The non-motile phenotype of Salmonella hha ydgT mutants is mediated through PefI-SrgD
© Wallar et al; licensee BioMed Central Ltd. 2011
Received: 11 February 2011
Accepted: 20 June 2011
Published: 20 June 2011
Two ancestral nucleoid-associated proteins called Hha and YdgT contribute to the negative regulation of several virulence-associated genes in Salmonella enterica serovar Typhimurium. Our previous work showed that Hha and YdgT proteins are required for negative regulation of Salmonella Pathogenicity Island-2 and that hha ydgT double mutants are attenuated for murine infection. Interestingly, hha ydgT mutant bacteria exhibited a non-motile phenotype suggesting that Hha and YdgT have a role in flagellar regulation.
In this study we show that the non-motile phenotype of hha ydgT mutants is due to decreased levels of the master transcriptional regulator FlhD4C2 resulting in down-regulation of class II/III and class III flagellar promoters and lack of surface flagella on these cells. The horizontally acquired pefI-srgD region was found to be partially responsible for this phenotype since deletion of pefI-srgD in a hha ydgT deletion background resulted in transient restoration of class II/III and III transcription, expression of surface flagella, and motility in the quadruple mutant.
These data extend our current understanding of the mechanisms through which Hha and YdgT regulate flagellar biosynthesis and further describe how S. Typhimurium has integrated horizontal gene acquisitions into ancestral regulatory networks.
The pathogenic nature of Salmonella enterica has been shaped by the horizontal acquisition of virulence determinants [1, 2]. In Salmonella enterica serovar Typhimurium (S. Typhimurium), many virulence genes are organized in mobile elements such as pathogenicity islands, prophages, and the Salmonella virulence plasmid [3, 4]. The increased pathogenic capacity conferred by such genes is dependent on their integration into ancestral regulatory networks of the cell, which can be accomplished by regulatory evolution following horizontal gene transfer .
The Hha/YmoA family of small nucleoid-associated proteins in Enterobacteriaceae  can participate in fine-tuning virulence gene expression in response to environmental cues [6, 7]. For example, YmoA regulates expression of Yop proteins, YadA adhesin, Yst enterotoxin and invasin in Yersinia enterocolitica [7–9]. Hha negatively regulates the α-hemolysin genes hlyCABD in Escherichia coli , hilA encoded within Salmonella pathogenicity island 1 (SPI-1) in S. Typhimurium  and the locus of enterocyte effacement in enterohemorrhagic E. coli . A third member, YdgT, similarly represses hlyCABD in E. coli . We and others have shown that Hha and YdgT are repressors of the type III secretion system (T3SS) encoded in Salmonella Pathogenicity island 2 (SPI-2), where they provide an important negative regulatory input required for virulence [14–16]. Within their role as modulators of gene expression, Hha and YdgT repress other genes in horizontally acquired regions in Salmonella including the pathogenicity islands SPI-1 through SPI-5 and genes on the Salmonella virulence plasmid .
Flagellar-based motility and surface flagellar expression is abolished in hha ydgTmutants
Transcriptional activity of class II/III and III promoters is decreased in a hha ydgTmutant
Loss of the fimbrial regulators PefI-SrgD restores motility in a hha ydgTbackground
Class II/III and class III promoters are transiently activated upon loss of PefI-SrgD in Δhha ΔydgTbacteria
We have shown that Hha and YdgT positively regulate flagellar biosynthesis through their influence on the horizontally acquired flagellar regulators PefI-SrgD. The ability of Hha and YdgT to act as positive regulators is manifested only in the presence of both proteins, as single deletions of hha and ydgT had no apparent effect on flagellar biosynthesis. A similar phenomenon has been observed in the regulation of α-haemolysin production in E. coli. Loss of both Hha and YdgT was required to dramatically de-repress α-haemolysin production which correlated with the ability of YdgT to attenuate the hha mutant phenotype . Similarly, Hha and YdgT may be able to compensate for any effect on flagellar biosynthesis in the single deletion mutants making it difficult to discern their individual roles in flagellar biosynthesis regulation.
PefI-SrgD were recently identified as negative regulators of flagellar gene expression as they inhibit class I activation at the top of the flagellar biosynthesis transcriptional hierarchy . PefI-SrgD is located within the pef fimbrial operon on the Salmonella virulence plasmid and PefI acts to regulate pef fimbriae expression [25, 26]. Pef fimbriae are involved in bacterial adherence and fluid accumulation in the murine small intestine . Phylogenetic data indicates that S. Typhimurium acquired pef as part of the serovar-specific virulence plasmid  which carries variable genetic elements required for virulence, fimbriae synthesis, plasmid transmission, innate immune resistance and antibiotic resistance [29, 30].
The dual regulatory action of PefI-SrgD on both pef and flagellar promoters is similar to that seen for the regulation of fimbriae and flagella in other pathogens. PapX in uropathogenic E. coli acts to reciprocally regulate the expression of type 1 fimbriae and flagella during urinary tract infection . MrpJ in Proteus mirabilis, an opportunistic urinary tract pathogen, activates MR/P fimbrial production while simultaneously repressing flagellar expression . FimZ in S. Typhimurium coordinates reciprocal expression of type 1 fimbriae and flagella . The existence of regulatory proteins able to dually control fimbriae and flagella production thus appears as an important evolutionary mechanism allowing tight modulation of adherence or motility phenotypes.
Although deletion of pefI-srgD in hha ydgT mutants de-represses the motility defect by re-establishing expression of surface flagella, it does not fully reconstitute class II/III and class III promoter activity to wild type levels suggesting the existence of other negative flagellar regulators. The protease ClpXP has been shown to degrade FlhD4C2 in S. Typhimurium , which may represent another negative regulatory mechanism in hha ydgT mutants.
The role of PefI-SrgD in the negative regulation of flagellar biosynthesis exemplifies the evolutionary significance of integrating horizontally acquired regulators into ancestral networks. For example, in S. Typhimurium, the horizontally acquired two-component regulatory system SsrA-SsrB regulates ancestral genes throughout the Salmonella genome [5, 35]. In extraintestinal pathogenic E. coli, the horizontally acquired regulator Hfp interacts with the nucleoid-associated protein H-NS to regulate ancestral genes . In Shigella flexneri, Sfh is located on a horizontally acquired virulence plasmid and regulates the expression of the ancestral proteins H-NS and StpA . Thus, horizontal acquisition of regulatory proteins can have a significant impact on ancestral gene expression often by interacting with other regulatory pathways.
We have shown that the non-motile phenotype of Δhha ΔydgT requires the loss of both Hha and YdgT and that this phenotype is partially mediated through PefI-SrgD. These data contribute to our understanding of Hha-and YdgT-dependent flagellar biosynthesis regulation and demonstrate the integration of the horizontally acquired regulators PefI-SrgD into the flagellar biosynthesis network.
Bacterial Strains and Mutant Construction
Bacteria were propagated in Luria-Bertani (LB) broth at 37°C with aeration unless otherwise indicated. Marked, in-frame deletions of clpXP and pefI-srgD were made in Salmonella enterica serovar Typhimurium SL1344 using the λ Red Recombinase method . Generation of Δhha ΔydgT was described previously  and this strain was used to generate mutants incorporating the pefI-srgD deletion using the primers pefI-srgDF: GTG ATA CTT ATC CGG CCT CCG GTC CGC ATT CCA GGC CGG CCA TAT GAA TAT CCT CCT TAG and pefI-srgDR ATT CCG GTT TAT GAG TGA ATC CAT TGT TAC AAA AAT TAT TGT GTA GGC TGG AGC TGC TTC.
Soft Agar Motility Assay
Two μl of overnight culture was inoculated into 0.25% LB Agar motility plates with antibiotic and incubated at 37°C for 6 h.
Wild type and mutant strains were cultured until the optical density at 600 nm (OD600) reached ~ 0.4-0.6. Whole cell lysates were collected and probed using anti-FlhC (1:5000), anti-FlhD (1:2500) and anti-DnaK (1:5000, Stressgen) antibodies. DnaK served as a loading control.
Transmission Electron Microscopy
Flagella were negatively stained using two different methods. In the first method, cells were cultured for 3-6 h. A carbon-stabilized Formvar support on 200-mesh copper TEM grid was floated for 30 seconds on a drop of culture, washed three times with water and stained for 10 seconds using 0.1% uranyl acetate. The second method involved staining copper grid-immobilized cells for 60 seconds with 2% phosphotungstic acid. Images were obtained using a JEOL-1200EX transmission electron microscope at the McMaster University Electron Microscopy Facility. For quantification, overnight cultures were diluted 1:50 or 1:100 in LB media with antibiotic and grown for at least 3 hours under static conditions. Flagella were stained as described above and quantified for at least 100 cells.
Transcriptional Reporter Assays
Wild type cells and the various mutants under study were transformed with the plasmid-based green fluorescent protein (GFP) reporter constructs pP flhD -GFP, pP fliA -GFP, pP fliC -GFP and pP less -GFP published previously . For reporter experiments, strains were either sub-cultured into culture tubes and propagated for 6 h at which point fluorescence intensity and OD600 were measured or strains were sub-cultured into 96-well plates in M9 media containing 0.1% casamino acids and antibiotic and grown with shaking at 37°C at 1080 cycles per minute. Fluorescence intensity and OD600 were measured at 15 minute intervals for 19 h using a Synergy 2 Multi-Mode Microplate Reader (Fisher Scientific Co).
We are grateful to Kazuhiro Kutsukake for providing FlhC and FlhD antibodies, Walid Houry for providing ClpP antiserum, and Brad Cookson for generously providing the GFP reporter constructs used in this study. LEW is supported by an Ontario Graduate Scholarship. BKC is a CIHR New Investigator and recipient of the Early Researcher Award from the Ontario Ministry of Research and Innovation. This work was supported by an operating grant from the Canadian Institutes of Health Research to BKC (MOP 82704).
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