1. Microbiology and Infectious Disease
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The horizontally-acquired response regulator SsrB drives a Salmonella lifestyle switch by relieving biofilm silencing

  1. Stuti K Desai
  2. Ricksen S Winardhi
  3. Saravanan Periasamy
  4. Michal M Dykas
  5. Yan Jie
  6. Linda J Kenney  Is a corresponding author
  1. National University of Singapore, Singapore
  2. Nanyang Technological University, Singapore
  3. University of Illinois-Chicago, United States
Research Article
Cite this article as: eLife 2016;5:e10747 doi: 10.7554/eLife.10747
7 figures and 2 tables

Figures

Figure 1 with 3 supplements
Loss of ssrB but not ssrA decreases Salmonella Typhimuirum biofilms.

(A) The defect in formation of biofilms in the ssrB null was complemented by the overexpression of SsrBc from plasmid pKF104 in trans as measured by crystal violet staining. (B) The typical rdar morphotype of the wild type strain was lost in the ssrB strain as shown on congo red plates. (C) A two day old macrocolony of the ssrB strain is not fluorescent under UV light with Fluorescent Brightener 28. (D) The wild type strain forms thick solid-surface biofilms, while the ssrB strain remains poor for biofilms as monitored for six days by SYTO-9 staining of flow cell biofilms; scale bar = 1 mm. (E) SEM images showing extensive mesh-like network of wild type biofilms and sparse extracellular matrix of the ssrB biofilms; scale bar = 1 µm. (F) The amount of biofilms formed by the wild type strain (solid black bars) increases after 24 hr but the ssrB null (hatched black bars) remains defective up to 84 hr. n = 2, Mean ± SD, p < 0.0001 between wild type and ssrB strains from 36 hr till 84 hr. (G) The amount of cholesterol-attached biofilms formed by the ssrB strain were significantly less than that produced by the wild type. n = 3, Mean ± SD, p < 0.0001. Source data file: Figure 1—source data 1.

https://doi.org/10.7554/eLife.10747.003
Figure 1—source data 1

Source data for crystal violet staining in Figure 1A,F and G.

https://doi.org/10.7554/eLife.10747.004
Figure 1—figure supplement 1
The ssrB mutant is not defective in growth compared to the wild type strain.

Number of colonies formed by the wild type, ssrA, ssrB and D56A strains were the same order of magnitude across all the time points tested. Source data file: Figure 1—figure supplement 1—source data 1.

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Figure 1—figure supplement 1—source data 1

Growth curves of wild type, ssrA, ssrB and D56A strains.

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Figure 1—figure supplement 2
The planktonic sub-population of the ssrB strain was higher by two orders of magnitude compared to the wild type, ssrA and D56A strains at 2 days.

n = 2, Mean ± SD, p < 0.05. Source data file: Figure 1—figure supplement 2—source data 1.

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Figure 1—figure supplement 2—source data 1

Number of cells in the planktonic sub-population of each strain.

https://doi.org/10.7554/eLife.10747.008
Figure 1—figure supplement 3
Total wet weight of the adherent sub-population was decreased by at least 50% in the ssrB strain compared to the wild type, ssrA and D56A strains at 2 days.

n = 2, Mean ± SD, p < 0.05 for the ssrB strain versus ssrA/D56A strains and p = 0.08 for the ssrB strain versus wild type. Source data file: Figure 1—figure supplement 3—source data 1.

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Figure 1—figure supplement 3—source data 1

Total wet weight of the adherent sub-population of each strain.

https://doi.org/10.7554/eLife.10747.010
Phosphorylation of SsrB is not required for biofilm formation.

Amount of biofilms formed as measured by crystal violet staining for (A) Strains ssrAH1, ssrAD, ssrAH2, ssrAH1AcP, ssrADAcP, ssrAH2AcP and (B) D56A SsrB shows similar levels to that of the wild type, and higher than the ssrB mutant. Source data file: Figure 2—source data 1. (C) Beta-galactosidase activity of a sifA-lacZ chromosomal fusion was significantly lower in the ssrB null and the D56A SsrB mutant compared to the wild type. n = 3, Mean ± SD, p < 0.0001. Source data file: Figure 2—source data 2.

https://doi.org/10.7554/eLife.10747.011
Figure 2—source data 1

Source data for crystal violet staining in Figure 2A and B.

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Figure 2—source data 2

Source data for the measurement of beta-galactosidase activity in Figure 2C.

https://doi.org/10.7554/eLife.10747.013
SsrB regulates biofilms by a CsgD-dependent mechanism.

(A) The SPI-2 needle, ssaC and ssaJ mutant strains were not affected in biofilm formation. (B) Over-expression of csgD from a plasmid (pBR328::csgD) in trans rescued biofilm formation in the ssrB mutant, as measured by crystal violet staining, n = 3. Source data file: Figure 3—source data 1. An estimate of csgD expression by (C) Real-time qRT-PCR showed a significant decrease in csgD transcription in the ssrB null, but not in the D56A SsrB and ssrA mutants. rrsA transcript levels were used as control; n = 2, Mean ± SD, p < 0.0001. Source data file: Figure 3—source data 2 and (D) Immunoblot analysis showing the absence of CsgD in the ssrB null strain in two day old biofilms, using GroEL as a loading control.

https://doi.org/10.7554/eLife.10747.014
Figure 3—source data 1

Source data for crystal violet staining in Figure 3A and B.

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Figure 3—source data 2

Source data for Real-time qRT-PCR in Figure 3C.

https://doi.org/10.7554/eLife.10747.016
hns deletion rescues biofilm formation in the ssrB mutant as shown by Crystal violet staining.

(A) The amount of biofilms formed is higher in the wild type, hns and ssrB hns strains compared to the ssrB null, n = 3, Mean ± SD, p < 0.0001. Source data file: Figure 4—source data 1. Macrocolony phenotype (B) ssrB hns forms a highly rugose and dry macrocolony, while the ssrB macrocolony was smooth and mucoidy. SEM imaging (C) ssrB hns biofilms were covered by a thick extra-cellular matrix; scale bar = 2 µm. (D) qRT-PCR: csgD levels were restored in the ssrB hns strain and were higher than the wild type (p < 0.03) and the ssrB mutant (p < 0.003) against rrsA transcripts as a control. Note that the normalized csgD levels in the ssrB null were 0.0009, too low for the scale. n = 2, Mean ± SD. Source data file: Source data file: Figure 4—source data 2.

https://doi.org/10.7554/eLife.10747.017
Figure 4—source data 1

Source data for crystal violet staining in Figure 4A.

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Figure 4—source data 2

Source data for Real-time qRT-PCR in Figure 4D.

https://doi.org/10.7554/eLife.10747.019
Figure 5 with 3 supplements
SsrB binds upstream of csgD.

(A) AFM images of the 755 bp csgD regulatory region (csgD755). (B) At 120 nM SsrB, distinct areas of SsrB binding were visualized as sharp bends (yellow arrows). (C) At 300 nM SsrB, areas of condensation (pink arrows) were observed. (D) Binding of SsrB bends the DNA by an average angle of 82º (for the naked DNA angle, refer to Figure 5—figure supplement 2 and for analysis refer to Supplementary method), Scale bar = 200 nm as in (A). (E) and (F) The SsrB mutant, K179A SsrB, which is defective in DNA binding, was unable to bind csgD755 both at 30 nM or 300 nM; Scale bar = 200 nm.

https://doi.org/10.7554/eLife.10747.020
Figure 5—figure supplement 1
AFM images of the 755 bp csgD regulatory region (csgD755) (A) with 120 nM D56ASsrB and (B) SsrBc.

Distinct areas of binding were visualized as sharp bends (yellow arrows) and as areas of condensation (pink arrows) at 300 nM D56A SsrB (C) and SsrBc (D); Scale bar = 200 nm as in Figure 5A.

https://doi.org/10.7554/eLife.10747.021
Figure 5—figure supplement 2
Bending angle of the naked csgD755 fragment.
https://doi.org/10.7554/eLife.10747.022
Figure 5—figure supplement 3
Electrophoretic mobility shift assay with the 122 bp csgD regulatory region, csgD122, showing a DNA-protein complex in the presence of SsrB.

The K179A SsrB mutant did not bind to csgD122. Addition of competitor unlabelled csgD122 fragment decreased the SsrB-DNA complex as apparent by an increase in free, labelled csgD122.

https://doi.org/10.7554/eLife.10747.023
Figure 6 with 4 supplements
SsrB condenses H-NS bound csgD DNA.

(A) (i) AFM imaging in the presence of 600 nM H-NS shows a straight and rigid filament on csgD755. (ii) Addition of 600 nM SsrB to the H-NS bound csgD DNA resulted in areas of condensation (pink arrows; an ‘SsrB signature’) along with a few areas where the straight H-NS bound conformation persisted (yellow line; an ‘H-NS signature’); Scale bar = 200 nm as in Figure 5A. (B) A model for the mechanism of anti-silencing by SsrB at csgD wherein SsrB likely displaces H-NS from the ends of a stiffened nucleoprotein filament and relieves the blockade on the promoter for RNA polymerase to activate transcription. For details refer to (Winardhi et al., 2015).

https://doi.org/10.7554/eLife.10747.024
Figure 6—figure supplement 1
Liquid AFM imaging of (A) the 755 bp csgD regulatory region.

(B) In the presence of 600 nM H-NS, a straight and rigid filament was observed (yellow line). (C) In the presence of 300 nM SsrB, areas of condensation were evident (pink arrow). (D) Addition of 600 nM SsrB to the H-NS bound csgD DNA resulted in areas of condensation (pink arrows; an ‘SsrB signature’) along with a few areas where the straight H-NS bound conformation persisted (yellow line; an ‘H-NS signature’); Scale bar = 200 nm.

https://doi.org/10.7554/eLife.10747.025
Figure 6—figure supplement 2
SsrB D56A and SsrBc condense H-NS-bound csgD DNA.

AFM imaging in the presence of H-NS shows areas of condensation upon addition of (A) 600 nM D56ASsrB and (B) 600 nM SsrBc; Scale bar = 200 nm as in Figure 5A.

https://doi.org/10.7554/eLife.10747.026
Figure 6—figure supplement 3
SsrB and H-NS form a complex on csgD.

Electrophoretic mobility shift assay with the 122 bp csgD regulatory region, csgD122 (left to right); in the presence of SsrB and H-NS, the DNA-protein complex (*) is super-shifted in the presence of anti-SsrBc serum (**). A DNA-protein complex is also observed when SsrB and H-NS were present alone. Note the absence of any complex in a control reaction with csgD122 and anti-SsrBc, while anti-SsrBc recognizes the SsrB-csgD122 complex.

https://doi.org/10.7554/eLife.10747.027
Figure 6—figure supplement 4
The sequence of the 755 bp csgD regulatory region indicating the H-NS binding region according to Gerstel et al. (2003); and the SsrB binding motif as found by Feng et al. (2004).
https://doi.org/10.7554/eLife.10747.028
DNA tracing and bending angle measurement.

(A) The original AFM image as processed with the Gwyddion software. (B) A Matlab code was used to trace the DNA in the AFM images. The digitized binary line represents the DNA backbone (red line), with the two end points marked by green dots. (C) For the bending angle measurement, a point along the red line was manually selected as the binding location of SsrB on DNA (green asterisk). The red asterisks indicate two points that are 15 nm upstream and downstream from this protein-binding site. Linear interpolation of points scattered along the green and red asterisk was used to plot a straight line between these points (yellow line). The angle between the yellow lines (α) is determined as the bending angle of SsrB; 81 such measurements were made. The same procedure was followed for the control AFM images of csgD755, 327 such measurements were made.

https://doi.org/10.7554/eLife.10747.031

Tables

Table 1

List of Bacterial strains and plasmids.

https://doi.org/10.7554/eLife.10747.029
StrainDescription/NomenclatureReference
wild typeSalmonella enterica serovar Typhimurium strain 14028sLab strain collection
ssrAssrA::TetRA derivative of 14028sThis work
ssrBDW85Don Walthers (originally from Stephen Libby)
ssaCssaC::TetRA derivative of 14028sChakraborty et al. (2015)
ssaJssaJ::TetRA derivative of 14028sHideaki Mizusaki unpublished
D56AD56A SsrB derivative of 14028sThis work
ssrAH1DW748Don Walthers unpublished
ssrADDW749Don Walthers unpublished
ssrAH2DW750This work
ssrAH1 sifA-LacZMade by transducing sifA-lacZ from DW636 to DW748This work
ssrAD sifA-lacZMade by transducing sifA-lacZ from DW636 to DW749This work
ssrAH2 sifA-lacZMade by transducing sifA-lacZ from DW636 to DW750Don Walthers; Lab strain collection
DW636sifA-lacZ at attP site in 14028sDon Walthers; Lab strain collection
DW637ssrB::Km derivative of DW636This work/Don Walthers (lab strain collection)
ssrAH1 AcPackA-pta::Km (from DW142) transduced in DW748This work/Don Walthers (lab strain collection)
ssrAD AcPackA-pta::Km (from DW142) transduced in DW749This work/Don Walthers (lab strain collection)
ssrAH2 AcPackA-pta::Km (from DW142) transduced in DW750This work/Don Walthers (lab strain collection)
hnshns::TetRA derivative of DW636This work/Don Walthers (lab strain collection)
hns ssrBhns::TetRA derivative of DW637This work/Don Walthers (lab strain collection)
Plasmid pKF46D56A His-SsrB pMpM-A5Ω constructFeng et al. (2004)
Plasmid pKF43His-SsrB pMpM-A5Ω constructFeng et al. (2004)
Transformant DW160DH5α harboring His-HNS (S. typhimurium) in pMpM-A5ΩWalthers et al. (2011)
Plasmid pKF104His-SsrBc pMpM-A5Ω constructFeng et al. (2004)
pBR328::csgDcsgD constructProf Iñigo Lasa’s group
Plasmid pRC24K179A His-SsrB pMpM-A5Ω constructCarroll et al. (2009)
Table 2

List of oligonucleotides.

https://doi.org/10.7554/eLife.10747.030
Purpose/nameSequence (5’-3’)
Digf (forward 755bp csgD regulatory region)tgatgaaactccacttttttta
Digr (reverse 755bp csgD regulatory region)tgctgtcaccctggacctggtc
ssrA knockout (forward)atgaatttgctcaatctcaagaatacgctgcaaacatctt ttaagacccactttcacatt
ssrA knockout (reverse)agccgatacggcattttcaatatcagccagcaagaggtcc ctaagcacttgtctcctg
csg1 (forward csgD internal)ggaagatatctcggccggttgc
csg2 (reverse csgD internal)tcagcctagggataatcgtcag
rrsA1 (forward rrsA internal)gcaccggctaactccgtgcc
rrsA2 (reverse rrsA internal)gcagttcccaggttgagcccg
PSsrBF (forward for pKF46)atgaaagaatataagatcttat
PSsrBTR (hybrid reverse for pKF46)ttaatactctaattaacctcattcttcgggcacagttaagtctaagcacttgtctcctg
TSsrBF (forward TetRA-ssrB)acttaactgtgcccgaagaatgaggttaatagagtattaattaagacccactttcacatt
TSsrBR (reverse TetRA- after ssrB stop)catcaaaatatgaccaatgcttaataccatcggacgcccctggctaagcacttgtctcctg
Digb (Forward for EMSA)Biotin- tgatgaaactccacttttttta
CsgDigRS (Reverse for EMSA)aatatttttctctttctggata
hns knockout (forward)gctcaacaaaccaccccaatataagtttggattactacattaagacccactttcacatt
hns knockout (reverse)atcccgccagcggcgggattttaagcatccaggaagtaaactaagcacttgtctcctg

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