Gene expression in Pseudomonas aeruginosa: evidence of iron override effects on quorum sensing and biofilm-specific gene regulation

Abstract

Prior studies established that the Pseudomonas aeruginosa oxidative stress response is influenced by iron availability, whereas more recent evidence demonstrated that it was also controlled by quorum sensing (QS) regulatory circuitry. In the present study, sodA (encoding manganese-cofactored superoxide dismutase [Mn-SOD]) and Mn-SOD were used as a reporter gene and endogenous reporter enzyme, respectively, to reexamine control mechanisms that govern the oxidative stress response and to better understand how QS and a nutrient stress response interact or overlap in this bacterium. In cells grown in Trypticase soy broth (TSB), Mn-SOD was found in wild-type stationary-phase planktonic cells but not in a lasI or lasR mutant. However, Mn-SOD activity was completely suppressed in the wild-type strain when TSB was supplemented with iron. Reporter gene studies indicated that sodA transcription could be variably induced in iron-starved cells of all three strains, depending on growth stage. Iron starvation induction of sodA was greatest in the wild-type strain and least in the lasR mutant and was maximal in stationary-phase cells. Reporter experiments in the wild-type strain showed increased lasI::lacZ transcription in response to iron limitation, whereas the expression level in the las mutants was minimal and iron starvation induction of lasI::lacZ did not occur. Studies comparing Mn-SOD activity in P. aeruginosa biofilms and planktonic cultures were also initiated. In wild-type biofilms, Mn-SOD was not detected until after 6 days, although in iron-limited wild-type biofilms Mn-SOD was detected within the initial 24 h of biofilm establishment and formation. Unlike planktonic bacteria, Mn-SOD was constitutive in the lasI and lasR mutant biofilms but could be suppressed if the growth medium was amended with 25 microM ferric chloride. This study demonstrated that (i) the nutritional status of the cell must be taken into account when one is evaluating QS-based gene expression; (ii) in the biofilm mode of growth, QS may also have negative regulatory functions; (iii) QS-based gene regulation models based on studies with planktonic cells must be modified in order to explain biofilm gene expression behavior; and (iv) gene expression in biofilms is dynamic.

FIG. 1

SOD activity in planktonic cells cultured in TSB. Cell extracts (50 μg of protein per lane) of each strain were prepared at different time points corresponding to different growth stages, 6 h (mid-log phase) and 10 h (early stationary phase). SOD activity stains of wild-type strain PAO1 cultured in TSB (A), PAO1 in TSB amended with the iron-specific chelator 2,2-dipyridyl (B), and the lasI mutant PAO-JP1 in TSB amended with the iron-specific chelator 2,2-dipyridyl (C) were prepared as described in Materials and Methods. Results are of one of three independent experiments demonstrating this response.

FIG. 2

Transcription of sodA in mid-log-phase and stationary-phase cells of PAO1, PAO-JP1, and PAO-R1 as affected by iron limitation. Relative transcription levels were determined by measuring the reporter enzyme β-galactosidase as described by Miller (38) in planktonic cells harvested at mid-log (6 h) and late stationary (16 h) phases. Open bars, cells grown in TSB; filled bars, cells grown in TSB containing the iron-specific chelator 2,2-dipyridyl. The data represent the mean of three independent experiments (two to three cultures per experiment). Where visible, error bars denote 1 standard error.

FIG. 3

Expression of lasI in response to iron deprivation. Wild-type strain PAO1 was transformed with pPCS223, which contains the lasI::lacZ reporter fusion (50), and then cultured to mid-log or stationary phase in TSB (open bars) or TSB amended with the iron-specific chelator 2,2-dipyridyl (filled bars). The data represent the average of the means of three independent experiments (two cultures per experiment). Error bars, where visible, represent 1 standard error of the three experimental means. Differences between iron treatments for each culture stage are statistically significantly different (P = 0.01).

FIG. 4

SOD activity stains of cell extracts (50 μg of protein per lane) of the wild-type strain PAO1 (A) and the lasI mutant PAO-JP1 (B) obtained from biofilms cultured with 1/10 TSB for up to 6 days (6d). The data are representative of duplicate experiments, and locations of the Mn-SOD and Fe-SOD bands are as shown.

FIG. 5

Mn-SOD expression in PAO1 biofilms as affected by iron starvation. SOD activity was detected using activity stains as described in Materials and Methods. Cell extracts (50 μg per lane) were obtained from PAO1 biofilms grown for 1 or 2 days in 1/10 TSB containing the iron-specific chelator 2,2-dipyridyl and from 1- and 2-day PAO-JP1 biofilms grown in 1/10 TSB. The data are representative of duplicate experiments, and locations of the Mn-SOD and Fe-SOD bands are as shown.

FIG. 6

A potential mechanism for dual control of sodA by iron-sensitive and QS circuitry. (A) Gene arrangement of the fagA-fumC-orfX-sodA operon. (B) DNA sequence directly upstream of fagA, which is promoter proximal in the depicted operon (24, 25). The Fur regulatory protein (encoded by fur, which is itself regulated by iron availability) utilizes Fe²⁺ as a corepressor (23). The Fe(II)-Fur complex directly binds to the promoter region of genes that contain a specific regulatory sequence known as the iron box (9). Sequence analysis suggests the presence of iron boxes (orientation of each shown as a dashed line), which is the binding site for Fe(II)-Fur. Note that Mn-SOD production is elevated in fur mutants (25). Iron boxes are located at nucleotide positions -18 to -37 and -21 to -42. Positions of putative Lux boxes, the binding site for the PAI-1–LasR complex, are also shown at -11 to -29 and at -228 to -247. Nucleotide positions that are homologous with the consensus Vibrio Lux box are indicated by the connecting lines. Under high iron conditions, binding of the iron box by the Fe(II)-Fur complex would inhibit the LasR–PAI-1 complex from binding to the Lux box 2 region and would also inhibit transcription possibly originating upstream due to potential activation from binding at Lux box 1. Upon iron starvation, the Fe(II)-Fur complex would not be present, and thus the LasR–PAI-1 complex would be free to activate transcription. The bold thick line indicates a potential ribosome binding site.