis and Salmonella pathogenicity island 1 (Spi1) genes. Of six potential transcriptional start-sites within the flhDC promoter region, only two, P1flhDC and P5flhDC, were functional in a wild-type background, while P6flhDC was functional in the absence of CRP. These promoters are transcribed differentially to control either flagellar or Spi1 virulent gene expression at different stages of cell growth. Transcription from P1flhDC initiates flagellar assembly and a negative autoregulatory loop through FlhD4C2-dependent transcription of the rflM gene, which encodes a repressor of flhDC transcription. Transcription from P1flhDC also initiates transcription of the Spi1 regulatory gene, hilD, whose product, in addition to activating Spi1 genes, also activates transcription of the flhDC P5 promoter later in the cell growth phase. The regulators of flhDC transcription (RcsB, LrhA, RflM, HilD, SlyA and RtsB) also exert their control at different stages of the cell growth phase and are also subjected to cell growth phase control. This dynamic of flhDC transcription separates the roles of FlhD4C2 transcriptional activation into an early cell growth phase role for flagellar production from a late cell growth phase role in virulence gene expression

The Effect of Cell Growth Phase on the Regulatory Cross-Talk between Flagellar and Spi1 Virulence Gene Expression

<div><p>The flagellar regulon controls <i>Salmonella</i> biofilm formation, virulence gene expression and the production of the major surface antigen present on the cell surface: flagellin. At the top of a flagellar regulatory hierarchy is the master operon, <i>flhDC</i>, which encodes the FlhD₄C₂ transcriptional complex required for the expression of flagellar, chemotaxis and <i>Salmonella</i> pathogenicity island 1 (Spi1) genes. Of six potential transcriptional start-sites within the <i>flhDC</i> promoter region, only two, P1<i>_flhDC</i> and P5<i>_flhDC</i>, were functional in a wild-type background, while P6<i>_flhDC</i> was functional in the absence of CRP. These promoters are transcribed differentially to control either flagellar or Spi1 virulent gene expression at different stages of cell growth. Transcription from P1<i>_flhDC</i> initiates flagellar assembly and a negative autoregulatory loop through FlhD₄C₂-dependent transcription of the <i>rflM</i> gene, which encodes a repressor of <i>flhDC</i> transcription. Transcription from P1<i>_flhDC</i> also initiates transcription of the Spi1 regulatory gene, <i>hilD</i>, whose product, in addition to activating Spi1 genes, also activates transcription of the <i>flhDC</i> P5 promoter later in the cell growth phase. The regulators of <i>flhDC</i> transcription (RcsB, LrhA, RflM, HilD, SlyA and RtsB) also exert their control at different stages of the cell growth phase and are also subjected to cell growth phase control. This dynamic of <i>flhDC</i> transcription separates the roles of FlhD₄C₂ transcriptional activation into an early cell growth phase role for flagellar production from a late cell growth phase role in virulence gene expression.</p></div

Model depicting the flagellar and Spi1 regulatory circuitry.

<p>RcsB and LrhA inhibit transcription of <i>flhDC</i> at early cell's growth phase. These two regulatory factors inhibit transcription from the P1<i>_flhDC</i> and P5<i>_flhDC</i> promoters. Under proper conditions CRP activates transcription from P1<i>_flhDC</i>. This activation produces enough FlhD₄C₂ to promote synthesis of flagellar proteins required for flagellum assembly and motility of <i>Salmonella</i>. There is a simultaneous activation of the FlhD₄C₂-dependent <i>rflM</i> gene. RflM feedback inhibits any further surge of transcription from P1<i>_flhDC</i>. This effect limits the <i>flhDC</i> expression resulting in differential expression of flagellar class 2 genes. RflM transcription appears to be short-lived as there is a quick decay of <i>rflM</i> transcription and RflM production. The mechanism by which transcription inhibition of <i>rflM</i> happens is unclear. It appears that if RflM expression is maintained, P5<i>_flhDC</i> transcription is not activated. On the other hand, FlhD₄C₂ promotes <i>fliZ</i> transcription, whose product activates HilD at the posttranslational level. HilD positively activates transcription from P5<i>_flhDC</i> and inhibits P1<i>_flhDC</i> transcription through the activation of <i>rtsB</i>. HilD also activates transcription of <i>slyA</i>, whose product inhibits transcription from P5<i>_flhDC</i>. In the wild-type strain, <i>flhDC</i> transcription from P5<i>_flhDC</i> does not affect motility, where only a threshold of <i>flhDC</i> transcription is required to promote motility. The timing at which transcription of <i>flhDC</i> takes place appears to be a signal for FlhDC regulation of motility. However, P5<i>_flhDC</i> also is able to promote motility in the absence of P1<i>_flhDC</i> and when appropriate conditions are met such overexpression of HilD that allows for an early transcription of <i>flhDC</i> operon from P5<i>_flhDC</i>.</p

Growth phase dependent transcription of the <i>flhDC</i> operon promoter in <i>Salmonella enterica</i> serovar Typhimurium is controlled by LrhA, RcsB, RflM, HilD, SlyA and RtsB.

<p>(<b>A</b>) Diagram depicting a duplicated chromosomal region that includes fusion of the <i>flhDC</i> promoter region (P<i>_flhDC</i>, a 728 bp upstream of the start codon of <i>flhD</i> and the first 272 nucleotides of <i>flhD</i> coding region) to the luciferase operon of <i>Photorhabdus luminescens</i> in addition to a wild-type <i>flhDC</i> promoter-operon region. (<b>B</b>) A time course plot showing P<i>_flhDC</i>-<i>lux</i> expression at increasing cell density of strain Pwt<i>_flhDC-luxCDBAE-</i>Pwt<i>_flhDCflhD⁺C⁺</i> (TH18684) grown in LB media at 30°C with shaking. Luciferase activity was measured along with the OD595. Plots represent the recorded luciferase activity divided by the OD595. (<b>C & D</b>) Time course plots showing Pwt<i>_flhDC</i>-<i>lux</i> expression at increasing cell density in the absence of <i>flhDC</i> regulators. Individual regulators of <i>flhDC</i> promoter (Pwt<i>_flhDC</i>) transcription were removed by deletion in the Pwt<i>_flhDC-luxCDBAE-</i>Pwt<i>_flhDCflhD⁺C⁺</i> background. Plots for specific individual strains are identified at the right of their corresponding plots (wt = wild-type (TH18684), <i>rcsB⁻</i> = Δ<i>rcsB</i>::<i>tetRA</i> (TH19230), <i>lrhA⁻</i> = Δ<i>lrhA</i>::<i>tetRA</i> (TH18722), <i>rflM⁻</i> = Δ<i>rflM</i>::FCF (TH18716), <i>rtsB⁻</i> = <i>rtsB</i>::T-POP (TH18724), <i>slyA⁻</i> = <i>slyA</i>::T-POP (TH18720) and <i>hilD⁻</i> = Δ<i>hilD</i>::<i>tetRA</i> (TH19654)). (<b>C</b>) Loss of RcsB, LrhA or RflM resulted in increased transcription of the <i>flhDC</i> operon at early growth phase. (<b>D</b>) Effect of removal of virulence-related genes <i>slyA</i>, <i>rtsB</i> or <i>hilD</i> differentially affected <i>flhDC</i> operon transcription. Deletion of either the <i>rtsB</i> or <i>slyA</i> gene resulted in increased <i>flhDC</i> operon transcription once cells reach stationary phase contrary to a deletion in the <i>hilD</i> gene, which resulted in increased <i>flhDC</i> transcription once bacterial cells enter mid exponential phase. The OD595 values are shown at the bottom of the chart. Values are the average of three independent experiments done in duplicate. Error bars represent standard deviation.</p

Effects of mutations in putative transcriptional start-sites within the <i>flhDC</i> promoter region on <i>flhDC</i> operon transcription.

<p>(<b>A</b>) DNA sequence and regulatory elements of the upstream regulatory region of S. <i>typhimurium</i>. Nucleotides are labeled respective to the start of the open reading frame of FlhD. The −10 box of the putative promoters are in bold and their respective transcription start site are indicated by arrowheads as determined by primer extension <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003987#ppat.1003987-Yanagihara1" target="_blank">[13]</a>. The transcriptional factors LrhA, RcsB, RtsB and CRP have been shown to bind directly to the <i>flhDC</i> promoter regulatory region. Experimental evidence and mutations analysis have been performed to delineate the exact binding of LrhA, RcsB and CRP (underlined). The exact binding of the RtsB has not been defined but it has been shown that RtsB binds directly to <i>flhDC</i> promoter region corresponding to a DNA fragment covering from +4 to +104 nucleotides respective to the P1 transcription start site <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003987#ppat.1003987-Ellermeier1" target="_blank">[17]</a>. The direct and exact binding site for SlyA, RflM and HilD transcriptional factors have not been defined yet. (<b>B</b>) DNA sequences of the −10 boxes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003987#ppat.1003987-Yanagihara1" target="_blank">[13]</a> of putative transcriptional start-sites (shown as P1⁻, P2⁻, P3⁻, P4⁻, P5⁻ and P6⁻) and mutant constructs that were made in each of these −10 boxes. The individual transcriptional start-sites promoter mutants were made separately at each single −10 box or were combined together leaving only one functional −10 box out of the six described promoter start-sites (shown as P1⁺, P2⁺, P3⁺, P4⁺, P5⁺ and P6⁺). Charts represent the luciferase activities of the Pwt<i>_flhDC-luxCDBAE</i>-Pwt<i>_flhDCflhD⁺C⁺</i> reporter construct in wild-type and isogenic strains carrying mutations in individual start-site −10 boxes. Cells were grown overnight in LB and diluted 1 to 500 in fresh media, and grown at 30°C with shaking and luciferase activities were recorded at two optical densities (0.5, black bars and 1, grey bars). Charts of luciferase activity in strains with mutations in the P1 (<b>C</b>) and P5 (<b>D</b>) promoters of <i>flhDC</i> operon compared to the wild-type <i>flhDC</i> promoter activity that was set at 100%. Each specific mutation is indicated under their corresponding bars. (<b>E</b>) Graph of luciferase activity in strains harboring only one single wild-type −10 box of the indicated putative promoter. P1⁺ represents a strain that has only a functional P1 promoter while the rest of the promoters are mutated, etc (<b>F</b>) Luciferase activity of a strain with mutations in both P1 and P5 (P1⁻P5⁻), compared to wild type promoter and to a construct with mutations in all six promoters: AP's. Results are the average of three independent experiments done in duplicate. Error bars represent standard deviation.</p

Precise transcriptional regulation of the <i>flhDC</i> operon is growth phase dependent.

<p>Transcription kinetics for the <i>flhDC</i> operon in various mutant backgrounds with the Pwt<i>_flhDC-luxCDBAE</i>-Pwt<i>_flhDCflhD⁺C⁺</i> reporter construct measured in a 96 well plate growth format. The luciferase activity was investigated in seven genetic backgrounds: (<b>A</b>) wild-type (TH18684) empty circles, Δ<i>rcsB</i>::<i>tetRA</i> (TH19230) filled squares, Δ<i>lrhA</i>::<i>tetRA</i> (TH18722) filled diamonds, (<b>B</b>) <b>Δ</b><i>rflM</i>::FCF (TH18716) filled triangle (<b>C</b>) <i>rtsB</i>::T-POP (TH18724) filled circles, <i>slyA</i>::T-POP (TH18720) filled squares and (<b>D</b>) <b>Δ</b><i>hilD</i>::<i>tetRA</i> (TH19654) filled diamonds. The genotypes of the strains are indicated in the left of their plots at the level of their maximum A.U's. Cells from overnight cultures were diluted 1 to 500 in LB and 200 µl was inoculated into 96 well dark plates that were sealed with a breath easy membrane and incubated at 30°C in a plate reader with 5 min orbital shaking at 150 rpm. After a pause of 5 second following shaking, luminescence and OD595 of the inoculated wells were read during 95 second. The luminescence was recorded with a 0.1 s integration time for normalization. Arbitrary units (A.U.) were calculated as luminescence reading divided by OD595. The average at each time point was normalized to the maximum A.U. of the wild-type strain. Each data point represents six experiments performed in triplicate in different days. Error-bars indicate standard deviations. A representative growth curve is shown in the second axis of the plots.</p

Effects of RcsB, LrhA, RtsB and SlyA on transcription of P1<i>_flhDC</i> and P5<i>_flhDC</i>.

<p>For these assays, we compared the transcription from the P1⁻<i>_flhDC</i> (defective in the P1 start-site) and the P5⁻<i>_flhDC</i> (defective in the P5 start-site) promoter constructs. Plots represent luciferase activity divided by the OD595 plotted against the OD595 values shown at the bottom of the chart. (<b>A</b>) RcsB, LrhA and RtsB but not SlyA repressed transcription from the P1<i>_flhDC</i> promoter. Luciferase activity of P5⁻<i>_fhlDC-luxCDBAE-</i>Pwt<i>_flhDCflhD</i>⁺<i>C</i>⁺ transcriptional fusion (P1-expressed) was investigated in five genetic backgrounds: wild-type (TH18895), Δ<i>rcsB</i>::<i>tetRA</i> (TH20237), <i>rtsB</i>::T-POP (TH19976), Δ<i>lrhA</i>::<i>tetRA</i> (TH19974), <i>slyA</i>::T-POP (TH19975). (<b>B</b>) RcsB, LrhA and SlyA but not RtsB are negative regulators of P5<i>_flhDC</i> promoter. Luciferase activity of P1⁻<i>_fhlDC-luxCDBAE-</i>P_wt<i>flhD</i>⁺<i>C</i>⁺ transcriptional fusion (P5-expressed) was measured in wild-type (TH18889), Δ<i>rcsB</i>::<i>tetRA</i> (TH20236), <i>rtsB</i>::T-POP (TH19972), Δ<i>lrhA::tetRA</i> (TH19970) and <i>slyA</i>::T-POP (TH19971)., (<b>C</b>) RcsB inhibits <i>hilD</i> transcription in an <i>flhDC</i> independent manner. Luciferase activities of the P<i>_hilD</i>-<i>luxCDBAE</i> transcriptional fusion in wild-type (<i>rcsC</i>⁺) (TH19425), <i>rcsC</i>::T-POP (TH19687) and Δ<i>fliZ</i>::FCF (TH19690) backgrounds were recorded as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003987#ppat-1003987-g001" target="_blank">Figure 1</a>. FliZ, a post-translational activator of HilD, promotes transcription of the auto-regulated <i>hilD</i> gene. Tetracycline (Tc) was used at 3 µg/ml to induce <i>rcsC</i> transcription in the <i>rcsC</i>::T-POP background resulting in activation of RcsB. Upon RscB acticvation (<i>rcsC</i>::T-POP +Tc), transcription of <i>hilD</i> was abolished. The inhibitory effect of RcsB on <i>hilD</i> transcrption (40-fold) is more dramatic than the four-fold decrease in the absence of FliZ. Results are the average of two independent experiments performed in duplicate. Error bars represent standard deviation. (<b>D</b>) RflM inhibits <i>hilD</i> transcription in an <i>flhDC</i> independent manner. Luciferase activity of strains harboring a <i>hilD</i> transcriptional fusion, P<i>_hilD</i>-<i>luxCDBAE</i>, was measured in four genetic backgrounds, P<i>_araBAD</i>::FCF (TH20541) (Column 1), P<i>_araBAD::rflM</i>⁺ (TH20542) (Column 2) and P<i>_araBAD</i>::FCF P<i>_flhDC</i>::T-POP (TH20543) (Column 3, 5 and 7) and P<i>_araBAD::rflM</i>⁺ P<i>_flhDC</i>::T-POP (TH20544) (Column 4, 6 and 8). P<i>_araBAD::rflM</i>⁺ strains, in the presence of arabinose (Ara and +) leads to the overexpression of <i>rflM and</i> P<i>_araBAD::FCF</i> serves as a control. Addition of tetracycline (Tet and +) to <i>P_flhDC::T-POP</i> strains allows the overexpression of <i>flhDC</i> and in the absence of tetracycline the cells are <i>flhDC⁻</i>. Cells were diluted 1 to 500 from an overnight culture into LB in the presence arabinose, tetracycline or arabinose and tetracycline. 0.2% arabinose (Ara) was added to induce transcription of <i>rflM</i> and 3 µg/ml tetracycline (Tet) to induce transcription of <i>flhDC</i>. At an OD595∼1, the luciferase activity was recorded as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003987#ppat-1003987-g004" target="_blank">Figure 4</a>.</p

Time-dependent transcription of <i>flhDC</i> operon controls motility of <i>Salmonella</i>.

<p>(<b>A</b>) A representative image of motility of the wild-type strain compared to (<b>A</b>) <i>slyA</i>, <i>rtsB</i>, <i>spi</i>1 and <i>hilD</i> null mutants. Null mutations in <i>slyA</i>, <i>rtsB</i>, <i>spi1</i> or <i>hilD</i> does not affect motility compared to the wild-type strain (<b>B</b>) A representative image of motility of the wild-type strain compared to constructs harboring single promoters mutations in the <i>flhDC</i> regulatory region. (<b>C &D</b>) Early transcription of P5<i>_flhDC</i> promotes motility (<b>C</b>) the motility defect of P5⁺ construct (only P5 is active and the promoters P1, P2, P3, P4 and P6 are mutated) was rescued by a mutation that overexpresses <i>hilD</i> (P5⁺<i>hilD</i>up) and (<b>D</b>) transcription of class 2 gene, <i>fliL</i>, of the P5⁺ construct in a wild-type strain compared to its isogenic strain <i>hilD</i>up (mutation that overexpress HilD). ß-galactosidase activity (Miller Units) of a lac fusion to <i>fliL</i> gene was investigated in three genetic backgrounds: wild-type, P5⁺<i>_flhDC</i> and P5⁺<i>_flhDC hilD</i>up strains. Values are average of two experiments done in duplicate at different ODs.</p

The PmrA/PmrB and RcsC/YojN/RcsB systems control expression of the Salmonella O-antigen chain length determinant

The lipopolysaccharide (LPS) is the outermost component of the cell envelope in Gram-negative bacteria. It consists of the hydrophobic lipid A, a short non-repeating core oligosaccharide and a distal polysaccharide termed O-antigen. We report here that the PmrA/PmrB and RcsC/YojN/RcsB two-component systems of Salmonella enterica serovar Typhimurium independently promote transcription of the wzzst gene, which encodes a protein that determines the chain length of the O-antigen. We show that the regulatory proteins PmrA and RcsB footprint partially overlapping regions of the wzz st promoter stimulating transcription from the same start site. Induction of the PmrA/PmrB or RcsC/YojN/RcsB systems increased the fraction of LPS molecules containing 16-35 O-antigen subunits, leading to heightened resistance to serum. The LPS of a rcsB null mutant exhibited an altered mobility in the O-antigen subunits attached to the lipid A-core region when separated on a SDS/PAGE gel, suggesting that RcsB may regulate additional LPS genes. Inactivation of the wzzst gene eliminated the enhanced swarming behaviour exhibited by the rcsB mutant. That multiple regulatory systems control wzzst expression suggests that the Wzzst protein is required under different environmental conditions.Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Mouslim, Chakib. University of Washington; Estados UnidosFil: Groisman, Eduardo A.. University of Washington; Estados Unido