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J Bacteriol. Nov 2006; 188(22): 7988–7991.
Published online Sep 15, 2006. doi:  10.1128/JB.00791-06
PMCID: PMC1636311

Transcription Modulation of Salmonella enterica Serovar Typhimurium Promoters by Sub-MIC Levels of Rifampin[down-pointing small open triangle]

Abstract

Promoter-lux fusions that showed rifampin-modulated transcription were identified from a Salmonella enterica serovar Typhimurium 14028 reporter library. The transformation of a subset of fusions into mutants that lacked one of six global regulatory proteins or were rifampin resistant showed that transcription modulation was independent of the global regulators, promoter specific, and dependent on the interaction of rifampin with RNA polymerase.

Antibiotics have been shown to alter global bacterial transcription patterns at concentrations below those that completely inhibit the growth of the bacterial cell (sub-MIC) (10, 29). Among the genes affected by antibiotics are those related to modes of action and bacterial stress responses. It has been suggested that characteristic gene expression patterns may be used to identify unknown small molecule inhibitors (2, 28). There have also been studies demonstrating antibiotic-induced transcription modulation of genes for accessory functions such as motility and virulence (reviewed in references 2 and 29).

Using a library of 6,528 promoter-reporter clones, we previously demonstrated dramatic up- and down-regulation of the transcription induced by the antibiotic rifampin (a transcription inhibitor [3, 14]) and the macrolide class of antibiotics on a global scale in Salmonella enterica serovar Typhimurium 14028 (10, 28). In this communication, we further examine rifampin-responsive promoter-luxCDABE fusion clones from our initial screen (~5% of library clones [10]) to explore the effects of known global transcription regulators on rifampin-induced transcription modulation (RITM).

Fusions whose activity was modulated by rifampin in our initial screens (10) were screened continuously by inoculating cultures grown at 37°C in a white, opaque microtiter plate (Costar; Fisher Scientific, Ottawa, Ontario, Canada) sealed with a breathable sealing membrane (Nalge Nunc, Naperville, IL). Luminescence was followed during 12 to 16 h of growth in the presence or absence of rifampin by using a Victor II multilabel counter (PerkinElmer, Boston, MA). Clones were discarded if luminescence readings were below 1,000 cps in medium with and without rifampin. This resulted in the identification of a subset of 22 moderately to strongly affected promoters that displayed between 200-fold down-regulation and 24-fold up-regulation (Table (Table1).1). Figure Figure1A1A shows the patterns of luminescence produced from two promoters that were sensitive to rifampin and from an unaffected promoter. The strain carrying the vector alone produced very low levels of luminescence (~100 cps [data not shown]). Note that both stimulation and inhibition of the transcription by the inhibitor were found and that the timings of the maximum response differed between promoters. As a result, promoters were screened for a minimum of 12 h to identify the maximum response. The effect of rifampin was observed to be concentration dependent and generally maximal at 5 μg/ml rifampin (Fig. (Fig.1B1B).

FIG. 1.
(A) Luminescence profiles of promoter-lux reporters. Luminescence patterns for serovar Typhimurium 14028 reporters STM3595::luxCDABE ([open diamond]), invF::luxCDABE ([open triangle]), and STM2091::luxCDABE (□) in LB supplemented with kanamycin at 25 μg/ml ...
TABLE 1.
Characteristics of rifampin-responsive promoters in S. enterica serovar Typhimurium 14028

Upon sequencing the promoters sensitive to RITM (Table (Table1),1), we noticed that a number of serovar Typhimurium virulence genes were included. The affected promoters were also grouped by the involvement of the associated genes in two distinct regulons. Promoters from virulence genes associated with intracellular growth and survival in macrophages, slyA (5, 13, 16), spvAB (11, 13, 17), somA (7), htrB (15), and SPI-2 genes (4, 12, 13), showed rifampin-induced up-regulation (RIUR). Promoters from genes involved in intestinal invasion, those associated with the type III secretion system encoded on SPI-1 and its secreted effectors, showed rifampin-induced down-regulation (RIDR). These genes included invF (6, 9, 18), sopA, and sopB (8, 24). Promoters from genes in SPI-4 (STM4255 to 4258), which have been shown to be coordinately regulated with SPI-1 by HilA (1), also displayed RIDR. Furthermore, rifampin also down-regulated the promoters from three operons involved in flagellum synthesis.

It seemed possible that RITM might be due to antibiotic effects on one of the known global transcription regulators or to the activation of one or more stress responses. To examine these possibilities, eight different representative rifampin-responsive promoter (RRP) fusions were transformed into serovar Typhimurium 14028 strains, each carrying a mutation in the gene for one of six major global regulators: CRP (25), FNR (27), FIS (22), H-NS (21), IHF (23), or σS (25). Mutant alleles were introduced into the 14028 background by using P22HTint-mediated generalized transduction (26) and confirmed by PCR or inverse PCR (20), followed by nucleotide sequencing and phenotypic analysis. The promoter activity of four rifampin-up-regulated and four rifampin-down-regulated lux fusions was examined in the presence or absence of rifampin (Table (Table2).2). The change in expression (n-fold) in response to rifampin was calculated by dividing the amount of luminescence observed with rifampin by the amount of luminescence observed without rifampin. In the majority of promoter-mutant combinations, although the magnitude of RITM may have changed, RITM levels were similar for both the wild-type and mutant host backgrounds. In four cases, the introduction of the mutations changed RITM from strong RIDR to no effect (Table (Table2).2). However, in these cases, the introduction of regulatory mutations may have reduced the basal expression of the lux reporter to the lower limit of detection, preventing a clear conclusion regarding its involvement in RIDR. Overall, in 44/48 of the combinations tested, rifampin modulation of transcription was not altered by the loss of one of the regulatory proteins. In four cases, the mutation of a global regulator either blocked RIUR or switched the response from RIUR to RIDR, indicating that the regulator was involved in RIUR for that promoter (Table (Table2).2). We note that Fis was involved in three of the four cases in which RITM was altered by the loss of a regulator. This involvement may indicate that Fis has a role in RIUR, but as it did not affect all RRPs, no model involving Fis regulation of RITM was readily apparent. We also tested the effects of rifampin in a rifampin-resistant host (resistance was conferred by a mutation in the β subunit of RNA polymerase). The mutation conferring rifampin resistance eliminated RITM in all of the RRP fusions tested (Fig. (Fig.2).2). Thus, RITM is a transcription-specific effect and is not due to separate effects on cell physiology. Further analysis of this novel regulation will require the identification of the specific nucleotide sequences at the RRP responsible for rifampin sensitivity.

FIG. 2.
Luminescence profiles of promoter-lux fusions in serovar Typhimurium 14028 and a rifampin-resistant isogenic mutant, R306, in response to rifampin (Rif). Two rifampin-up-regulated promoters (STM3595 and slyA), two rifampin-down-regulated promoters (fliA ...
TABLE 2.
Summary of rifampin-induced transcription modulationa

Acknowledgments

We thank the Canadian Bacterial Diseases Network and the Natural Sciences and Engineering Research Council of Canada for providing financial support. During his sabbatical visit to the University of British Columbia, F.D.L.C. was supported by a fellowship from the Spanish Ministry of Education, Culture, and Sports (PR2003-0258). Work in the F.D.L.C. laboratory was financed by grant BFU2005-03477 from the Spanish Ministry of Education.

Footnotes

[down-pointing small open triangle]Published ahead of print on 15 September 2006.

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