FabR regulates Salmonella biofilm formation via its direct target FabB

Abstract

Background: Biofilm formation is an important survival strategy of Salmonella in all environments. By mutant screening, we showed a knock-out mutant of fabR, encoding a repressor of unsaturated fatty acid biosynthesis (UFA), to have impaired biofilm formation. In order to unravel how this regulator impinges on Salmonella biofilm formation, we aimed at elucidating the S. Typhimurium FabR regulon. Hereto, we applied a combinatorial high-throughput approach, combining ChIP-chip with transcriptomics.

Results: All the previously identified E. coli FabR transcriptional target genes (fabA, fabB and yqfA) were shown to be direct S. Typhimurium FabR targets as well. As we found a fabB overexpressing strain to partly mimic the biofilm defect of the fabR mutant, the effect of FabR on biofilms can be attributed at least partly to FabB, which plays a key role in UFA biosynthesis. Additionally, ChIP-chip identified a number of novel direct FabR targets (the intergenic regions between hpaR/hpaG and ddg/ydfZ) and yet putative direct targets (i.a. genes involved in tRNA metabolism, ribosome synthesis and translation). Next to UFA biosynthesis, a number of these direct targets and other indirect targets identified by transcriptomics (e.g. ribosomal genes, ompA, ompC, ompX, osmB, osmC, sseI), could possibly contribute to the effect of FabR on biofilm formation.

Conclusion: Overall, our results point at the importance of FabR and UFA biosynthesis in Salmonella biofilm formation and their role as potential targets for biofilm inhibitory strategies.

Keywords: Biofilm; ChIP-chip; FabB; FabR; Salmonella; Unsaturated fatty acids.

Fig. 1

Biofilm formation by an isogenic fabR mutant. The level of biofilm formation at the indicated temperatures is expressed as a percentage of wildtype SL1344 biofilm formation at the respective temperatures. The data are representative of three independent experiments (n = 3), with at least 4 replicates each. The error bars denote standard deviations between the independent experiments. For each temperature a one-sample t-test was performed to compare the mean biofilm formation of the mutant (expressed as a percentage of wildtype SL1344 biofilm formation) to 100 %. Significant changes (p-value <0.05) in the level of biofilm formation as compared to the wildtype at the same temperature are indicated with an asterix (*). SL1344: S. Typhimurium SL1344 wildtype strain; ∆fabR: S. Typhimurium SL1344 ∆fabR mutant (CMPG5624); ∆fabR-pFAJ1708-fabR trans complemented S. Typhimurium SL1344 ∆fabR mutant (pCMPG5678/CMPG5624)

Fig. 2

ChIP-qPCR validation of the ChIP-chip data. The validation of the ChIP-chip results by ChIP-qPCR analysis was performed as elaborated in Materials & Methods with dnaG as endogenous control. Values represent enrichment ratios of ChIP over mock ChIP samples, generated under free-living TSB conditions, and are averages of triplicate ChIP-qPCRs. The enrichment ratios of ChIP over mock ChIP samples were calculated as RQ = 2^{-(∆Ct ChIP -∆Ct mock ChIP)}, in which ∆Ct_ChIP is Ct_{gene test} – Ct_dnaG for the ChIP samples and ∆Ct_{mock ChIP} is Ct_{gene test} – Ct_dnaG for the mock ChIP samples. The presented values are representative for two biological repeats and standard deviations of the three technical repeats are indicated

Fig. 3

Alignment of the putative FabR binding site in the ChIP-qPCR verified in vivo FabR targets. a Alignment of the already known E. coli FabR targets [51] in S. Typhimurium SL1344; b Alignment of the ChIP-chip identified and ChIP-qPCR verified FabR targets in S. Typhimurium SL1344. All alignments were performed using MotifSampler [47]. Sequences upstream of the coding sequences of the indicated genes were taken from the complete genome sequence of S. Typhimurium SL1344. These input sequences comprised the full intergenic region, i.e. the region between the coding sequence of the FabR target gene and the upstream coding sequence, with hpaR and ddg indicating the hpaR/hpaG and ddg/yfdZ intergenic sequences, respectively. White letters with black background denote identical bases and black letters on a white background denote differing bases

Fig. 4

Functional classification of the differentially expressed genes in the isogenic fabR deletion mutant as compared to the wildtype under free-living TSB conditions. The bars represent the percentage of genes belonging to each group that were altered for absolute expression > 1.3 fold with a p-value < 0.02. The functional classes defined by the Welcome Trust Sanger Institute were used for this classification and the numbers behind each class represent the number of genes in this class

Fig. 5

Comparison between the microarray and qRT-PCR data. The expression of a number of genes was determined using qRT-PCR for the S. Typhimurium SL1344 wildtype and SL1344 ∆fabR mutant under free-living TSB conditions. The log₂-transformed mean value of at least three qRT-PCR technical repeats (representative for each of the two assayed biological replicates) for each gene was plotted on the X-axis and compared to the respective log₂-transformed microarray fold change (Y-axis). All depicted qRT-PCR tested genes had a p-value < 0.02 under their respective microarray conditions and their qRT-PCR primers are listed in Additional file 1: Table S2. The dotted lines represent arbitral boundaries (y = x + 1.2 and y = x – 1.2) between which the corresponding qRT-PCR and microarray results show good correspondence (i.e. not more than a 1.2 fold divergence on log₂ scale) with y = x being the ideal situation

Fig. 6

Effect on biofilm formation of individually overexpressing fabA, fabB and yqfA. The level of biofilm formation at the indicated temperatures is expressed as a percentage of wildtype SL1344 biofilm formation. The data are representative of three independent experiments (n = 3), with at least 4 replicates each. The error bars denote standard deviations between the independent experiments. For each temperature a one-sample t-test was performed to compare the mean biofilm formation of the different strains (expressed as a percentage of wildtype SL1344 biofilm formation) to the wildtype level of 100 %. Significant changes (p-value <0.05) in the level of biofilm formation as compared to the wild type at the same temperature are indicated with an asterix (*)SL1344: S. Typhimurium SL1344 wildtype strain; ∆fabR: S. Typhimurium SL1344 ∆fabR mutant (CMPG5624); SL1344/pFAJ1708-fabA: S. Typhimurium SL1344 overexpressing fabA (pCMPG10118/SL1344); SL1344/pFAJ1708-fabB: S. Typhimurium SL1344 overexpressing fabB (pCMPG10119/SL1344); SL1344/pFAJ1708-yqfA: S. Typhimurium SL1344 overexpressing yqfA (pCMP5553/SL1344)