migA, a quorum-responsive gene of Pseudomonas aeruginosa, is highly expressed in the cystic fibrosis lung environment and modifies low-molecular-mass lipopolysaccharide

Hongjiang Yang1, Mauricia Matewish2, Isabelle Loubens3, Douglas G. Storey3, Joseph S. Lam2 and Shouguang Jin1

Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA1
Department of Microbiology, University of Guelph, Guelph, Ontario, Canada2
Department of Biological Sciences, University of Calgary, Calgary, Canada3

Author for correspondence: Shouguang Jin. Tel: +1 352 392 8323. Fax: +1 352 392 3133. e-mail: sjin{at}mgm.ufl.edu


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INTRODUCTION
METHODS
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DISCUSSION
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Pseudomonas aeruginosa is an opportunistic human pathogen which poses a major threat to patients with cystic fibrosis (CF). Excessive amounts of mucus present in the lungs of CF patients promotes the colonization of P. aeruginosa. The migA gene, encoding a putative glycosyltransferase, has been shown to be highly inducible by respiratory mucus derived from CF patients. In this study, it is further demonstrated by population transcript analysis that the migA gene is highly expressed in the CF lung environment. Deletion analysis of the migA promoter identified a las-box-like sequence commonly found in promoters that are responsive to quorum sensing regulation. Further analysis of migA expression in quorum-sensing-defective strains, as well as its expression in response to autoinducer molecules, demonstrated that migA is regulated by the RhlI/RhlR quorum sensing regulatory system. Functionally, as the MigA sequence homology data suggested, the migA gene indeed affects the structure of LPS in P. aeruginosa. Increased expression of the migA gene results in a loss of core-plus-one LPS, while having no obvious effect on the long-chain O-antigen-bearing LPS. Although the exact biological role of the core-plus-one LPS is not clear, these experimental results suggest that migA up-regulation in the CF lung environment is part of the adaptive response which confers on P. aeruginosa a survival advantage.

Keywords: migA, mucus, LPS and quorum response

Abbreviations: CF, cystic fibrosis; C12-HSL, 3-oxo-dodecanoyl homoserine lactone; C4-HSL, butanoyl homoserine lactone


   INTRODUCTION
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INTRODUCTION
METHODS
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DISCUSSION
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Pseudomonas aeruginosa is an opportunistic pathogen which poses a major threat to immunocompromised patients, burn victims and cystic fibrosis (CF) patients (Bodey et al., 1983 ; Gilligan, 1991 ; Holder, 1993 ). More than 90% of mortality among CF patients is caused by lung infection with P. aeruginosa. The presence of excessive amounts of viscous mucus in the lungs of CF patients is thought to be a major contributing factor to their susceptibility to infection by P. aeruginosa. The mucus not only affects the host defence system by blocking the movement of alveolar macrophages and inhibiting cilia movement-associated bacterial clearance, but it also provides a unique growth environment, allowing P. aeruginosa to persist (Carnoy et al., 1993 ; Marshall & Carroll, 1991 ). The host immune response mounted against the infecting bacteria causes lung tissue damage and eventually leads to the failure of lung function (Gilligan, 1991 ).

As an opportunistic pathogen, P. aeruginosa relies on a combination of virulence factors to infect its host. Production and secretion of many of these virulence factors are increased at high cell density through a quorum sensing mechanism. Two such regulatory systems in P. aeruginosa, las and rhl, have been well characterized (Latifi et al., 1996 , 1995 ; Pearson et al., 1994 , 1995 ; Pesci et al., 1997 ; Pesci & Iglewski, 1997 ; Winson et al., 1995 ). Each is composed of two components, an inducer locus, lasI or rhlI, that controls the synthesis of diffusible autoinducer molecule 3-oxo-dodecanoyl homoserine lactone (C12-HSL; PAI-1) or butanoyl homoserine lactone (C4-HSL; PAI-2), respectively, and a response locus, lasR or rhlR, that encodes transcriptional factors which become active upon binding to the respective autoinducers (Passador et al., 1993 ). The PAI-1-bound form of LasR enhances the expression of genes encoding secreted proteases, siderophore, ADP-ribosylating enzymes and genes that control secretion of virulence factors (Passador et al. 1993 ; Seed et al., 1995 ; Toder et al., 1994 , 1991 ; Winson et al., 1995 ). PAI-2-bound RhlR controls some genes affected by LasR and, in addition, activates the expression of loci responsible for the production of toxic chemicals, including hydrogen cyanide, rhamnolipids and phenazines (Latifi et al., 1996 , 1995 ; Ochsner & Reiser, 1995 ; Ochsner et al., 1994 ; Pearson et al., 1997 ).

Chronic CF lung infections by P. aeruginosa start from acute infections early in the life of CF patients. The chronic infection has interspersed episodes of acute lower respiratory tract symptoms. As the chronic stage of the infection develops, there is a gradual transition of the P. aeruginosa isolates from a predominately non-mucoid, smooth LPS, piliated and motile phenotype to a predominately mucoid, rough LPS, non-piliated and non-motile phenotype (Govan, 1988 ; Luzar & Montie, 1985 ; Mahenthiralingam et al., 1994 ; Hancock et al., 1983 ; Woods et al., 1986 ). These morphological changes reflect bacterial adaptation to the CF lung environment, resulting from changes in gene regulation as well as mutation and selection. The mechanism for conversion to the mucoid phenotype was shown to be due to mutations in the anti-{sigma} factor gene, mucA, which alleviate repression of a 22 kDa {sigma} factor encoded by algU (or algT) that is required for the expression of the alginate biosynthetic pathway genes, in combination with environmental signals that activate genes under the control of two transcriptional regulators, AlgR and AlgB (Deretic et al., 1995 ; Govan & Deretic, 1996 ; Ma et al., 1998 ). Alginate is the predominant exopolysaccharide moiety of the mucoid substance in CF isolates. In the case of pili and motility losses, mutation in the rpoN gene, which is required for pilus and flagellum assembly, accounts for the majority of the cases (Mahenthiralingam et al., 1994 ). However, the molecular mechanism for the conversion from smooth LPS to rough LPS is not as clear. P. aeruginosa LPS consists of distinct A and B bands; the high-molecular-mass B-band determines the O-antigenic specificity of the bacterium, whereas the antigenically conserved A-band is a common antigen consisting of {alpha}-D-rhamnose trisaccharide repeating units. Wild-type P. aeruginosa strains express a proportion of their LPS as long-chain molecules, containing up to 50 repeating units (smooth LPS). In addition, these strains will have LPS that is devoid of O-polysaccharide (rough LPS) and LPS containing only one O chain repeating unit (core-plus-one LPS) (Rocchetta et al., 1999 ).

To understand the molecular mechanism of bacterial adaptation to the CF lung environment, we have previously isolated and characterized P. aeruginosa genes that are specifically inducible by respiratory mucus derived from CF patients (Wang et al., 1996 ). One of these encodes a putative glycosyltransferase which was predicted to be involved in LPS or exopolysaccharide modification. In this report, we show that the migA gene product indeed modifies bacterial LPS structure, predominantly the core-plus-one LPS. Furthermore, we demonstrate that the migA gene is regulated by the RhlI/RhlR quorum sensing regulatory system and is highly expressed in the CF lung environment. The significance of these observations is discussed.


   METHODS
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INTRODUCTION
METHODS
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DISCUSSION
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Bacterial strains and plasmids.
Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli and P. aeruginosa were grown in L-agar or L-broth at 37 °C. Minimal medium A (Min A; Davis & Mingioli, 1950 ) was also used for the growth of P. aeruginosa. Antibiotics were used at the following final concentrations (µg ml-1): for E. coli, ampicillin, 100; spectinomycin, 50; streptomycin, 25; tetracycline, 20; for P. aeruginosa, carbenicillin, 150; spectinomycin, 200; streptomycin, 200; tetracycline, 100. P. aeruginosa isolates from soil, blood of infected individuals and CF sputa were designated as environmental, non-CF and CF isolates, respectively. Both CF and non-CF clinical isolates were obtained from the Medical Center of the University of Arkansas for Medical Sciences, USA.


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Table 1. Strains and plasmids used in this study

 
To generate the migA insertional mutant, a 2 kb {Omega} fragment was inserted into the SalI site of the 5' migA coding region in pMSB3A (Wang et al., 1996 ). The resulting plasmid, pMSB3A{Omega}, was linearized by EcoRI digestion and electroporated into PAK as described previously (Jin et al. 1994 ). Colonies with resistance to spectinomycin/streptomycin were selected, followed by screening for sensitivity to carbenicillin. Chromosomal double crossovers were further confirmed by Southern hybridization.

To generate migA promoter deletion constructs, either available restriction sites upstream of the migA promoter or EcoRI sites generated in desired positions by site-directed mutagenesis (Kunkel et al., 1987 ) were used in combination with the BamHI site in pMS18 (Wang et al., 1996 ). Isolated migA promoter fragments were fused to a promoterless lacZ by cloning into the EcoRI/BamHI sites of pDN19lac{Omega} (Totten & Lory, 1990 ). To introduce an EcoRI site at positions -200, -89 and -38 relative to the ATG translational start codon of migA in pMS18, the following three oligonucleotides were used: migA-200, 5'-GGCCTACCCTCTAGAATTCCGACGGTATACTG-3'; migA-100, 5'-CCAGCCCGCGCCGAATTCCGATCGCCGGAACG-3'; and migA-50, 5'-GACGGCCCCCTGGAATTCCTACCGGGCAAGACG-3', respectively.

Sputum collection and extraction of total RNA.
Sputum samples from nine paediatric patients were utilized in this study. These patients were part of a larger cohort of patients currently being followed (Storey et al., 1992 , 1997 , 1998 ). The patients in this study all attend the Alberta Children’s Hospital Cystic Fibrosis Clinic, Canada. Voluntary consent was obtained from all patients and their guardians. Ethical approval of the study design was given by the Conjoint Research Ethics Board of the University of Calgary, Canada. All samples used in this study were collected from patients who were in the moderate to severe disease categories. Also, the selected patients had acquired solely or predominantly P. aeruginosa infections.

Sputum from the CF patients was collected as described previously (Storey et al., 1998 ). RNA was extracted from the sputum samples, blotted onto Nylon membrane, hybridized and signal intensity was measured as described by Storey et al. (1992) . A 613 bp internal HincII fragment of migA from pIL17 was used as a probe to measure migA-specific transcripts. A 700 bp internal EcoRI fragment of algD from plasmid pCC27 (Chitinis & Ohman, 1990 ) and a 450 bp PstI fragment of lasA from pPS1816 (Schad & Iglewski, 1988 ) were used to examine algD-specific and lasA-specific transcripts, respectively.

Primer extension.
Total bacterial RNA was isolated from strain PAK that was grown in minimal medium A with or without 10% respiratory mucus from CF patients. For primer extension experiments, an oligonucleotide, migA-down (5'-CTTCTTCCAGGTACTTTTCCGCGTTG-3') was used as primer and according to the protocol described by Maniatis et al. (1982) . The same primer was also used to sequence the migA promoter region to identify the transcription initiation site.

LPS preparation, SDS-PAGE and Western blot analysis.
LPS was prepared from whole-cell lysates of P. aeruginosa by the method of Hitchcock & Brown (1983) . LPS samples were run on standard discontinuous 12·5% glycine SDS-PAGE gels (Hancock & Carey, 1979 ) or by commercially prepared 16% Tricine SDS-PAGE gels (Novex). LPS was visualized by the silver-staining methods of Dubray & Bezard (1982) for glycine SDS-PAGE and Tsai & Frasch (1982) for Tricine gels. For Western immunoblots, the procedure was essentially as described by de Kievit & Lam (1994) . LPS separated by SDS-PAGE was transferred onto nitrocellulose and then blocked with 3% skim milk. For detection of P. aeruginosa LPS, blots were immersed in hybridoma-culture supernatants containing mAb O25G3D6 (specific for B-band LPS serotype O6; Emara et al., 1995 ), mAb N1F10 (specific for A-band LPS; Lam et al., 1989 ) or mAb 7-4 (specific for LPS inner core; de Kievit & Lam, 1994 ). After incubation with the corresponding first antibodies, blots were washed and then incubated with the appropriate second antibody. The second antibody for anti-P. aeruginosa LPS mAbs was goat anti-mouse F(ab')2–alkaline phosphatase conjugate (Jackson ImmunoResearch Laboratories) at the dilutions suggested by the manufacturer. Following a series of washes, the blots were developed using a substrate consisting of 30 mg nitro blue tetrazolium and 15 mg 5-bromo-4-chloro-3-indolyl phosphate (toluidine salt; Sigma) in 100 ml 0·1 M sodium bicarbonate buffer (pH 9·8).

ß-Galactosidase assay.
P. aeruginosa strains harbouring various lacZ fusion constructs were grown in MinA with or without 10% CF respiratory mucus and measured for ß-galactosidase activities as described by Wang et al. (1996) . To induce migA and lasB, 100 nM of their respective autoinducers, C12-HSL (PAI-1) or C4-HSL (PAI-2) was added at the start of culture and ß-galactosidase activities were measured at various cell densities.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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migA of P. aeruginosa is highly expressed in the CF lung environment
The migA gene was identified by virtue of its inducibility in vitro by respiratory mucus derived from CF patients (Wang et al., 1996 ). To test if this gene is indeed expressed in vivo during infection of CF lungs, we conducted population transcript analysis. Sputum samples were collected from CF patients who had confirmed P. aeruginosa infections. Total RNA was isolated from 25 sputum samples obtained from nine CF patients over a period of 3 years and blotted onto a nitrocellulose membrane. When the membrane was hybridized with the migA probe, all 25 samples showed a hybridization signal above background levels of approximately 100- to 400-fold relative intensity (Fig. 1). In the samples that we selected, P. aeruginosa was either the sole or the predominant pathogenic organism in the sample. Thus, hybridization of the migA probe is not likely to be due to hybridization of the probe to RNA from other species. This suggested that in the lungs of patients with CF, migA was commonly expressed. We have also probed these RNA samples with algD, encoding a key enzyme in the biosynthesis of alginate, which is known to hybridize strongly to these RNA samples (Storey et al., 1997 ). Our second probe, lasA, encoding an elastase enzyme, usually hybridizes at an intermediate level to these RNA samples (Storey et al., 1997 , 1998 ). The hybridization signals with the migA probe appear to be at a level nearer to the algD probe, suggesting abundant transcript accumulation in the bacterial populations found in the sputa from CF patients (Fig. 1).



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Fig. 1. Comparison of population transcript accumulation of migA (white bars), algD (hatched bars) and lasA (black bars) in P. aeruginosa cells chronically colonizing the lungs of CF patients. Total RNA was isolated from sputum samples of CF patients and blotted onto a nitrocellulose membrane. Relative intensities of radioactive signals after hybridization with individual probes are shown (see Methods).

 
The data in Fig. 1 indicate that migA transcript levels positively correlate to that of algD. This suggests that these genes might be controlled by the same regulatory system. This possibility seems more likely when we consider the fact that both genes are possibly involved in polysaccharide biosynthesis or modification. To test this, we examined the effect of algR, a known regulator of algD, on migA expression. A migA::lacZ fusion construct, pMS19lac{Omega}, was introduced into wild-type PAO1 and its algR mutant derivative, and the levels of ß-galactosidase activity were compared. Results showed that mucus-mediated migA gene activation was not altered in the algR mutant background compared to the wild-type strain (data not shown), indicating that mucus-mediated expression of migA is independent of algR.

Deletion analysis of the migA promoter
To identify functional promoter elements of the migA gene, a series of promoter deletion constructs were generated using both existing restriction sites as well as sites generated by site-directed mutagenesis. The promoter deletion fragments were fused to the promoterless lacZ gene in pDN19lac{Omega}, and PAK cells harbouring the resulting constructs were tested for ß-galactosidase activities with or without the addition of CF respiratory mucus. As shown in Fig. 2, promoter deletion up to -200 (relative to the putative ATG start codon) did not affect wild-type promoter activity but deletion to -89 totally abolished the inducibility by mucus. These results indicate that important migA promoter elements reside within the 111 bp region between -200 and -89.



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Fig. 2. Promoter deletion analysis of the migA gene. DNA fragments containing various lengths of the migA promoter region were fused with the promoterless lacZ reporter gene and introduced into the wild-type PAK strain. ß-Galactosidase activities (Miller units) were measured after overnight growth in MinA with (+) or without (-) 10% CF respiratory mucus. B, BamHI; K, KpnI; R, EcoRI site introduced by site-directed mutagenesis (see Fig. 3 for sequence). A chloramphenicol acetyltransferase homologue (cat) and two exotoxin A regulatory genes (regA and regB) reside upstream of migA.

 


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Fig. 3. Determination of the transcription initiation site of the migA gene by primer extension. (a) DNA sequence of the migA promoter region. EcoRI sites introduced by site-directed mutagenesis which were used to construct plasmids pSJ9727, pSJ9728 and pSJ9729 are shown. Lower case letters represent mismatch mutations introduced to generate EcoRI sites. The transcription initiation site ‘A’ is shown in bold and underlined. (b) Autoradiogram of primer extension experiment. Lanes marked with - and + contain RNA samples prepared from PAK grown in the absence or presence, respectively, of 10% CF mucus. Equal amounts of the two RNA samples were used. DNA sequencing termination reactions for A, C, G and T are marked. The arrow indicates the extended primer and the DNA sequence of the anti-sense strand is shown next to it.

 
The migA transcription initiation site was further determined. Based on the above promoter deletion analysis, an oligonucleotide that hybridizes downstream of the ATG start codon was used as a primer to conduct a primer extension experiment. Total RNA was prepared from the same number of PAK cells grown in MinA medium with or without 10% CF respiratory mucus. Under both growth conditions, a single transcription initiation site corresponding to the A at position -23 upstream of the ATG start codon was identified (Fig. 3). A higher intensity of signal was detected in the RNA sample prepared from cells grown in the presence of CF mucus, representing a higher transcript level. An identical size transcript was detected under non-inducing conditions after a longer exposure (data not shown). Comparison of sequences between -200 and -23 to binding sites of known transcriptional factors revealed two las-box-like sequences (Fig. 3) that are found among quorum-responsive gene promoters (Whiteley et al., 1999 ). The consensus las box consists of invariant CT and AG that are separated by 12 variable nucleotides CT-(N12)-AG and the two las-box-like sequences of migA have CT-(N11)-AG sequences. This finding suggests that the migA gene might be regulated by a quorum sensing system. However, no obvious recognition sequence for {sigma}70 or {sigma}54 was found in the sequence upstream of the transcription initiation site. Since pSJ9729 contains the smallest functional migA promoter, we utilized this plasmid construct for further analysis of migA promoter activity.

migA is regulated by the RhlI/RhlR quorum sensing system
To investigate whether migA is indeed regulated by the quorum sensing system, cell-density-dependent expression of the migA::lacZ fusion was first tested in a PAK background. PAK(pSJ9729) was grown in L-broth with appropriate antibiotics at 37 °C. As shown in Fig. 4(a), ß-galactosidase activity increased proportional to cell density and more than fourfold greater ß-galactosidase activity was observed at high cell density than that at low cell density (OD600 4·5 vs 1·0), demonstrating cell-density-dependent expression, a characteristic of genes under the control of quorum sensing systems. We further examined whether migA is regulated by either one or both of the two well characterized quorum sensing systems in P. aeruginosa, lasI/R or rhlI/R. Expression of migA::lacZ in lasI and rhlI single mutants, as well as in a lasI/rhlI double mutant background was compared to that in the wild-type PAO1 background. At high cell density (OD600 4·5), migA expression levels were much lower in either the rhlI or lasI/rhlI double mutant background than in wild-type strain. However, the migA expression level in the lasI mutant background was not different from that in the wild-type PAO1 background (Fig. 4b), suggesting that migA is regulated by the RhlI/R system. Furthermore, migA expression in a lasI/rhlI double mutant background is stimulated by the addition of C4-HSL (PAI-2) but not by C12-HSL (PAI-1). Interestingly, addition of both PAI-1 and PAI-2 did not induce migA expression (Fig. 4c), which is similar to other RhlI/RhlR-regulated genes described by Pesci et al. (1997) . It was postulated in these cases that PAI-1 inhibits the binding of PAI-2 to RhlR. As a control, the expression of lasB, which is known to be regulated by LasR, was stimulated by PAI-1 but not by PAI-2 in the same mutant background (Fig. 4c).



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Fig. 4. RhlI/RhlR-mediated activation of migA::lacZ expression. (a) Cell-density-dependent expression of the migA gene. Strain PAK containing the migA::lacZ fusion construct pSJ9729 ({blacksquare}) or vector pDN19lac{Omega} ({blacktriangleup}) was cultured in L-broth at 37 °C and ß-galactosidase activity was plotted against cell density. (b) rhlI is required for the cell-density-dependent migA expression. Plasmid pSJ9729 was introduced into wild-type PAO1 as well as isogenic lasI, rhlI and lasI/rhlI mutant derivatives PDO100, PAO-JP1 and PAO-JP2, respectively. ß-Galactosidase activities were measured at low (OD600 1·0; black bars) or high (OD600 4·5; white bars) cell density after growth in L-broth. (c) Effect of PAI-1 and PAI-2 autoinducers on the expression of migA. ß-Galactosidase activities of the lasI/rhlI double mutant (PAO-JP2) harbouring migA::lacZ and lasB::lacZ fusion constructs pSJ9729 and pTS400, respectively, were grown to an OD600 of 3·5 in L-broth in the absence (white bars) or presence of 100 nM C12-HSL (shaded bars), 100 nM C4-HSL (black bars) or 100 nM each of C12-HSL and C4-HSL (hatched bars).

 
migA affects low-molecular-mass LPS production
The migA-encoded protein shows homology to glycosyltransferases associated with LPS biosynthesis in other Gram-negative bacteria (Wang et al., 1996 ), suggesting a possible role of migA in LPS core synthesis. To determine the role of migA in LPS biosynthesis, LPS banding patterns were compared among strains of wild-type PAK, a migA::{Omega} mutant and PAK harbouring a plasmid that constitutively expresses the migA gene (pSJ9610). Two gel systems were used to examine the LPS banding patterns, Tricine SDS-PAGE and the standard glycine discontinuous SDS-PAGE system followed by silver-staining and Western immunoblotting with appropriate mAbs. The Tricine SDS-PAGE system allows for significantly improved resolution of low-molecular-mass LPS of the core region (Lesse et al., 1990 ) while standard SDS-PAGE provides good resolution of high-molecular-mass LPS, such as O antigen. High-molecular-mass LPS of wild-type P. aeruginosa strain PAK, which includes A-band and B-band LPS, was analysed by silver-stained SDS-PAGE gels and Western immunoblots using B-band-specific mAb O25G3D6 and A-band-specific mAb N1F10. No significant difference in the production of high-molecular-mass LPS was observed between the parent strain PAK and the migA::{Omega} mutant (Fig. 5). Results from the Tricine gel analysis showed, however, that the expression of migA clearly affected core LPS production. Wild-type PAK exhibits two LPS bands corresponding to ‘complete-core’ (band 2) and ‘core-plus-one O antigen repeat’ (band 1) (Fig. 6, lane 1). For comparison, LPS from a PAK wbpL mutant, deficient in the initial glycosyltransferase required for both A-band and B-band LPS production, was included as a control in lane 4. As expected, only band 2 (complete core) was detected. PAK(pJS9610) produced only band 2 (Fig. 6, lane 2) which co-migrated with the complete core band of the wbpL mutant (Fig. 6, lane 4). Thus high-level expression of migA in PAK(pSJ9610) affects core biosynthesis and resulted in the loss of the core-plus-one LPS molecules. The inhibitory role of migA in the formation of core-plus-one LPS correlates with the observation that the migA mutant strain produces an increased amount of the core-plus-one LPS (Fig. 6, lane 3).



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Fig. 5. Effect of migA on the production of high-molecular-mass LPS. (a) Silver-stained SDS-PAGE. (b, c) Western immunoblots reacted with the A-band-specific mAb N1F10 and B-band-specific mAb O25G3D6, respectively. The migA gene was constitutively expressed by a lac promoter in pSJ9610. Lanes: 1, wild-type PAK; 2, PAK(pSJ9610); 3, PAK(migA::{Omega}).

 


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Fig. 6. Visualization of low-molecular-mass LPS by Tricine SDS-PAGE analysis. LPS was purified from the four bacterial strains and subjected to separation by Tricine SDS-PAGE. (a) Silver-stained Tricine SDS-PAGE gel. (b) Western immunoblot reacted with LPS inner-core-specific mAb 7-4 which reacts with low-molecular-mass LPS. Lanes: 1, wild-type PAK; 2, PAK(pSJ9610); 3, PAK(migA::{Omega}); 4, PAK(wbpL-). Band 1, core-plus-core; band 2, complete core.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The initial screen for migA used mucus from CF sputa to induce the expression of various genes (Wang et al., 1996 ). Although the migA gene was shown to be inducible by CF respiratory mucus in vitro, it was difficult to demonstrate its expression in vivo due to the lack of an animal model which mimics the CF lung infection. Population transcript analysis, first described by Storey et al., (1992) can best predict the in vivo situation and was therefore adopted in this study. We detected a high level of transcript accumulation in all 25 RNA samples extracted from the bacterial population found in CF sputum samples. The level of migA transcript accumulation was almost as abundant as algD transcript accumulation (Fig. 1). These results argue that migA is commonly transcribed at high levels by the P. aeruginosa populations in the lungs of patients with CF.

Quorum sensing gene regulation plays an important role in the pathogenesis of P. aeruginosa. In response to an increased bacterial cell density, the quorum sensing system not only mediates an increased synthesis and secretion of extracellular enzymes to meet the nutritional requirement of the pathogen, but also mediates biofilm formation to overcome adverse environmental conditions (Pesci & Iglewski, 1997 ; Fuqua & Greenberg, 1998 ). Whiteley et al. (1999) have recently reported on the identification of a large number of genes that are under the control of quorum sensing systems. By comparing their promoter sequences, a consensus las box promoter element (CT-N12-AG), potentially the LasR and or RhlR recognition sequence, has been deduced. However, the migA gene was not included in that study. Our experimental data clearly demonstrate that migA expression is dependent on the rhlI/rhlR regulatory system and its promoter also contains two las-box-like sequences (CT-N11-AG) that fall into the essential promoter region identified by the promoter deletion analysis. Whether one or both of the las-box-like sequences are required for migA expression is not known at this point.

Since the CF respiratory mucus that we used was derived from CF patients who had acquired P. aeruginosa infection, the mucus should contain high concentrations of autoinducers. However, the mucus was subjected to dialysis against distilled water before use and therefore it is unlikely that residual autoinducer molecules are responsible for the activation of migA gene expression. Recently, our group has observed that addition of CF respiratory mucus into minimal medium actually promotes P. aeruginosa growth which results in a higher cell density (unpublished results). It is therefore possible that this higher cell density triggers the quorum sensing response which activates migA expression. The growth promoting activity of the CF mucus is consistent with the reported observations of high bacterial cell densities in CF lungs (Potts et al., 1995 ). The exact components of the mucus that promote the growth of P. aeruginosa deserve further study, since they represent important host factors that promote P. aeruginosa colonization and the bacterial genes mediating the utilization of these mucus components could potentially serve as targets in antimicrobial strategy.

The observation that MigA shares amino acid sequence homology with glycosyltransferases, implies a potential role of this protein in LPS modification. We have shown here that migA indeed affects the modification of low-molecular-mass LPS. High-level expression of migA leads to the loss of the core-plus-one LPS band, whereas migA mutation results in an increase in this band. We hypothesize that MigA is a glycosyltransferase that transfers a sugar residue onto the core region and consequently modifies the attachment point for O antigen on the outer-core oligosaccharide. The MigA modified outer-core molecule would therefore be less able to serve as an acceptor for O antigen. A ligase enzyme, WaaL, joins the O antigen polysaccharide to the lipid A core and there is a strong evidence that the nature of the acceptor molecule is important for this ligation reaction. Evidence to support this hypothesis is as follows. (i) In P. aeruginosa, the structure of the outer-core region in a fully assembled O chain containing LPS differs from that of the O-chain-deficient rough LPS. The point of attachment of the O chain to the outer core is at the 1,3-linked rhamnose (Sadovskaya et al., 2000 ). When this residue is missing, the core molecule is less able to serve as an acceptor for O antigen attachment. Genetic evidence suggests that the rhamnose residue in the outer-core region is necessary for O antigen attachment (Rahim et al., 2000 ). (ii) A rhamnosyltransfersase, required for the addition of the 1,3-linked rhamnose which serves as the attachment point for O antigen to the outer core, shares amino acid sequence homology with MigA (Rahim et al., 2000 ). (iii) MigA is homologous to glycosyltransferases, including WaaV of E. coli, possessing a type R1 core (Heinrichs et al., 1998 ). The proposed function of WaaV is that of a UDP-glucosyltransferase required for the addition of a 1,3-linked glucose, which is a side-branch substituent in the outer core and acts as the attachment site for long-chain O antigen to an R1-type core.

Recently, Ernst et al. (1999) have demonstrated that the lipid A portion of the P. aeruginosa LPS undergoes specific modification during chronic colonization of CF lungs. The CF-specific lipid A apparently contains palmitate and aminoarabinose which are associated with bacterial resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease. Since the migA gene is highly expressed in the CF lung environment and interferes with the formation of core-plus-one LPS, it is possible that suppression of such low-molecular-mass LPS renders P. aeruginosa a survival advantage in the CF lung environment. If indeed this is the case, a prolonged colonization in the CF lung environment could result in enrichment of adaptive mutants in which migA gene expression become constitutive, similar to the enrichment of mucoid isolates of P. aeruginosa. Our survey of CF- and non-CF-derived P. aeruginosa strains indicates that a high proportion of CF isolates lost core-plus-one LPS, whereas all of the non-CF isolates retained it (unpublished results). However, it is not clear if this is due to migA up-regulation or a defect in the ligase which mediates core-plus-one LPS synthesis. Further studies are underway to understand this as well as the role of migA up-regulation in the adaptation of P. aeruginosa to the CF lung environment.


   ACKNOWLEDGEMENTS
 
We would like to thank Dr R. Ramphal for providing respiratory mucus from CF patients, Dr B. Iglewski and Dr P. Greenberg for providing lasI, rhlI and lasI/rhlI mutant strains and Dr D. Wozniak for providing the algR mutant. We would also like to acknowledge the helpful comments of the two anonymous referees. This work was supported by NIH grant R29AI39524 (to S.J.) and Canadian Cystic Fibrosis Foundation (CCFF) operating grants (to D.S. and J.L., respectively). M.M. is a recipient of a CCFF studentship.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Belanger, M., Burrows, L. L. & Lam, J. S. (1999). Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide. Microbiology 145, 3505-3521.[Abstract/Free Full Text]

Bodey, G. P., Bolivar, R., Fainstein, V. & Jadeja, L. (1983). Infections caused by Pseudomonas aeruginosa. Rev Infect Dis 5, 279-313.[Medline]

Brint, J. M. & Ohman, D. E. (1995). Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family. J Bacteriol 177, 7155-7163.[Abstract]

Carnoy, C., Ramphal, R., Scharfman, A., Lo, G. J. M., Houdret, N., Klein, A., Galabert, C., Lamblin, G. & Roussel, P. (1993). Altered carbohydrate composition of salivary mucins from patients with cystic fibrosis and the adhesion of Pseudomonas aeruginosa. Am J Respir Cell Mol Biol 9, 323-334.[Medline]

Chitinis, C. E. & Ohman, D. E. (1990). Cloning of Pseudomonas aeruginosa algG, which controls alginate structure. J Bacteriol 172, 2894-2900.[Medline]

Davis, B. D. & Mingioli, E. S. (1950). Mutants of Escherichia coli requiring vitamin B12. J Bacteriol 60, 17-28.

Deretic, V., Schurr, M. J. & Yu, H. (1995). Pseudomonas aeruginosa, mucoidy and the chronic infection phenotype in cystic fibrosis. Trends Microbiol 3, 351-356.[Medline]

Dubray, G. & Bezard, G. (1982). A highly sensitive periodic acid-silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels. Anal Biochem 119, 325-329.[Medline]

Emara, M. G., Tout, N. L., Kaushik, A. & Lam, J. S. (1995). Diverse VH and V kappa genes encode antibodies to Pseudomonas aeruginosa LPS. J Immunol 155, 3912-3921.[Abstract]

Ernst, R. K., Yi, E. C., Guo, L., Lim, K. B., Burns, J. L., Hackett, M. & Miller, S. I. (1999). Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa. Science 286, 1561-1565.[Abstract/Free Full Text]

Fuqua, C. & Greenberg, E. P. (1998). Self perception in bacteria: quorum sensing with acylated homoserine lactones. Curr Opin Microbiol 1, 183-189.[Medline]

Gilligan, P. H. (1991). Microbiology of airway disease in patients with cystic fibrosis. Clin Microbiol Rev 4, 35-51.[Medline]

Govan, J. R. W. (1988). Alginate biosynthesis and unusual characteristics associated with the pathogenesis of Pseudomonas aeruginosa in cystic fibrosis. In Bacterial Infections of Respiratory and Gastrointestinal Mucosae , pp. 67-96. Edited by E. Griffiths, W. Donachie & J. Stephen. Oxford:IRL Press.

Govan, J. R. & Deretic, V. (1996). Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 60, 539-574.[Abstract]

Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580.[Medline]

Hancock, R. E. W. & Carey, A. M. (1979). Outer membrane of Pseudomonas aeruginosa: heat- and 2-mercaptoethanol-modifiable proteins. J Bacteriol 140, 902-910.[Medline]

Hancock, R. E. W., Mutharia, L. M., Chan, L., Darveau, R. P., Speert, D. P. & Pier, G. B. (1983). Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains. Infect Immun 42, 170-177.[Medline]

Heinrichs, D. E., Yethon, J. A., Amor, P. A. & Whitfield, C. (1998). The assembly system for the outer core portion of R1- and R4-type lipopolysaccharides of Escherichia coli. The R1 core-specific beta-glucosyltransferase provides a novel attachment site for O-polysaccharides. J Biol Chem 273, 29497-29505.[Abstract/Free Full Text]

Hitchcock, P. J. & Brown, T. M. (1983). Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol 154, 269-277.[Medline]

Holder, I. A. (1993). Pseudomonas aeruginosa virulence-associated factors and their role in burn wound infections. In Pseudomonas aeruginosa, the Opportunist, pp. 235–245. Edited by R. B. Fick, Jr. Boca Raton, FL: CRC Press.

Holloway, B. W., Krishnapillai, V. & Morgan, A. F. (1979). Chromosomal genetics of Pseudomonas. Microbiol Rev 43, 73-102.

Jin, S., Ishimoto, K. & Lory, S. (1994). Nucleotide sequence of the rpoN gene and characterization of two downstream open reading frames in Pseudomonas aeruginosa. J Bacteriol 176, 1316-1322.[Abstract]

de Kievit, T. R. & Lam, J. S. (1994). Monoclonal antibodies that distinguish inner core, outer core, and lipid A regions of Pseudomonas aeruginosa lipopolysaccharide. J Bacteriol 176, 7129-7139.[Abstract]

Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987). Rapid and efficient site-specific mutagenesis without phenotypic selection. Method Enzymol 154, 367-382.[Medline]

Lam, M. Y., McGroarty, E. J., Kropinski, A. M., MacDonald, L. A., Pedersen, S. S., Hoiby, N. & Lam, J. S. (1989). Occurrence of a common lipopolysaccharide antigen in standard and clinical strains of Pseudomonas aeruginosa. J Clin Microbiol 27, 962-967.[Medline]

Latifi, A., Winson, M. K., Foglino, M., Bycroft, B. W., Stewart, G. S., Lazdunski, A. & Williams, P. (1995). Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1. Mol Microbiol 17, 3333-3343.

Latifi, A., Foglino, M., Tanaka, K., Williams, P. & Lazdunski, A. (1996). A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol Microbiol 21, 1137-1146.[Medline]

Lesse, A. J., Campagnari, A. A., Bittner, W. E. & Apicella, M. A. (1990). Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Immunol Methods 126, 109-117.[Medline]

Luzar, M. A. & Montie, T. C. (1985). Avirulence and altered physiological properties of cystic fibrosis strains of Pseudomonas aeruginosa. Infect Immun 50, 572-576.[Medline]

Ma, S., Selvaraj, U., Ohman, D. E., Quarless, R., Hassett, D. J. & Wozniak, D. J. (1998). Phosphorylation-independent activity of the response regulators AlgB and AlgR in promoting alginate biosynthesis in mucoid Pseudomonas aeruginosa. J Bacteriol 180, 956-968.[Abstract/Free Full Text]

Mahenthiralingam, E., Campbell, M. E. & Speert, D. P. (1994). Nonmotility and phagocytic resistance of Pseudomonas aeruginosa isolates from chronically colonized patients with cystic fibrosis. Infect Immun 62, 596-605.[Abstract]

Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Marshall, B. C. & Carroll, K. C. (1991). Interaction between Pseudomonas aeruginosa and host defenses in cystic fibrosis. Semin Respir Infect 6, 11-18.[Medline]

Nunn, D., Bergman, S. & Lory, S. (1990). Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili. J Bacteriol 172, 2911-2919.[Medline]

Ochsner, U. A. & Reiser, J. (1995). Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 92, 6424-6428.[Abstract]

Ochsner, U. A., Koch, A. K., Fiechter, A. & Reiser, J. (1994). Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. J Bacteriol 176, 2044-2054.[Abstract]

Passador, L., Cook, J. M., Gambello, M. J., Rust, L. & Iglewski, B. H. (1993). Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260, 1127-1130.[Medline]

Pearson, J. P., Gray, K. M., Passador, L., Tucker, K. D., Eberhard, A., Iglewski, B. H. & Greenberg, E. P. (1994). Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci U S A 91, 197-201.[Abstract]

Pearson, J. P., Passador, L., Iglewski, B. H. & Greenberg, E. P. (1995). A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 92, 1490-1494.[Abstract]

Pearson, J. P., Pesci, E. C. & Iglewski, B. H. (1997). Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179, 5756-5767.[Abstract]

Pesci, E. C. & Iglewski, B. H. (1997). The chain of command in Pseudomonas quorum sensing. Trends Microbiol 5, 132-135.[Medline]

Pesci, E. C., Pearson, J. P., Seed, P. C. & Iglewski, B. H. (1997). Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J Bacteriol 179, 3127-3132.[Abstract]

Potts, S. B., Roggli, V. L. & Spock, A. (1995). Immunohistologic quantification of Pseudomonas aeruginosa in the tracheobronchial tree from patients with cystic fibrosis. Pediatr Pathol Lab Med 15, 707-721.[Medline]

Rahim, R., Burrows, L. L., Monteiro, M. A., Perry, M. B. & Lam, J. S. (2000). Involvement of the rml locus in core oligosaccharide and O polysaccharide assembly in Pseudomonas aeruginosa. Microbiology 146 (in press).

Rocchetta, H. L., Burrows, L. L. & Lam, J. S. (1999). Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa. Microbiol Mol Biol Rev 63, 523-553.[Abstract/Free Full Text]

Sadovskaya, I., Brisson, J. R., Thibault, P., Richards, J. C., Lam, J. S. & Altman, E. (2000). Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5. Eur J Biochem 267, 1640-1650.[Abstract/Free Full Text]

Schad, P. A. & Iglewski, B. H. (1988). Nucleotide sequence and expression in Escherichia coli of the Pseudomonas aeruginosa lasA gene. J Bacteriol 170, 2784-2789.[Medline]

Seed, P. C., Passador, L. & Iglewski, B. H. (1995). Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy. J Bacteriol 177, 654-659.[Abstract]

Storey, D. G., Ujack, E. E. & Rabin, H. R. (1992). Population transcript accumulation of Pseudomonas aeruginosa exotoxin A and elastase in sputa from patients with cystic fibrosis. Infect Immun 60, 4687-4694.[Abstract]

Storey, D. G., Ujack, E. E., Mitchell, I. & Rabin, H. R. (1997). Positive correlation of algD transcription to lasB and lasA transcription by populations of Pseudomonas aeruginosa in the lungs of patients with cystic fibrosis. Infect Immun 65, 4061-4067.[Abstract]

Storey, D. G., Ujack, E. E., Rabin, H. R. & Mitchell, I. (1998). Pseudomonas aeruginosa lasR transcription correlates with the transcription of lasA, lasB, and toxA in chronic lung infections associated with cystic fibrosis. Infect Immun 66, 2521-2528.[Abstract/Free Full Text]

Toder, D. S., Gambello, M. J. & Iglewski, B. H. (1991). Pseudomonas aeruginosa LasA: a second elastase under the transcriptional control of lasR. Mol Microbiol 5, 2003-2010.[Medline]

Toder, D. S., Ferrell, S. J., Nezezon, J. L., Rust, L. & Iglewski, B. H. (1994). lasA and lasB genes of Pseudomonas aeruginosa: analysis of transcription and gene product activity. Infect Immun 62, 1320-1327.[Abstract]

Totten, P. A. & Lory, S. (1990). Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK. J Bacteriol 172, 7188-7199.[Medline]

Tsai, C. M. & Frasch, C. E. (1982). A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119, 115-119.[Medline]

Wang, J., Lory, S., Ramphal, R. & Jin, S. (1996). Isolation and characterization of Pseudomonas aeruginosa genes inducible by respiratory mucus derived from cystic fibrosis patients. Mol Microbiol 22, 1005-1012.[Medline]

Whiteley, M., Lee, K. M. & Greenberg, E. P. (1999). Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 96, 13904-13909.[Abstract/Free Full Text]

Winson, M. K., Camara, M., Latifi, A. & 8 other authors (1995). Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 92, 9427–9431.[Abstract]

Woods, D. E., Schaffer, M. S., Rabin, H. P., Campbell, G. D. & Sokol, P. A. (1986). Phenotypic comparison of Pseudomonas aeruginosa strains isolated from a variety of clinical sites. J Clin Microbiol 24, 260-264.[Medline]

Wozniak, D. J. & Ohman, D. E. (1994). Transcriptional analysis of the Pseudomonas aeruginosa genes algR, algB, and algD reveals a hierarchy of alginate gene expression which is modulated by algT. J Bacteriol 176, 6007-6014.[Abstract]

Received 2 June 2000; revised 21 July 2000; accepted 24 July 2000.