Detection of conversion to mucoidy in pseudomonas aeruginosa infecting cystic fibrosis patients

Compositions and methods for detecting the conversion to mucoidy in Pseudomonas aeruginosa are disclosed. Chronic respiratory infections with mucoid Pseudomonas aeruginosa are the leading cause of high mortality and morbidity in cystic fibrosis. The initially colonizing strains are nonmucoid but in the cystic fibrosis lung they invariably convert into the mucoid form causing further disease deterioration and poor prognosis. The molecular basis of this conversion to mucoidy is also disclosed. The algU gene encodes a protein homologous to an alternative sigma factor regulating sporulation and other developmental processes in Bacillus, and along with the negative regulators mucA and mucB comprises the gene cluster controlling conversion to mucoidy. The switch from nonmucoid to mucoid state is caused by frameshift deletions and duplications in the second gene of the cluster, mucA. Inactivation of mucA results in constitutive expression of genes, such as algD, dependent on algU for transcription. Insertional inactivation of mucB on the chromosome of the standard genetic strain PAO also resulted in mucoid phenotype, and in a strong transcriptional activation of algD. Activation of algD results in increased synthesis of the exopolysaccharide alginate rendering P. aeruginosaa mucoid.

BACKGROUND OF THE INVENTION 
Cystic Fibrosis (CF) is the most common inheritable lethal disease among 
caucasians. There are approximately 25,000 CF patients in the U.S.A. The 
frequency of CF in several other countries (e.g., Canada, United Kingdom, 
Denmark) is high (ranging from 1 in 400 to 1 in 1,600 live births). There 
are numerous CF centers in the U.S.A. and Europe--specialized clinical 
facilities for diagnosing and treating children and adolescents with CF. 
Chronic respiratory infections caused by mucoid Pseudomonas aeruginosa are 
the leading cause of high morbidity and mortality in CF. The initially 
colonizing P. aeruginosa strains are nonmucoid but in the CF lung they 
inevitably convert into the mucoid form. The mucoid coating composed of 
the exopolysaccharide alginate leads to the inability of patients to clear 
the infection, even under aggressive antibiotic therapies. The emergence 
of the mucoid form of P. aeruginosa is associated with further disease 
deterioration and poor prognosis. 
The microcolony mode of growth of P. aeruginosa, embedded in 
exopolysaccharide biofilms in the lungs of CF patients (Costerton et al., 
1983), among other functions, plays a role in hindering effective 
opsonization and phagocytosis of P. aeruginosa cells (Pier et al., 1987; 
Pier 1992). Although CF patients can produce opsonic antibodies against P. 
aeruginosa antigens, in most cases phagocytic cells cannot effectively 
interact with such opsonins (Pressler et al., 1992; Pier et al., 1990; 
Pier 1992). Physical hindrance caused by the exopolysaccharide alginate 
and a functionally important receptor-opsonin mismatch caused by chronic 
inflammation and proteolysis are contributing factors to these processes 
(Pedersen et al., 1990; Tosi et al., 1990; Pier, 1992). Under such 
circumstances, the ability of P. aeruginosa to produce alginate becomes a 
critical persistence factor in CF; consequently, selection for alginate 
overproducing (mucoid) strains predominates in the CF lung. 
Synthesis of alginate and its regulation has been the object of numerous 
studies (Govan, 1988; Ohman et al., 1990; Deretic et al., 1991; May et 
al., 1991). It has been shown that several alginate biosynthetic genes 
form a cluster at 34 min of the chromosome (Darzins et al., 1985), and 
that the algD gene, encoding GDPmannose dehydrogenase, undergoes strong 
transcriptional activation in mucoid cells (Deretic et al., 1987; 1991). 
GDP mannose dehydrogenase catalyzes double oxidation of GDP mannose into 
its uronic acid, a reaction that channels sugar intermediates into 
alginate production. The transcriptional activation of algD has become a 
benchmark for measuring molecular events controlling mucoidy (Deretic et 
al., 1991; Ohman et al., 1990; May et al., 1991). Studies of these 
processes have lead to the uncovering of several cis- and trans-acting 
elements controlling algD promoter activity: (i) The algD promoter has 
been shown to consist of sequences unusually far upstream of the mRNA 
start site (Mohr et al., 1990). These sequences (termed RB1 and RB2), as 
well as a sequence closer to the mRNA start site (RB3) are needed for the 
full activation of algD (Mohr et al., 1990; 1991; 1992). (ii) AlgR, a 
response regulator from the superfamily of bacterial signal transduction 
systems (Deretic et al., 1989), binds to RB1, RB2, and RB3, and is 
absolutely required for high levels of algD transcription (Mohr et al., 
1990; 1991; 1992). (iii) Another signal transduction factor, AlgB, also 
contributes to the expression of genes required for alginate synthesis 
(Wozniak and Ohman, 1991). (iv) The peculiar spatial organization of AlgR 
binding sites imposes steric requirements for the activation process. The 
conformation of the algD promoter appears to be affected by histone like 
proteins [e.g. Alg (H.sub.p 1) (Deretic et al., 1992) and possibly IHF 
(Mohr and Deretic, 1992)], and perhaps by other elements controlling 
nucleoid structure and DNA topology. (v) The algD promoter does not have a 
typical -35/-10 canonical sequence (Deretic et al., 1989). It has been 
proposed that RpoN may be the sigma factor transcribing this promoter; 
however, several independent studies have clearly ruled out its direct 
involvement (Mohr et al., 1990; Totten et al., 1990). The present 
inventors have cloned and characterized a new gene, algU, which plays a 
critical role in algD expression (Martin et al., 1993). The algU gene 
encodes a polypeptide product that shows sequence and domainal 
similarities to the alternative sigma factor Spo0H from Bacillus spp. 
(Dubnau et al., 1988). Spo0H, although dispensable for vegetative growth, 
is responsible for the initial events in the triggering of the major 
developmental processes in Bacillus subtilis, viz. sporulation and 
competence (Dubnau et al., 1988; Dubnau, 1991). These findings suggest 
that activation of alginate synthesis may represent a cell differentiation 
process participating in interconversions between planktonic organisms and 
biofilm embedded forms in natural environments (Martin et al., 1993; 
Costerton et al., 1987). 
Inactivation of algU abrogates algD transcription and renders cells 
nonmucoid, further strengthening the notion that algU plays an essential 
role in the initiation of mRNA synthesis at algD (Martin et al., 1993). 
algU maps in the close vicinity of muc markers that have been demonstrated 
in the classical genetic studies by Fyfe and Govan (1980) to cause the 
emergence of mucoid strains constitutively overproducing alginate. The 
mucoidy-causing property of muc mutations has been based on the ability of 
different muc alleles (e.g. muc-2, muc-22, and muc-25) to confer mucoidy 
in genetic crosses (Fyfe and Govan, 1980; 1983). The present application 
describes the presence of additional genes immediately downstream of algU, 
termed mucA and mucB, which also play a role in the regulation of mucoidy. 
Detection of mucoid P. aeruginosa is a standard practice, however, due to 
the variability in expression of mucoidy on standard clinical media, more 
objective detection methods are needed. An early detection of conversion 
to mucoidy will be possible by using the present invention. 
SUMMARY OF THE INVENTION 
The present invention provides compositions and methods for the early 
detection and diagnosis of the conversion to mucoidy of Pseudomonas 
aeruginosa. 
The present invention also provides a molecular mechanism for the 
conversion from the nonmucoid to the mucoid state, including specific 
sequence alterations that occur in the mucA gene that cause the conversion 
and molecular probes for the early detection of this disease state. 
The present invention provides a composition of matter comprising a first 
polynucleotide having the sequence of FIG. 9A, FIG. 9B, and FIG. 9C or 
FIG. 14, a second polynucleotide complementary to the first polynucleotide 
or a polynucleotide differing from the first or second polynucleotide by 
codon degeneracy. Also claimed is a polynucleotide which hybridizes with 
the first or second polynucleotide, or an oligonucleotide probe for the 
first or second polynucleotide which hybridizes with said polynucleotide. 
A further composition of matter of the present invention is a Pseudomonas 
aeruginosa mucA or mucB gene in substantially pure form. The mucA gene is 
defined as substantially comprising the sequence of FIG. 14. Also claimed 
is a mucA gene defined further as having an altered sequence. The 
alteration may be an insertion or deletion of at least one nucleotide or 
it may be a frameshift mutation. In alteration of the gene results in an 
inactive mucA gene product. In a preferred embodiment of the invention, 
the altered gene sequence has a deletion of nucleotide "A" from position 
371 of the sequence of FIG. 14. 
The polynucleotide may be a polydeoxyribonucleotide or a 
polyribonucleotide. The oligonucleotide may be an oligodeoxyribonucleotide 
or an oligoribonucleotide. A further composition of matter is an 
oligonucleotide useful as a probe for and which hybridizes with the 
polynucleotide sequence of FIG. 14 or sequence alterations thereof. A 
preferred embodiment of the invention is an oligonucleotide comprising a 
sequence complementary to a region spanning the deletion at position 371. 
In particular, the oligonucleotide comprises the sequence 
5'-GGGACCCCCCGCA-3'SEQ ID NO: 2. The altered sequence of the mucA gene may 
have a deletion of the nucleotide "G" from position 439 or 440. One 
skilled in the art would see that since there is a "G" in both positions 
439 and 440 it is not possible to know which "G" is deleted in this 
altered sequence. 
A further embodiment of the present invention is an oligonucleotide 
comprising a sequence complementary to a region spanning the deletion at 
position 439 or 440. In particular, this oligonucleotide comprises the 
sequence 5'-GAGCAGGGGCGCC-3'. 
A further composition of matter of the present invention is a mucA gene 
having an altered sequence wherein the altered sequence is an insertion of 
nucleotides 5'-CAGGGGGC-3' between positions 433 and 434. Also claimed is 
an oligonucleotide comprising a sequence complementary to a region 
spanning the insertion of the nucleotides 5'-CAGGGGGC-3'. A preferred 
embodiment is an oligonucleotide comprising 5'-CAGGGGGCCAGGGGGC-3'. 
A further embodiment of the present invention is the use of these 
compositions of matter for a method of detecting conversion to mucoidy in 
Pseudomonas aeruginosa comprising detecting a loss of mucA or mucB 
function. In particular, a method of detecting conversion to mucoidy in 
Pseudomonas aeruginosa having an inactive mucA or mucB gene product 
comprising the detection of an altered sequence in the mucA or mucB gene 
is claimed. A preferred embodiment is a method of detecting conversion to 
mucoidy in Pseudomonas aeruginosa having an inactive mucA gene product 
comprising the detection of an altered sequence in the mucA gene. In this 
case, the altered sequence encodes an inactive product and the altered 
sequence is detected by hybridization with a complementary 
oligonucleotide. The complementary oligonucleotide may be 
5'-GGGACCCCCCGCA-3'SEQ ID NO: 2, 5'-GAGCAGGGGCGCC-3', or 
5'-CAGGGGGCCAGGGGGC-3'. One skilled in the art could see that an altered 
sequence may comprise nucleotide changes, insertions or deletions anywhere 
within this locus between about positions 300 and 500 of the sequence of 
FIG. 14. 
A further embodiment of the present invention is a method of detecting 
conversion to mucoidy in Pseudomonas aeruginosa having an inactive mucA 
gene comprising the steps of: 1) obtaining Pseudomonas aeruginosa 
suspected of conversion to mucoidy to provide a test sample, and 2) 
hybridizing the test sample with an oligonucleotide 5'-GGGACCCCCCGCA-3'SEQ 
ID NO: 2, 5'-GAGCAGGGGCGCC-3', or 5'-CAGGGGGCCAGGGGGC-3'. Positive 
hybridization indicates conversion to mucoidy in Pseudomonas aeruginosa. 
A further embodiment of the present invention is a Pseudomonas aeruginosa 
algU gene in substantially pure form. The algU gene comprises the sequence 
of FIG. 6. 
ABBREVIATIONS 
PAO1=nonmucoid strain of P. aeruginosa, mucA+ 
PAO568=mucoid strain of P. aeruginosa carrying the muc-2 mutation 
TC.sup.r =tetracycline resistance 
PAO578=mucoid derivative of PAO carrying the muc-22 mutation 
oligO 568 (mucA2)=5'CAGGGGGCCAGGGGGC-3' 
oligo 578 (mucA22)=5'-GAGCAGGGGCGCC-3' 
oligo CFl=5'-GGGACCCCCCGCA-3'SEQ ID NO: 2

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Mucoidy in Pseudomonas aeruginosa is a critical virulence factor associated 
with chronic infections in cystic fibrosis (CF). The initially colonizing 
strains are nonmucoid but once in the CF lung, they almost inevitably 
convert into mucoid phenotype. Three tightly linked genes algU, mucA, and 
mucB have been identified with a chromosomal region shown by genetic means 
to represent the site where mutations cause conversion to mucoidy. 
Mutations causing mucoidy occur in mucA. The complete nucleotide sequence 
of the mucA gene is shown in FIG. 14. The positions of mutations in PAO568 
(muc-2), PAO578 (muc-22), and CF isolates (CF1, CF14, and CF23) are also 
indicated in FIG. 14. The oligonucleotides designed to detect such 
mutations by hybridization are shown in FIG. 12. 
The algU gene plays a positive regulatory role in the transcription of 
algD, a gene encoding GDPmannose dehydrogenase. The algD gene must be 
expressed at high levels in order for cells to attain mucoid phenotype 
(FIG. 17A and FIG. 17B). mucA and mucB play a negative regulatory role, 
and, when active, these genes suppress mucoidy. When either mucA or mucB 
are inactivated, this results in derepression of algD transcription and 
conversion to mucoidy (FIG. 17A and 17B). The present inventors have 
isolated, sequenced, and characterized the entire region containing algU, 
mucA, and mucB. When a clone of algU, mucA and mucB, isolated from 
nonmucoid cells, is placed into mucoid derivatives of the standard genetic 
strain PAO and in CF isolates, it can cause suppression of mucoidy, viz. 
the cells become phenotypically nonmucoid and the algD promoter is 
silenced. Using gene replacements on the chromosome and phage-mediated 
generalized transduction, the present inventors have shown that algU and 
the downstream genes described here as mucA and mucB map at about 67.5 
minutes on the P. aeruginosa chromosome. These genetic markers represent a 
site where mutations causing conversion from nonmucoid to mucoid phenotype 
occur, and have not been previously isolated or characterized. Mutations 
(deletions and insertions) causing frameshift mutations and premature 
termination of the mucA open reading frame have been identified through 
the work described herein (FIGS. 12A, 12B, and FIG. 14). 
Even though the invention has been described with a certain degree of 
particularity, it is evident that many alternatives, modifications, and 
variations will be apparent to those skilled in the art in light of the 
foregoing disclosure. Accordingly, it is intended that all such 
alternatives, modifications, and variations which fall within the spirit 
and the scope of the invention be embraced by the defined claims. 
The following examples are presented to describe preferred embodiments and 
utilities of the present invention, but should not be construed as 
limiting the claims thereof. 
Example 1 describes the characterization of algU, example 2 describes the 
characteristic of mucB and example 3, mucA. 
EXAMPLE 1 
Characterization of a Locus Determining the Mucoid Status of Pseudomonas 
aeruginosa: algU Shows Sequence Similarities with a Sigma Factor from 
Bacillus 
MATERIALS AND METHODS 
Media and bacterial growth. E. coli was grown on LB supplemented with 10 
.mu.g/ml tetracycline (Tc), 40 .mu.g/ml ampicillin (Ap), and 25 .mu.g/ml 
kanamycin (Km) when required. P. aeruginosa was grown on LB, minimal media 
(12,44), and Pseudomonas isolation agar (PIA) (DIFCO). The nitrogen free 
medium (P), used to test the ability to utilize proline (supplemented at 
the concentration of 20 mM) as the sole carbon and nitrogen source, has 
been previously described (44). Other amino acids were supplied as 1 mM 
when necessary. Media for environmental modulation by different nitrogen 
sources (nitrate or ammonia) have been described previously (12,50). 300 
mM NaCl was added to LB when required (12). Antibiotics supplements for P. 
aeruginosa were: 300 .mu.g/ml Tc for PIA, 50 .mu.g/ml Tc for LB and 
minimal media, and 300 .mu.g/ml carbenicillin (Cb) for all media. 
Plasmids and bacterial strains. Strains of P. aeruginosa and plasmids used 
in this study are shown in Table 1. 
TABLE 1 
______________________________________ 
Bacterial strains, plasmids, and bacteriophages. 
Species, 
strain, 
plasmid, 
or phage Relevant properties.sup.a 
Reference 
______________________________________ 
P. aeruginosa 
PAO1 prototroph Alg.sup.- 31 
PAO1293 prototroph Alg.sup.- 55 
PAO568 FP2.sup.+ muc-2 (Alg.sup.+i) leu-38 
24 
PAO578 FP2.sup.+ muc-22 (Alg.sup.+) leu-38 
24 
PAO579 FP2.sup.+ muc-23 (Alg.sup.+) leu-38 
24 
PAO581 FP2.sup.+ muc-25 (Alg.sup.+) leu-38 
24 
PAO540 cys-5605 his-5075 argA171 Alg.sup.- 
24 
PAO669 FP2.sup.+ muc-2 (Alg.sup.+i) leu-38 Cb.sup.4 
This work 
algD.sup.+ algD::xylE 
(Derived from PAO568) 
PAO670 FP2.sup.+ algU::Tc.sup.4 (Alg.sup.-) 
This work 
(Derived from PAO568) 
PAO964 pru-354 ami-151 hut C107 Alg.sup.- 
44 
PAM425 muc-3739 (Alg.sup.+) lys-13 
43 
Plasmids 
pLA2917 IncP1 mob.sup.+ tra cos.sup.+ Tc.sup.r Km.sup.r 
1 
pCMob ColE1 mob.sup.+ (RK2) tra cos.sup.+ 
47 
Ap.sup.4 (Cb.sup.4) Tc.sup.r 
pSF4 Ori (p15A) mob.sup.+ (RK2) cos.sup.+ Tc.sup.r 
57 
pRK2013 ColE1 mob.sup.+ tra.sup.+ (RK2) Km.sup.r 
21 
pT7-5 ColE1 Ap.sup.r .phi.10 promoter-EcoRI- 
60 
polylinker- HindIII 
pT7-6 ColE1 Ap.sup.r .phi.10 promoter-HindIII- 
60 
polylinker-EcoRI 
pGP1-2 Ori (p15A) P.sub.L T7 gene 1 
60 
(T7 RNA polymerase)P .sub.lac -c1857 
Km.sup.r 
pVDZ'2 IncP1 mob.sup.+ tra lacZ' 
9 
(lacZ.sub.--) Tc.sup.r 
pCMR7 algR as 827 bp HindIII-BamHI 
48 
in pT7-6 
pPAOM3 pVDX18 IngQ/P4 algD::xylE Ap.sup.r 
37 
(Cb.sup.4) 
pMO011809 
hisI.sup.+ (cosmid clone in pLA2917) 
55 
pMO012046 
algU.sup.+ (cosmid clone in pLA2917) 
This work 
pDMU1 algU.sup.+ (a 6 kb HindIII-EcoRI 
This work 
fragment from pMO012046 
subcloned on pVDZ'2) 
pDMU4/76 algU.sup.+ as .DELTA.4/76 subcloned 
This work 
on pVDZ'2 
pRCW1 a 6 kb HindIII-NsiI subclone 
This work 
from the cosmid pMO011809 
pDMU100 pUC12 mob.sup.+ algU::Tc.sup.r Ap.sup.r 
This work 
(Cb.sup.4) 
pDMDX pCMobB algD::xylE mob.sup.+ Ap.sup.r 
(Cb.sup.4) 
Phages 
F116L Generalized transduction phage 
40 
______________________________________ 
.sup.a Alg.sup.+i, inducible production of alginate resulting in mucoid 
phenotype (12). Alg.sup.+, mucoid phenotype, Alg.sup.-, nonmucoid 
phenotype. 
Strains PAO669 and PAO670 were derivatives of P. aeruginosa PAO568 (muc- 
2). The strain PAO669 was generated by integration of a nonreplicative 
plasmid carrying an algD::xylE fusion on the chromosome of PAO568. An 11.5 
kb HindIII fragment carrying algD with xylE inserted in the XhoI site of 
algD, was cloned in the HindIII site of pCMobB (47), and the resulting 
plasmid pDMDX conjugated into PAO568. pCMobB and its derivative pDMDX 
cannot replicate in Pseudomonas but can be effectively mobilized into this 
bacterium (47). Cb.sup.r exconjugants were obtained and tested for the 
presence of other plasmid markers [development of a yellow color when 
sprayed with a solution of catechol (37)] and insertions on the chromosome 
verified by Southern blot analysis. The strain PAO669 was mucoid and 
produced alginate on inducing media. PAO670, a strain used to determine 
effects of the inactivation of algU on the chromosome, was constructed by 
gene replacement of the chromosomal algU with an insertionally inactivated 
algU (algU::Tc.sup.r). This was accomplished as follows: A 2.4 kb 
HindIII-EcoRI fragment from .DELTA.U4/76 was inserted into pUC12. The 
resulting construct was digested with EcoRV, and NotI linkers were added. 
A NotI modified Tc.sup.r cassette (32) was inserted, and the resulting 
plasmid digested with EcoRI. Into this site an 1.4 kb EcoRI fragment with 
mob from pCMobA (originating from pSF4) (47,57) was inserted to produce 
pDMU100. This plasmid was transferred into P. aeruginosa PAO568 by 
conjugation and exconjugants selected on PIA supplemented with Tc. Since 
pUC12 and its derivative pDMU100 cannot replicate in Pseudomonas, Tc.sup.r 
strains had this plasmid integrated on the chromosome via homologous 
recombination. Double cross-over events were identified as Tc.sup.r 
Cb.sup.s strains, chromosomal DNA extracted, digested with appropriate 
enzymes, and gene replacements verified by Southern blot analysis. CF 
strains were from a combined collection of mucoid isolates from CF 
patients in Edinburgh, Scotland, and San Antonio, Texas. Cosmid clones not 
shown in Table 1 are described in Results. The source of regA was a 1.9 kb 
PstI-XhoI subclone in mp18 (30). The use of E. coli strains for subcloning 
in pVDZ2 (JM83), triparental conjugations (HB101 harboring pRK2013), and 
deletion subcloning (WB373) has been described elsewhere (14,38). 
Nucleic acids manipulations and recombinant DNA methods. All DNA 
manipulations and Southern blot analyses were according to the previously 
published methods (14,38,50,55) or standard recombinant DNA procedures 
(3). Radiolabeled probes (3) were generated using random priming labeling 
method and [.alpha.-.sup.32 P]dCTP (3,000 Ci/mmol; DuPont NEN). RNA 
extraction and S1 nuclease analysis have been previously published 
(14,38). The construction of the cosmid clone library has been reported 
(55). Overlapping deletions of the clones in M13 were generated as 
previously described (14). DNA was sequenced by a modification of the 
chain termination method with the substitution of dGTP by its analog 
7-deaza-dGTP to avoid compressions as previously described (38), and using 
17 bp or custom made primers when needed. Similarity searches were 
performed using FASTA program (52) and GenBank databases, as well as 
through NBRF-PIR protein identification resource network server. 
Genetic methods. clones made in broad host-range plasmids (pVDX18 and 
pVDZ'2) were transferred into P. aeruginosa by triparental filter matings 
as described previously (37), using E. coli harboring pRK2013 as the 
helper. Cosmid clones were mobilized into P. aeruginosa from E. coli S17-1 
(58) as previously reported (55). Generalized transduction using Fl16L 
(40) was performed as follows: Serially diluted (to achieve near 
confluency) single plaque preparations of Fl16L were grown mixed with the 
donor strain in top agar for 17 h at 37.degree. C. The top agar was 
scraped and phage eluted in equal volume of TNM (10 mM Tris-HCl pH 7.4, 
150 mM NaCl, 10 mM MgSO.sub.4), centrifuged at 9000 rpm in SM24 rotor, and 
supernatant filtered through a 0.45 .mu.m membrane to generate transducing 
phage stock (used within one month). 500 .mu.l of freshly grown overnight 
recipient cells was incubated with 500 .mu.l of transducing phage stock 
(diluted to 5.times.10.sup.9 ; multiplicity of infection 5:1) for 20 min 
at 37.degree. C. Cells were centrifuged for 1 min in a microcentrifuge and 
resuspended in 1 ml of TNM. Aliquots were plated on selective media and 
incubated for 1 to 2 days, strains purified on selective media, and then 
spot tested for coinheritance of unselected markers. 
Enzyme and alginate assays and scoring of suppression of mucoidy. Catechol 
2,3-dioxygenase (CDO), the gene product of xylE, was assayed in cell-free 
sonic extracts as previously described (37). The activity was monitored in 
50 mM phosphate buffer (pH 7.5)-0.33 mM catechol by following the increase 
of A.sub.375 in a Shimadzu UV160 spectrophotometer. The molar extinction 
coefficient of the reaction product, 2-hydroxymuconic semialdehyde, is 
4.4.times.10.sup.4 at 375 nm. Suppression of mucoidy by plasmid borne 
genes was monitored on PIA plates unless specified otherwise, and the 
phenotypic appearance of the colonies scored as mucoid or nonmucoid. A 
control strain harboring the vector without an insert was always used for 
comparison. Alginate was assayed by a colorimetric method (36). 
Visualization of gene products using T7 RNA polymerase/promoter system. 
Polypeptides encoded by cloned genes were visualized by expression in E. 
coli using a temperature-inducible T7 expression system (plasmid vectors 
pT7-5 and pT7-6 and T7 RNA polymerase encoded by pGP1-2) (60) and protein 
labeling with [.sup.35 S]methionine and [.sup.35 S]cysteine (Expre.sup.35 
S.sup.35 S protein labeling mix; 1000 Ci/mmol; DuPont NEN) with previously 
described modifications (38,47). Proteins were separated on 12% sodium 
dodecyl sulfate-polyacrylamide gels. .sup.14 C-labeled methylated proteins 
(Amersham) were used as molecular weight standards. The gels were fixed in 
10% acetic acid, washed with H.sub.2 O, impregnated with 1M salicylic 
acid, and bands representing radiolabeled polypeptides detected by 
autofluorography at -70.degree. C. 
Pulsed-field gel electrophoresis and Southern blot analysis. Localization 
of genes on the SpeI map of P. aeruginosa PAO was performed by previously 
published methods (55,59). Identification of SpeI fragments was done by 
comparison to the lambda phage concatameric ladder ranging in size from 
48.5 to 582 kb (55) as well as based on the hybridization to the 
previously mapped genes (55,59). 
Nucleotide Sequence accession number. The sequence reported here has been 
deposited in GenBank (accession number LO2119). 
RESULTS 
Isolation of cosmid clones affecting mucoidy in trans. Several genetic 
studies have indicated that muc loci have the property to affect mucoidy 
when present in trans. For example, it has been observed that R' 
derivatives of R68.45, which carry pruAB.sup.+ and an adjacent muc locus 
from a nonmucoid PAO strain, are capable of switching off (suppressing) 
alginate production in mucoid strains PAO568, PAO578, and PAO581 (23). 
This effect appeared to be specific since another mucoid PAO derivative, 
strain PAO579, was not affected (23). This suggested to the present 
inventors that changes in mucoidy could be used as a screening tool to 
clone and isolate additional regulatory genes. A generation of a 
comprehensive genomic library from P. aeruginosa has been reported 
previously (55). Several cosmids from this library have been successfully 
used for construction of a combined physical and genetic map of P. 
aeruginosa PAO (55). This cosmid library was constructed in pLA2917 (which 
can replicate in P. aeruginosa) using DNA from a derivative of the strain 
PAO1 (nonmucoid) (31,55). The library was introduced into several mucoid 
strains by conjugation and ten independent and nonoverlapping clones 
capable of altering the mucoid character were isolated: pMO010533, 
pMO010921, pMO011021, pMO011537, pMO011644, pMO011744, pMO011801, 
pMO011809, pMO011920, and pMO012046. Two of the clones had previously been 
described as carrying other genetic markers (55). pMO011809 contains hisI 
and has been used to demonstrate that this locus resides on the SpeI 
fragment E (FIG. 1A and FIG. 1B, 360 kb) in the late region of the 
chromosome (55). In the same study, pM0011644 was shown to carry the oruI 
gene, also mapping in the late region of the chromosome, but hybridizing 
to a different SpeI fragment (FIG. 1A and FIG. 1B, 330 kb; fragment F). 
One of the clones, pMO012046, rendered a significant number of strains 
completely nonmucoid, and was chosen for further study. The locus 
affecting alginate production on this chromosomal fragment was designated 
algU. 
Deletion mapping of the algU locus. In order to facilitate molecular 
characterization of algU, this locus was examined by deletion mapping. The 
subcloning of the ability of algU to suppress alginate production and 
mucoid phenotype was done using the broad host range subcloning vector 
pVDZ'2 (9). Initially, a 6 kb HindIII-EcoRI fragment from pMO012046 was 
found to carry the suppressing activity, and was subjected to further 
deletion mapping. Two series of consecutive overlapping deletions were 
produced from each end of the 6 kb fragment (FIG. 2A and FIG. 2B), using 
the previously described deletion-subcloning strategy (14). Subclones of 
these deletion products in pVDZ'2 were transferred by conjugation into 
PAO568, a mucoid derivative of the standard genetic strain PAO (24). The 
exconjugants were screened for the loss of mucoid character. A summary of 
this analysis is shown in FIG. 2A. All deletion clones which retained the 
suppressing activity caused phenotypically indistinguishable effect; all 
negative deletions completely lost the ability to affect mucoidy. The 
activity was delimited to a region demarcated by the endpoints of 
deletions .DELTA.U4/76 and .DELTA.UM9. 
algU has a strain-specific effect on suppression of mucoidy. It has been 
shown that different mucoid PAO derivatives and clinical CF isolates 
display significant differences in algD promoter activity and alginate 
production in response to modulation by environmental stimuli, such as the 
salt concentration in the medium or growth on nitrate (12). For example, 
the algD promoter in strains PAO568 and PAO578 is induced by salt or 
growth on nitrate (12), although the effects differ in magnitude. PAO568 
and PAO578 carry muc determinants designated muc-2 and muc-22 (24), 
respectively, which map close to each other and to pruAB (23,25). PAO579 
has a different muc locus (designated muc-23) which maps between hisI and 
proB (FIG. 1A and FIG. 1B) and displays a completely opposite response to 
increased salt concentration in the medium when compared to PAO568 and 
PAO578 (12). Another possibly different muc locus is represented by 
muc-3739 (strain PAM425) (43). When the plasmid pDMU1, containing an 
active algU locus on the 6 kb HindIII-EcoRI insert in pVDZ'2 was 
introduced into a panel of strains representative of different mucoid PAO 
derivatives and CF clinical isolates, a specific pattern of suppression of 
mucoidy was observed (Table 2). 
TABLE 2 
______________________________________ 
Strain specific suppression of mucoidy by algU. 
Plasmid.sup.b 
pRCW1 
Suppression of 
Strain.sup.a 
pVDZ'2 pDMU1 mucoidy.sup.c 
______________________________________ 
PAO568 (muc-2) 
- + - 
PAO578 (muc-22) 
- + - 
PAO581 (muc-25) 
- + - 
PAO579 (muc-23) 
- - - 
PAM425 (muc-3739) 
- - + 
CF strains - (18/18).sup.d 
+ (7/18).sup.e 
+ (3/8).sup.f 
______________________________________ 
.sup.a PAO strains are isogenic mucoid derivatives of P. aeruginosa PAO38 
carrying different mapped muc markers (24) (FIG. 1A and FIG. 2). PAM425 i 
a cross between PAO and a mucoid clinical P. aeruginosa isolate, Ps3739 
(43); the corresponding muc3739 locus has been mapped (43) (FIG. 1A and 
FIG. 1B). CF strains were mucoid P. aeruginosa isolates from different 
cystic fibrosis patients. 
.sup.b pDMU1 is algU from PAO1 cloned as a 6 kb HindIIIEcoRI fragment on 
the broad host range vector pVDZ'2 (9). pRCW1 is a subclone of a 6 kb 
HindIIINsiI fragment (see Results) from pMO011809 in pVDZ'2. 
.sup.c Suppression was scored on PIA supplemented with Tc as + (transitio 
from mucoid to nonmucoid status when harboring the plasmid) or - (the 
strain remained mucoid when harboring the plasmid). 
.sup.d Of 18 strains tested none were affected by the vector pVDZ'2. 
.sup.e,f Of 18 strains tested (denominator), 7 lost mucoidy when harborin 
PDMU1; of 8 strains (denominator) in which pRCW1 was introduced, 3 lost 
mucoidy. The strains affected by pDMU1 were different from those affected 
by pRCW1, except in one case with variable results. Not all strains teste 
with pRCW1 were tested with pDMU1 and vice versa. 
pDMU1 rendered muc-2, muc-22 and muc-25 strains (PAO568, PAO578, and 
PAO581) nonmucoid. In contrast, it had no detectable effect on the muc-23 
strain PAO579 and a muc-3739 strain (PAM425). It also affected a 
substantial number of mucoid clinical isolates (7 out of 18 tested). 
Congruent with these results was the finding that the mucoid phenotype of 
some of the strains not affected by algU were affected by a different 
clone. For example, the strain PAM425 which was not affected by pDMU1 lost 
its mucoid character when pRCW1, containing a 6 kb HindIII-NsiI subclone 
from the cosmid pMO011809 (55), was introduced (Table 2). pRCW1 affected 3 
out of 8 CF isolates tested. Thus, the CF strains fell into three 
categories: (i) Those affected by pDMU1, (ii) those affected by pRCW1, and 
(iii) those not affected by either of the plasmids. 
The results presented in this section indicated that: (i) The suppression 
of mucoidy in trans was strain dependent; (ii) algU affected a significant 
number of CF isolates; and (iii) there was a correlation between different 
muc linkage groups and different clones exerting effects. 
Two polypeptides, P27 and P20, are encoded by the region affecting mucoidy 
in muc-2, muc-22, and muc-25 strains. Since deletion inactivation of the 
algU locus from either end had similar effects, suppression of mucoidy was 
unlikely to be due to the titration of a diffusable factor (e.g. AlgR) by 
its binding to DNA. Whether this locus had a coding capacity for a 
possible trans-acting factor was tested by analysis of [.sup.35 S] 
methionine and [.sup.35 S] cysteine labeled polypeptides encoded by the 
insert in a T7 expression system. The results of these studies are 
illustrated in FIG. 3A and FIG. 3B. Two polypeptides, with apparent 
M.sub.r of 27.5 kDa (P27) and 20 kDa (P20) were observed as encoded by the 
algU containing DNA fragment. The consecutive deletions were then used to 
establish the order of genes and their importance for the suppressing 
activity (FIG. 3A and FIG. 3B). Deletions extending from the HindIII end 
abolished P27 synthesis while not affecting P20, thus establishing the 
order of genes as P27 followed by P20. The gene encoding P27 was 
designated algU. Deletion .DELTA.U4/33, which lost the ability to produce 
P27, but still directed the synthesis of P20, was no longer capable of 
suppressing mucoidy. Thus, algU was necessary for the activity of this 
region. 
Suppression of mucoidy by algU is exerted at the level of algD 
transcription. Both algD and algR undergo transcriptional activation in 
mucoid cells (14). The difference in transcription is very profound at the 
algD promoter, which remains silent in nonmucoid cells and is highly 
active in mucoid strains (11,12,14). algR is transcribed from two 
promoters, one distal and constitutive (47,50), and the other proximal and 
induced in mucoid cells (14). We investigated whether the presence of algU 
affected transcription of algD and algR. To assay algD transcription under 
different conditions in the presence of algU on a plasmid, we first 
constructed a transcriptional fusion of algD and xylE [used as a reporter 
gene (37)] on the chromosome of PAO568. The strain was constructed as a 
merodiploid for algD, with one intact copy of algD while the other was 
inactivated due to the fusion with xylE (strain PAO669; for construction 
details see Materials and Methods). 
The parental strain PAO568 (24) has a remarkable feature in that it 
displays a broad dynamic range of algD expression (12). Both algD 
transcription and colony morphology (changing from nonmucoid to mucoid) 
respond dramatically to inducing conditions (high salt concentration in 
the medium or growth on nitrate) (12). The strain PAO669 retained these 
properties (since PAO669 was merodiploid for algD it could synthesize 
alginate). The induction of algD on the chromosome of PAO669 was analyzed 
to verify the previously established parameters of algD response to 
environmental conditions (12,37,50). The results of xylE fusion assays and 
phenotypic induction of mucoidy indicated that the chromosomal fusion 
reacted to environmental modulation in the same manner previously reported 
for algD-xylE fusions on plasmids (Table 3). 
TABLE 3 
__________________________________________________________________________ 
Effects of plasmid borne algU from PAO1 on algD transcription in the 
muc-2 
background. 
Growth conditions.sup.c 
Strain and LB LB + NaCl 
NH.sub.4 
NO.sub.3 
plasmids.sup.a 
Phenotype.sup.b 
CDO (U/mg).sup.d 
__________________________________________________________________________ 
PAO669 [None] 
M 0.43 (ND) 
2.84 (ND) 
0.22 (.+-.0.02) 
5.69 
(.+-.1.19) 
PAO669 [pVDZ'2] 
M 0.76 (.+-.0.14) 
4.61 (.+-.1.19) 
0.59 (.+-.0.10) 
3.25 
(.+-.0.47) 
PAO669 [pDMU4/76] 
NM 0.39 (.+-.0.08) 
0.40 (.+-.0.08) 
0.20 (.+-.0.03) 
0.20 
(.+-.0.02) 
__________________________________________________________________________ 
.sup.a PAO669 is a derivative of PAO568 (muc2) in which an algDxylE fusio 
has been placed on the chromosome. The plasmid pDMU4/76 was constructed b 
cloning the deletion product U4/76 (FIG. 2A and 2B) into pVDZ'2. This 
plasmid suppresses mucoidy in muc2, muc22, and muc25 PAO derivatives. 
.sup.b Phenotype was scored on inducing media (PIA, LB + NaCl and 
NO.sub.3). M, mucoid; NM, nonmucoid. 
.sup.c Growth conditions and media were as previously reported (12). LB + 
NaCl, LB supplemented with 300 mM NaCl. NH.sub.4 and NO.sub.3, minimal 
media with ammonia or nitrate as the nitrogen source, respectively. The 
composition and the use of these media for algD induction have been 
previously described (12,50). 
.sup.d Activity of catechol 2,3 dioxygenase (CDO), the xyle gene product, 
was determined in cell free extracts as previously described (37). One 
unit of CDO is defined as the amount of enzyme that oxidizes 1 .mu.mol of 
catechol per min at 24.degree. C. .+-., standard error; ND, not 
determined. 
When plasmid pDMU4/76, carrying algU and capable of suppressing mucoidy, 
was introduced into PAO669, this resulted in a complete loss of alginate 
synthesis and algD transcription. No induction was observed in response to 
environmental stimuli known to induce algD in PAO568 (12) (Table 3). When 
PAO669 harboring pDMU4/76, which displayed nonmucoid colony morphology, 
was transferred to a medium that no longer supplied selective pressure for 
plasmid maintenance, colonies segregated into outgrowing mucoid and 
nonmucoid sectors (data not shown). This was accompanied by a loss of the 
plasmid in mucoid segregants, as evidenced by the loss of Tc.sup.r in such 
cells. The Tc.sup.s bacteria (devoid of pDMU4/76) had algD activity 
restored, as indicated by activities of the chromosomal algD- xylE fusion 
in strains purified from the corresponding sectors. The mucoid segregants 
grown on PIA showed CDO (the xylE gene product) activities ranging from 
1.76-2.01 U/mg, while the nonmucoid strains originating from the same 
colonies had CDO activities ranging from 0,401-0.445 U/mg of protein in 
crude cell extracts. The effects of algU on algD was confirmed by S1 
nuclease protection analysis of algD mRNA levels (data not shown). The S1 
nuclease protection experiments also indicated that neither of the algR 
promoters were affected in PAO568 harboring algU on a plasmid (not shown). 
These results strongly suggested that the effect of algU on mucoidy was at 
the level of algD transcription. 
Insertional inactivation of the algU locus on the chromosome of PAO568 
renders cells nonmucoid and abrogates algD transcription. The experiments 
presented in the previous sections were not sufficient to conclude that 
algU participates in algD promoter regulation under normal circumstances. 
In order to investigate this possibility, and to explore whether algU is a 
positive or a negative regulator of algD transcription, we insertionally 
inactivated this locus on the chromosome. Transposon mutagenesis of algU 
on a plasmid using Tn5 proved to be uninterpretable, possibly due to the 
reported instability of Tn5 in Pseudomonas (26) and was not pursued 
further. Instead, a Tc.sup.r cassette was inserted into a conveniently 
located restriction site within the algU region. These experiments were 
performed as follows: (i) The presence of two closely spaced EcoRV sites 
(FIG. 2A and FIG. 2B) was noted in the region where the gene encoding P27 
(algU) resided. This was based on the estimated size of the gene needed to 
encode a 27.5 kDa polypeptide, the detailed mapping of the coding region 
of algU using T7 expression studies (summarized in FIG. 2B), and was 
further confirmed by DNA sequence analysis (see later sections). Since the 
endpoint of the last positive deletion still producing P27 was located 540 
bp upstream from the first EcoRV site, we concluded that this site must be 
within the algU coding region. (ii) A suicide plasmid (pDMU100) was 
constructed (see Materials and Methods) in which the 2.4 kb HindIII-EcoRI 
fragment from .DELTA.U4/76 was placed on pUC12 which cannot replicate in 
P. aeruginosa. EcoRV sites within the algU insert were converted into NotI 
specificity, and a Tc.sup.r cassette (32), modified as a NotI fragment, 
was inserted. After addition of a DNA fragment with the mob functions to 
facilitate plasmid mobilization into Pseudomonas (57), the final construct 
(pDMU100) was conjugated into PAO568 and Tc.sup.r exconjugants were 
selected. These strains were expected to have the plasmid with 
algU::Tc.sup.r integrated on the chromosome via homologous recombination. 
Two possible types of recombinants were anticipated: (i) Merodiploids for 
algU, retaining an active algU copy, which would have an insertion of the 
entire plasmid as the result of a single cross-over event; and (ii) true 
gene replacements, products of double cross-overs, in which case the 
plasmid moiety and the associated markers would be lost. We have observed 
in other gene replacement studies using this procedure that double 
cross-over events on the P. aeruginosa chromosome are frequent and that 
they range from 10% to 70% for various genes studied (unpublished 
results), obviating in all cases examined the need for a positive 
selection against markers encoded by the plasmid moiety. In 9 independent 
experiments with algU::Tc.sup.r, 1663 Tc.sup.r exconjugants were examined. 
Of these 29% lost Cb.sup.r encoded by the plasmid moiety, indicative of 
double cross-over events. All such Tc.sup.r Cb.sup.s strains were 
nonmucoid and did not produce alginate under any of the conditions tested. 
Most of the colonies with Tc.sup.r and Cb.sup.r markers (results of single 
cross-over events and thus expected to have a functional copy of algU) 
were mucoid, while a portion of such strains showed a delayed mucoid 
phenotype (mucoidy was developing after 3-4 days as compared with 48 hours 
needed for the parental strain PAO568). Further experiments with Tc.sup.r 
Cb.sup.s recombinants using Southern blotting analysis confirmed that 
these nonmucoid strains had a true gene replacement with the chromosomal 
copy of algU disrupted by the Tc.sup.r cassette (FIG. 4A and FIG. 4B). 
Moreover, when the mutation in such strains was purified by transduction 
(using the generalized transducing phage F116L) into the parental strain 
PAO568, all Tc.sup.r transductants displayed nonmucoid phenotype. One of 
the algU::Tc.sup.r derivatives characterized in these experiments (strain 
PAO670) was used to investigate algD transcription. This time, the 
previously characterized algD-xylE fusion plasmid pPAOM3 (37) was 
introduced into PAO670, and algD promoter activity assayed. 
TABLE 4 
______________________________________ 
Analysis of algD transcription in PAO670 (algU::Tc.sup.r). 
Growth conditions.sup.b 
PIA LB + NaCl NO.sub.3 
Strain and plasmid.sup.a 
CDO (U/mg).sup.c 
______________________________________ 
PAO568 [pPAOM3] 
12.10 11.54 10.95 
PAO670 [pPAOM3] 
1.02 1.85 1.40 
______________________________________ 
.sup.a PAO568 (muc2) is the mucoid parental strain of PAO670. PAO670 has 
algU insertionally inactivated on the chromsome. Both strains harbored th 
algDxylE transcriptional fusion plasmid pPAOM3. 
.sup.b PIA is a rich medium on which all mucoid strains, including PAO568 
present their mucoid phenotype. Other media induce mucoidy and algD 
transcription in PAO568 (12) and are defined in Table 3. 
.sup.c CDO, catechol 2,3 dioxygenase. Relative error did not exceed 20%. 
These results (Table 4) indicated that inactivation of the algU locus on 
the chromosome resulted in a loss of algD transcription, and strongly 
suggested a positive role for algU in algD expression. 
Genetic and physical mapping of algU indicates its close linkage or 
identity with a subset of muc loci. Plasmid borne algU showed specific 
suppression of mucoidy in strains containing muc- 2 and muc-22. These and 
other muc loci have been suggested to participate in the emergence of 
mucoid strains (24,43), although their nature and the mechanism of action 
have not been studied. Extensive information is available on the linkage 
of muc to genetic markers within the late region of the PAO chromosome 
(23,24,25,43) (FIG. 1A and FIG. 1B). Of particular significance is the 
cotransducibility of muc-2 and muc-22 with the pru-354 marker [a mutation 
in pruAB, genes required for the utilization of proline as the sole carbon 
and nitrogen source (44)] demonstrated by F116L bacteriophage mediated 
genetic exchange (23,25). This indicates that these muc loci and the pruAB 
genes are very close, since F116L can transduce regions in the range of 
one min of the chromosome. 
The present inventors proceeded to localize algU on the chromosome: The 
first approach was based on the recently determined physical map of P. 
aeruginosa PAO (55); in these experiments algU was used as a probe for 
Southern hybridization analysis of SpeI fragments separated by pulsed 
field gel electrophoresis. The second approach was to map algU via F116L 
transduction; in this case we took the advantage of having a strain 
(PAO670) with the algU gene on the chromosome tagged with the Tc.sup.r 
cassette and monitored the coinheritance of pruAB with Tc.sup.r. 
The results of the Southern blot analyses with SpeI digested PAO chromosome 
subjected to separation by pulse field gel electrophoresis are illustrated 
on a gel in FIG. 5. As explained in the figure legend, several 
consecutively applied probes were used to confirm identification of the 
SpeI fragments. The algU gene hybridized to the 330 kb SpeI fragment (#6, 
F) known to carry two genetic markers linked to muc-2 and muc-22, viz. 
pur-70 at 66 min, and pruAB at 67.5 min (55). This indicated that algU may 
be close to the muc-2 and muc-22 markers. To explore this possibility, 
cotransducibility of pruAB with algU::Tc.sup.r was tested. The results of 
transductional crosses between PAO670 [algU::Tc.sup.r on the chromosome of 
PAO568 (muc-2)] and PAO964 (pru-354), a mutant in pruAB which cannot grow 
on proline as the sole carbon and nitrogen source, revealed a high degree 
of coinheritance of pruAB with algU::Tc.sup.r (Table 5). 
TABLE 5 
______________________________________ 
Cotransduction of algU and pruAB.sup.a. 
% coinheritance of 
Selected the unselected marker.sup.c 
Donor .times. Recipient 
marker.sup.b 
Tc.sup.r Mucoidy 
______________________________________ 
PAO670 .times. PAO964 
pru-354.sup.+ 
20.3 0 (&lt;0.3%) 
PAO670 .times. PAO540 
hisI.sup.+ 
0 (&lt;0.25%) 0 (&lt;0.25%) 
______________________________________ 
.sup.a F116L transduction was performed using an algU::Tc.sup.r derivativ 
of PAO568 (muc2) (strain PAO670) as the donor, and PAO964 (pru354) or 
PAO540 (cys5605 his5075 argA17l) as recipients. PAO670 is nonmucoid due t 
the inactivation of algU by the insertion of Tc.sup.r cassette. PAO964 an 
PAO540 are nonmucoid. 
.sup.b pru354 is a mutant allele of pruAB (44). PAO964 (pru354) cannot 
grow on proline as the sole carbon and nitrogen source. The selection was 
performed for pruAB.sup.+ or hisI as described in Materials and Methods. 
.sup.c pruAB.sup.+ transductants (300 colonies) and hisI transductants 
(400 colonies) were tested for coinheritance of Tc.sup.r. Tc.sup.r in 
transduction crosses originates from algU::Tc.sup.r on the PAO670 
chromosome. No strain displayed mucoid character in at least two 
independent transduction experiments. In a reciprocal experiment, in whic 
Tc.sup.r was the selected marker, a 50% coinheritance of pruAB.sup.+ with 
Tc.sup.r was observed (not shown). 
The % coinheritance of Tc.sup.r with pruAB corresponded closely to the 
values previously reported for muc-2 and muc-22 (20-49%) (23,25). In a 
control experiment, no coinheritance of hisI and Tc.sup.r was observed 
using the same transducing phage lysates (Table 5). Significantly, no 
mucoid transductants (expected from the transfer of muc-2) among over 700 
colonies examined were observed in these crosses regardless whether the 
selection was for pru.sup.+ or Tc.sup.r. This was in sharp contrast with 
the results obtained with the recipient strain PAO964 and the donor strain 
PAO568 (muc-2; the strain parental to PAO670). Normally, 49% of the 
pru.sup.+ colonies are mucoid in transductions involving PAO568 and 
PAO964 (23,25). Although PAO568 in our hands had the capacity to transfer 
the muc-2 marker conferring mucoidy upon the recipient cells, its 
algU::Tc.sup.r derivative PAO670 completely lost this ability. This effect 
could be attributed to the insertional inactivation of algU in PAO670. 
These results indicate that algU is in the close vicinity of the muc loci 
represented by muc-2 and muc-22 and may even be allelic with these 
determinants. 
AlgU shows sequence similarity with SpoOH, a sigma factor required for 
developmental processes in Bacillus subtilis. In order to gain information 
about the nature and possible function of genetic elements within the algU 
region, the nucleotide sequence of the DNA region from the endpoint of the 
deletion ?U4/76 (the last 5' deletion positive for suppression of mucoidy 
and synthesis of P27) and extending through one of the EcoRV sites used 
for insertional inactivation of algU was determined (FIG. 6). An open 
reading frame was identified within the region defined as algU by deletion 
and functional mapping. This sequence contained translational initiation 
signals, conformed with Pseudomonas codon usage (63), and was in the 
direction of transcription determined in T7 expression studies. When a 
global homology search was performed using the translated sequence of algU 
with GenBank and NBRF databases, two known proteins showed statistically 
significant similarity with AlgU: SpoOH from B. licheniformis and B. 
subtilis. SpoOH is dispensable for growth, and is primarily required for 
initiation of sporulation and other developmental processes (competence) 
in B. subtilis (20,62). The sequence similarity observed (24.9% identity 
over the entire length of both sequences, and the optimized score of 155), 
although limited, was equivalent to the extent of similarity of 
sigma.sup.H to other known sigma factors (ranging between 22% and 31% 
identity with optimized scores between 113 and 145) (20). All regions 
noted in several sequence compilations and alignments of sigma factors 
(29,41) were represented in the regions of homology between Spo0H and 
AlgU. The predicted pI of AlgU was 5,315, similar to the pI of SpoOH 
(5.052-5.146). A relatively low pI is characteristic of sigma factors (45) 
and is known to cause anomalous mobility of several members of this class 
of proteins during SDS-PAGE (45). This may help explain a discrepancy in 
the observed electrophoretic mobility corresponding to 27.5 kDa and 
predicted M.sub.r of AlgU from the sequence (22,194 Da) which is in the 
range of discrepancies reported for several sigma factors (45). B. 
subtilis sigma.sup.H shows electrophoretic mobility corresponding to 30 
kDa, while its predicted M.sub.r is 25,331 (5). The pertinent parts of the 
following references for this example are incorporated by reference 
herein. 
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EXAMPLE 2 
Differentiation of Pseudomonas aeruginosa into the Alginate Producing Form: 
Inactivation of mucB Causes Conversion to Mucoidy 
This example further characterizes genes within the chromosomal region at 
67.5 min which play a critical role in determining the mucoid status of P. 
aeruginosa. Two new genes within this locus, mucA and mucB, have been 
identified, characterized, and demonstrated to participate in the control 
of mucoidy. 
EXPERIMENTAL PROCEDURES 
References cited herein are listed at the end of the specification. 
Bacterial strains. plasmids and growth conditions. All strains of 
Pseudomonas aeruginosa used in this study are derivatives of the standard 
genetic strain PAO1. PAO671 was generated by the insertional inactivation 
of mucB (mucB::Tc.sup.r) on the chromosome of the nonmucoid parental 
strain PAO381 (FP2.sup.+ leu-38 mucA.sup.+ mucB.sup.+ ; Fyfe and Govan, 
1980). This was accomplished using a previously described procedure for 
allele replacement (Martin et al., 1993). A 2.4 HindIII-EcoRI fragment 
(U4/76) was inserted into pUC12. A BglII fragment containing the Tc.sup.r 
cassette was cloned into the unique BglII site within the mucB open 
reading frame. Next a 1.4-kb EcoRI fragment with mob from pCMobA (Mohr and 
Deretic 1990; Selvaray et al., 1984) was inserted into a unique EcoRI site 
resulting in pDMB100. This plasmid was transferred by triparental 
conjugations into PAO381 to generate PAO671, and additionally into three 
other nonmucoid PAO strains. Exconjugants were selected on PIA 
supplemented with tetracycline and double crossovers were identified as 
Tc.sup.r and Cb.sup.s. In all cases, Tc.sup.r Cb.sup.s exconjugants were 
mucoid, while Tc.sup.r Cb.sup.r (single crossovers) were nonmucoid. Gene 
replacements in Tc.sup.r Cb.sup.s strains (all mucoid) were verified by 
Southern blot analysis. 
P. aeruginosa was grown on LB and Pseudomonas Isolation agar (PIA, Difco). 
Antibiotic supplements for P. aeruginosa were 300 .mu.g/ml tetracycline 
for PIA, 50 .mu.g/ml of tetracycline for LB and 300 .mu.g/ml carbenicillin 
for all media. Escherichia coli was grown on LB supplemented with 
tetracycline (10 .mu.g/ml), ampicillin (40 .mu.g/ml) and kanamycin (25 
.mu.g/ml) when required. All incubation were at 37.degree. C. 
Nucleic acid manipulations and recombinant DNA techniques. All recombinant 
DNA manipulations and Southern blot analysis were carried out using 
standard procedures (Ausubel et al., 1989; Martin et al., 1993). DNA 
sequencing was carried out using the United States Biochemical Sequenase 
kit with 7-deaza GTP. 
Labeling and detection of the mucB gene product. The gene product of mucB 
was specifically labeled and expressed in E. coli using a 
temperature-inducible T7 RNA polymerase/promoter expression system 
(plasmid vectors pT7-6 or pT7-5 and T7 RNA polymerase encoded by pGP1-2) 
(Tabor and Richardson 1985). Nascent polypeptides were labeled with 
[.sup.35 S]methionine and [.sup.35 S]cysteine (Expre.sup.35 S.sup.35 S 
protein labeling mix; 1,000 Ci/mmol; DuPont NEN). Proteins were separated 
on a sodium dodecyl sulfate (SDS)-12% polyacrylamide gel. .sup.14 
C-labeled methylated proteins (Amersham) were used as molecular weight 
standards. Gels were fixed in 10% acetic acid, washed with H.sub.2 O, 
impregnated with 1M salicylic acid, and bands representing radiolabeled 
peptides were detected by autofluorography at -70.degree. C. 
Phenotypic scoring, enzyme assays and alginate measurements. Suppression of 
mucoidy by plasmid-borne genes was monitored on PIA plates, and the 
phenotypic appearance of the colonies was scored as mucoid or nonmucoid. 
Alginate was assayed as previously described (Knutson and Jeanes, 1976). 
Various deletion products of the region containing the genes algU, mucA, 
and mucB were placed in the broad host range vector pVDZ'2 (Martin et al., 
1993) and introduced into PAO568 and PAO581 to test their ability to 
suppress mucoidy. The plasmid pPAOM3 (Cb.sup.r ; Konyecsni and Deretic 
1988), containing an algD::xylE transcriptional fusion, was introduced 
into PAO671 carrying mucB::Tc.sup.r and the parental strain PAO381 (Table 
6) by triparental conjugation (Konyecsni and Deretic, 1988). Cell- free 
sonic extracts were assayed for catechol 2,3-dioxygenase (CDO) activity 
using previously described methods (Konyecsni and Deretic 1988). The 
activity was monitored in 50 mM phosphate buffer (pH 7.5)-0.33 mM catechol 
by following the increase of A.sub.375 in a Shimadzu UV160 
spectrophotometer. The molar extinction coefficient of the reaction 
product, 2-hydroxymuconic semialdehyde, is 4.4.times.10.sup.4 at 375 nm. 1 
unit of CDO is defined as the amount of enzyme that oxidizes 1 .mu.mol of 
catechol per min at 24.degree. C. 
RESULTS 
Complementation of muc-25 requires two genes downstream of algU. The 
present inventors have discovered that the chromosomal muc mutations can 
be suppressed to nonmucoidy (Martin et al., 1993). This can be 
accomplished by in trans complementation with a cosmid clone and its 
derivatives carrying DNA from a nonmucoid PAO strain (Martin et al., 
1993). It has also been shown that this suppression activity was at the 
level of reducing algD transcription (Martin et al., 1993). The region 
needed for complementation includes algU, but this process also requires 
additional sequences downstream of algU (Martin et al., 1993). These 
studies have also indicated the presence of at least one additional gene, 
termed mucA, immediately following algU, which encodes a polypeptide (P20) 
with an apparent M.sub.r of 20 kDa (Martin et al., 1993). algU and mucA 
are necessary to exert detectable suppression of mucoidy in the PAO568 
(muc-2) strain (Fyfe and Govan, 1980). Finer analyses indicated that a 
region further downstream of mucA was also needed to completely abrogate 
mucoidy in this strain (FIG. 7). Moreover, another isogenic mucoid strain, 
PAO581, carrying a different muc mutation (muc-25) known to map close to 
muc-2, was not affected by the DNA fragment containing only algU and mucA 
unless downstream sequences were included. The present inventors further 
defined this additional region. The results of these experiments are shown 
in FIG. 7. Based on the size of additional DNA required for the 
suppression activity, it seemed likely that there was another gene, 
located downstream of the mucA gene, that was needed for suppression of 
mucoidy in PAO581. To test this hypothesis we first determined whether a 
polypeptide product encoded by this DNA region could be detected. The 
results of these studies are shown in FIG. 8. A polypeptide with an 
apparent M.sub.r of 32.8 kDa (P33) was encoded by the insert required for 
the suppression of the muc-25 mutation. No polypeptide product was 
observed when the same inserts were expressed in the opposite direction. 
However, P33 was expressed relatively poorly when compared to algU and 
mucA (not shown) (Martin et al., 1993). The gene encoding P33 was 
designated mucB. 
Complete nucleotide sequence of the mucA and mucB genes. In order to 
further characterize the mucA and mucB genes, the complete nucleotide 
sequence of this region from the prototype PAO strain PAO1 (nonmucoid), 
parental to PAO381 and its mucoid derivatives PAO568 and PAO581, was 
determined. The sequence of algU has been reported previously (Martin et 
al., 1993). The assignment of open reading frames for mucA and mucB in 
this region was facilitated by protein expression and other analyses. The 
only two open reading frames compatible with: (i) the order of genes (mucA 
followed by mucB); (ii) direction of transcription; (iii) apparent M.sub.r 
of gene products (P20 and P33); (iv) the endpoints of deletions 
encroaching on the mucB open reading frame that abrogate the suppression 
activity in PAO581; and (v) conforming with the codon usage typical of 
Pseudomonas (West and Iglewski, 1988) are shown in FIG. 9A, 9B, and 9C. 
The mucA open reading frame, encoding a polypeptide with predicted M.sub.r 
of 20,997, immediately follows algU. The mucB open reading frame, within 
the region necessary for suppression of mucoidy in PAO581 (FIG. 9A, 9B, 
and 9C) encodes a polypeptide with predicted M.sub.r of 34,471 kDa. To 
further confirm the correct assignment of the genes, this same region was 
cloned using PCR from several different strains, including PAO381, and in 
each case the complete nucleotide sequence was determined in multiple PCR 
clones confirming the one presented in FIG. 9A, 9B, and 9C. 
Insertional inactivation of mucB on the chromosome of the nonmucoid strain 
PAO381 results in mucoid phenotype. Any explanation of the requirement for 
all three genes (algU, mucA, and mucB) for suppression of mucoidy must 
take into account that algU plays a positive regulatory role in algD 
expression, possibly as the sigma factor required for mRNA initiation at 
the algD promoter (Martin et al., 1993). One of the models compatible with 
this function of algU in conjunction with the requirement for mucA and 
mucB (from a nonmucoid strain) to complement muc mutations and suppress 
mucoidy, is that mucA and mucB counteract the activity of algU and are 
needed for the maintenance of nonmucoid phenotype. If this is the case, 
then inactivation of mucB on the chromosome of P. aeruginosa should result 
in the mucoid phenotype. 
To test this hypothesis we inactivated mucB on the chromosome of the 
nonmucoid strain PAO381. This strain is parental to the mucoid derivatives 
PAO568 (muc-2), and PAO581 (muc-25) that have muc mutations mapping in the 
same chromosomal region (67.5 min) as the algU-mucAB cluster (Fyfe and 
Govan, 1980; 1983; Martin et al., 1993). Thus, PAO381 is capable of 
conversion to mucoidy via mutations in the muc genes. To inactivate mucB, 
the algU- mucAB cluster was first cloned on pUC12. The conveniently 
located BglII site (FIG. 9) within the coding region of mucB was used to 
insert a Tc.sup.r cassette (Totten, et al., 1990), resulting in the 
disruption of mucB, as described in Experimental Procedures. To this 
construct was added a fragment containing mob functions (to facilitate its 
mobilization into P. aeruginosa), resulting in the plasmid pDMB100. Since 
pUC12 and its derivative pDMB100 cannot replicate in Pseudomonas, upon a 
transfer of this plasmid into P. aeruginosa via triparental conjugation 
(see Experimental Procedures), any Tc.sup.r exconjugants must carry this 
marker integrated on the chromosome. This can occur via homologous 
recombination involving the algU-mucAB region through single crossover or 
double crossover events. In the case of single crossovers, the 
exconjugants are expected to be merodiploids, and should also display 
Cb.sup.r (Ap.sup.r ; encoded by the vector moiety); in the case of double 
crossovers, a true gene replacement is expected to take place with the 
vector moiety of the plasmid being lost, and thus the resulting strains 
should be sensitive to carbenicillin (Cb.sup.s). Of 129 Tc.sup.r P. 
aeruginosa exconjugants obtained from 4 independent crosses between E. 
coli JM83 harboring pDMB100 and PAO381, 28% of exconjugants were Cb.sup.r, 
indicative of a single crossover event. As expected, all such strains were 
nonmucoid, since they were merodiploids, and contained a functional copy 
of mucB. These strains were indistinguishable from the parental strain 
PAO381. In contrast, all Tc.sup.r exconjugants that were Cb.sup.s (72% of 
total Tc.sup.r exconjugants), thus indicative of double crossover events, 
became mucoid. Thus, a complete and stable conversion to mucoidy was 
achieved by inactivating mucB on the chromosome of a previously nonmucoid 
strain. A true gene replacement event of mucB with mucB::Tc.sup.r on the 
chromosome of such strains was further confirmed by Southern blot 
hybridization (FIG. 10A and 10B). 
To determine whether inactivation of mucB resulted in transcriptional 
activation of algD, one such mucB::Tc.sup.r strain (PAO671) was further 
examined. A plasmid containing algD-xylE transcriptional fusion was 
introduced into PAO671 and the levels of algD transcription in the 
parental strain PAO381 (mucB.sup.+) and its mucoid derivative PAO671 
(mucB::Tc.sup.r) were compared. The results of these experiments indicated 
a 26-fold activation of algD in PAO671 vs PAO381, under identical growth 
conditions (Table 6). Thus, inactivation of mucB is an event that results 
in increased algD transcription, alginate overproduction, and the 
establishment of mucoid phenotype. 
TABLE 6 
______________________________________ 
Effects of mucB inactivation on algD promoter activity 
algD::xylE activity.sup.c 
Strain.sup.a Phenotype.sup.b 
(U/mg of CDO) 
______________________________________ 
PAO381 (mucB.sup.+) 
NM 0.4 
PAO671 (mucB::Tc.sup.r) 
M 10.5 
______________________________________ 
.sup.a All strains harbored the algD::xylE transcription fusion plasmid 
pPAOM3 (Konyecsni and Deretic, 1989; Mohr et al., 1990). 
.sup.b Phenotype was scored as mucoid (alginate producing) or nonmucoid 
after growth for 24 h on PIA. 
.sup.c The activity was expressed as units of catechol 2,3 deoxygenase 
(CDO; the xyle gene product) per milligram of total protein in crude 
extracts. Standard error did not exceed 20%. Growth conditions, extract 
preparation, activity measurements, and unit definitions (see Experimenta 
Procedures) are as previously described (Martin et al., 1992; Konyecsni 
and Deretic, 1989). 
The experiments presented here demonstrate that inactivation of genes such 
as mucB can lead to a derepression of the algD promoter and conversion to 
mucoid (alginate overproducing) status. More importantly, using an 
isogenic series of strains, different frameshift mutations within the 
mucAB region that were present in several mucoid strains including CF 
isolates and absent in the nonmucoid strains have been detected, see 
Example 3. 
A model founded on recently reported evidence (Martin et al., 1993), the 
results presented in this work, and studies by others (Fyfe and Govan, 
1980; 1983; Costerton et al., 1983), is based on the premise that the 
synthesis of alginate and the emergence of alginate overproducing strains 
may be a developmental or a cell-differentiation process. Signal 
transduction involving response regulators such as AlgR and AlgB (Deretic 
et al., 1989; 1991; Wozniak and Ohman, 1991), nucleoid structure (Deretic 
et al., 1992; Mohr and Deretic, 1992), and activation of the specific 
sigma factor(s) (Martin et al., 1993) are most likely different 
contributing mechanisms for activation of alginate synthesis in natural 
environments. In the CF lung, while this environment may also be conducive 
to the induction of the alginate system, due to strong selective pressures 
(e.g. increased resistance of mucoid forms to phagocytosis, physical 
clearance mechanisms, antibiotic treatments, etc.) mutants are being 
selected that overproduce alginate and render cells constitutively mucoid. 
Such mutants, once extracted from the CF lung, retain mucoid character 
(Govan, 1988; Martin et al., unpublished results). Mutations in the 
algU-mucAB region, e.g. inactivation of mucA by frameshift mutations (see 
Example 3), or mutations affecting mucB activity, represent major pathways 
for conversion into the mucoid phenotype. Understanding of the principles 
of signal transduction processes activating the alginate system at several 
levels, as well as the precise definition of the mutations causing mucoidy 
in CF strains which is currently in progress, will provide improved 
diagnostic tools and present potential targets for therapeutic 
interventions. 
EXAMPLE 3 
Mechanism of Conversion to Mucoidy in P. aeruginosa Infecting Cystic 
Fibrosis Patients: Frameshift Mutations of mucA Cause Conversion to 
Mucoidy 
The references referred to in this example are listed at the end of the 
specification. 
METHODS 
In addition to those methods presented in Examples 1 and 2, the following 
methods were followed in the experiments described in this example. 
Amplification of algU-mucA-mucB sequences, and hybridizations with allele 
specific oligonucleotides. The algU-mucA-mucB region was amplified using 
the following pairs of oligonucleotides: (i) UL5 GCCGCACGTCACGAGC and UR16 
GAGTCCATCCGCTTCG for sequences containing mostly algU and a 5' portion of 
mucA; and (ii) UL3 CTGTCCGCTGTGATGG and UR12 CGCCCCTGCTCCTCGA for 
sequences containing most of mucA and the entire mucB gene. For 
amplification of genomic sequences, a loopful of bacteria from a P. 
aeruginosa colony was washed in 0.85% saline, centrifuged, resuspended in 
500 .mu.l H.sub.2 O, boiled for 10 min, and stored at -20.degree. until 
use. One .mu.l of boiled preparations is sufficient to obtain necessary 
amounts of products for amplification by polymerase chain reaction (PCR). 
PCR was carried out in 50 .mu.l volumes using standard procedures. 
Amplification products were tested by electrophoresis on agarose gels. 
Equal amounts of amplification products were electrophoretically separated 
on 1% agarose gels and then blotted onto a nitrocellulose filter using 
standard methods. After the transfer, and crosslinking using UV light (254 
nm), blots were prehybridized in 10.times.SSC, 5.times.Denhardt solution 
(without BSA) for at least 30 min. Allele specific oligonucleotides were 
kinased with .sup.32 p following standard methods, purified using 
chromatography on Sep-Pak C.sub.18 columns (Waters) and lyophilized by 
evaporation in a Savant SpeedVac apparatus. Hybridization with 
radiolabeled allele specific oligonucleotides was performed in 10 ml of 
10.times.SSC, 5.times.Denhardt solution for 12 h at 42.degree. C. 
Membranes were washed 3.times.for 10 min at 42.degree. C. and 
autoradiograms taken overnight at -70.degree. C. The blots were boiled for 
3 min between hybridizations with different probes. 
A simplified version of differential hybridization was also carried out 
using dot blots. In this case, 5 .mu.l taken directly from the PCR mixture 
was blotted onto a nitrocellulose or nylon membrane presoaked in 
10.times.SSC, and after denaturation, neutralization, crosslinking (by 
standard methods or as described above), hybridized and processed as 
explained for Southern blots. 
RESULTS 
In the course of performing gene replacements with algU in the mucoid 
strain PAO568 (carrying the muc-2 mutation), the present inventors 
discovered an informative class of recombinants. The gene replacements on 
the chromosome were carried out via homologous recombination with 
algU::Tc.sup.r on a plasmid that cannot replicate in Pseudomonas. A set of 
experiments was performed using a deletion that simultaneously removed the 
3' end of algU and the 5' end of the downstream gene mucA (FIG. 11). Two 
types of recombinants were expected: (i) Nonmucoid strains containing true 
gene replacements with inactivated algU (results of double crossovers); 
and (ii) mucoid strains that were merodiploids (results of single 
crossovers). As expected, all double crossovers were nonmucoid since they 
lost a functional algU. The majority of merodiploids were mucoid, since 
they retained a functional copy of algU. However, a third class of 
recombinants was also observed which consisted of nonmucoid merodiploids. 
Since the plasmid DNA came from the nonmucoid strain PAO1, parental to the 
PAO568 lineage, a plausible explanation for the existence of nonmucoid 
merodiploids was that the crossover took place between the deletion in 
mucA on the plasmid and a putative mutation (muc- 2) in mucA on the 
chromosome of the mucoid strain PAO568. Only such a crossover could 
restore a wild type copy of mucA resulting in nonmucoid merodiploids (FIG. 
11). The mutation had to be located between the EcoRV site of mucA, where 
the 5' deletion in the plasmid construct ended (FIG. 11), and the 3' end 
of mucA. In order to test this hypothesis, the present inventors cloned 
the corresponding region from the strain PAO568 by PCR and determined its 
complete nucleotide sequence in multiple independent clones. A duplication 
of 8 nucleotides at position 433 was observed within mucA in all PCR 
clones from PAO568 (FIG. 12A). The existence of this mutation was further 
confirmed by hybridization with allele specific oligonucleotides, oligo 
381 and oligo 568 (see FIG. 12A and 12B), to PCR amplified chromosomal 
sequences from PAO568 (muc-2) and its direct nonmucoid parental strain 
PAO381 (FIG. 13). Next, the entire algU-mucAB region was cloned by PCR 
from PAO568 and its parental strain PAO381, and the complete nucleotide 
sequence of this region was determined in at least three independent 
clones. FIG. 14 contains the sequence of the mucA gene and encoded 
protein. The only difference between PAO381 (muc.sup.+) and PAO568 (muc-2) 
was the octanucleotide duplication in mucA. The present inventors 
concluded that this was the muc-2 mutation and the mucA allele was 
designated mucA2. The muc-2 mutation results in a frameshift causing 
premature termination of mucA at a downstream TGA codon (see FIG. 14). 
The present inventors examined whether the allele specific oligonucleotides 
could be used to screen other mapped muc mutations in PAO and mucoid CF 
isolates. Although the oligonucleotide probe 568 (specific for the mucA2 
allele) did not hybridize with the PCR amplified sequences from several 
strains, the control oligonucleotide (381) did hybridize but with a 
reduced intensity relative to PAO381. This suggested that although the 
tested strains did not have the octanucleotide duplication observed in 
mucA2, there were other alterations within the region complementary to the 
oligonucleotide probe. The corresponding regions from several strains 
hybridizing weakly with the oligonucleotide 381 were cloned and examined. 
The following strains were included: PAO578 (mucoid derivative of PAO381 
with the mutation muc-22 mapping close to muc-2 as determined by 
transduction) and representative clinical mucoid isolates obtained from 
different cystic fibrosis patients. Following the procedure outlined for 
cloning and sequencing of the region encompassing the muc-2 mutation, the 
corresponding nucleotide sequences in the strain PAO578, and the clinical 
isolates tested were determined. Instead of the duplication of the 
octanucleotide sequence in PAO568, there was a deletion of a G residue 
within a string of 5 Gs within the same general region (see FIG. 15A and 
15B). Since this was a deletion of one nucleotide, the net result was a 
similar frameshift as in PAO568, placing the same TGA termination codon in 
frame with the mucA sequence (see FIG. 14). The results of these analyses 
were additionally confirmed by designing an allele specific 
oligonucleotide designed for this mutation (oligo 578, see FIG. 12). The 
mutant allele in PAO578 was designated mucA22. 
Next, the strains that were complemented by plasmids carrying the 
algU-mucAB region from PAO1 but did not show reduced hybridization with 
the oligonucleotides were examined. One such cystic fibrosis isolate was 
subjected to the same cloning and sequencing procedure as outlined above. 
No changes were detected within the location of the mucA2 and mucA22 
mutations. Instead, a deletion of a single nucleotide at the position 371 
was detected (see FIG. 16). This deletion was confirmed by sequencing 
multiple clones and by hybridizations with an allele specific 
oligonucleotide CF1 (see FIG. 12A and FIG. 12B). This frameshift mutation 
also results in a premature termination of mucA although at an upstream 
termination codon (see FIG. 14). Another strain, PAO581, that did not show 
differential hybridization with the allele specific oligonucleotides was 
also examined. PAO581 carries a muc mutation (muc-25) which has not be 
mapped by transduction. In this case the present inventors could not find 
any sequence differences between PAO581 and PAO381 within the region 
examined here. Similar to PAO581, several mucoid CF strains did not show 
detectable alterations in mucA. 
The work described herein identifies a major site of mutations causing 
mucoidy in P. aeruginosa. The mucA gene and a tightly linked downstream 
gene, mucB are both required for suppression of mucoidy. When these 
functions are lost by insertional inactivation on the chromosome of 
previously nonmucoid strains, provided that the first gene of the cluster 
(algU) is intact, this results in a strong activation of algD 
transcription and conversion to mucoidy. 
Mucoidy in P. aeruginosa has received attention mainly due to its 
association with CF. However, almost all strains of P. aeruginosa have the 
genetic capacity to synthesize alginate suggesting that this system must 
play a role in other ecological niches. The vast majority of P. aeruginosa 
biomass in nature exists as the form embedded in the exopolysaccharide 
biofilm attached to surfaces. It has been shown that P. aeruginosa 
undergoes interconversions between the free floating planktonic form and 
the sessile form in biofilms, a process which has been viewed as a 
developmental or cell differentiation phenomenon. Regulation of alginate 
production by a factor (algU) homologous to an alternative sigma factor 
Spo0H, controlling the initial stages of development in Bacillus spp. 
(e.g. sporulation and competence), may reflect the nature of regulatory 
processes controlling development of biofilms. The genetic data indicate 
that mucA and mucB suppress the function of algU. There are now ample 
examples of accessory factors associated with or linked to alternative 
sigma factors in Bacillus and other organisms that post-translationally 
modify (e.g. inhibit) their function. By analogy, MucA and MucB may play a 
similar role. This system, along with signal transduction regulators and 
histone-like elements, is likely designed to control development of 
biofilms in response to appropriate environmental cues. Mutations in mucA 
that lock the system in its constitutive state, which is favorable due to 
the antiphagocytic properties of the mucoid coating, are being selected in 
the course of chronic respiratory infection in CF. 
In addition to the improved understanding of the molecular mechanisms 
controlling an important bacterial virulence factor, several aspects of 
the regulation of mucoidy presented here shed light on developmental 
processes in Gram negative organisms. The finding that algU shows 
similarities with a sigma factor specializing in developmental processes 
of a Gram positive sporulating organism, suggests that bacterial cell 
differentiation phenomena (e.g. sporulation, biofilm development, and 
bacterial encystment) may share common regulatory mechanisms. 
The following references are specifically incorporated by reference herein 
in pertinent part. 
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__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 19 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 595 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AGGTATCGCTATGAGTCGTGAAGCCCTGCAGGAAACTCTGTCCGCTGTGATGGATAACGA60 
AGCGGATGAACTCGAGTTGCGGCGGGTGCTCGCAGCTTGCGGCGAGGATGCCGAGCTGCG120 
TTCCACCTGGTCGCGTTACCAGTTGGCGCGGTCCGTCATGCACCGCGAGCCTACCCTGCC180 
GAAGCTGGATATCGCTGCGGCGGTCTCTGCTGCCCTGGCCGACGAGGCCGCTCCGCCGAA240 
AGCGGAGAAGGGACCGTGGCGGATGGTCGGTCGCCTGGCGGTCGCTGCCTCGGTGACCCT300 
GGCGGTGCTGGCCGGCGTGCGTCTGTACAACCAGAACGACGCCCTGCCGCAAATGGCGCA360 
ACAGGGGACCACCCCGCAGATCGCCCTGCCTCAGGTGAAAGGCCCGGCCGTGCTGGCCGG420 
CTACAGCGAAGAGCAGGGGGCGCCGCAGGTGATCACCAACTCCTCGTCCAGCGATACCCG480 
CTGGCATGAGCAGCGTCTGCCGATCTACCTGCGTCAGCACGTGCAACAATCCGCCGTCAG540 
TGGTACAGAGAGCGCGCTGCCCTACGCTCGGGCAGCCAGCCTGGAAAACCGCTGA595 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GGGACCCCCCGCA13 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GAGCAGGGGCGCC13 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CAGGGGGCCAGGGGGC16 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GCCGCACGTCACGAGC16 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GAGTCCATCCGCTTCG16 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
CTGTCCGCTGTGATGG16 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CGCCCCTGCTCCTCGA16 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 647 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GTCTATCTTGGCAAGACGATTCGCTGGGACGCTCGAAGCTCCTCCAGGTTCGAAGAGGAG60 
CTTTCATGCTAACCCAGGAACAGGATCAGCAACTGGTTGAACGGGTACAGCGCGGAGACA120 
AGCGGGCTTTCGATCTGCTGGTACTGAAATACCAGCACAAGATACTGGGATTGATCGTGC180 
GGTTCGTGCACGACGCCCAGGAAGCCCAGGACGTAGCGCAGGAAGCCTTCATCAAGGCAT240 
ACCGTGCGCTCGGCAATTTCCGCGGCGATAGTGCTTTTTATACCTGGCTGTATCGGATCG300 
CCATCAACACCGCGAAGAACCACCTGGTCGCTCGCGGGCGTCGGCCACCGGACAGCGATG360 
TGACCGCAGAGGATGCGGAGTTCTTCGAGGGCGACCACGCCCTGAAGGACATCGAGTCGC420 
CGGAACGGGCGATGTTGCGGGATGAGATCGAGGCCACCGTGCACCAGACCATCCAGCAGT480 
TGCCCGAGGATTTGCGCACGGCCCTGACCCTGCGCGAGTTCGAAGGTTTGAGTTACGAAG540 
ATATCGCCACCGTGATGCAGTGTCCGGTGGGGACGGTACGGTCGCGGATCTTCCGCGCTC600 
GTGAAGCAATCGACAAAGCTCTGCAGCCTTTGTTGCGAGAAGCCTGA647 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1800 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
TTTGTTGCGAGAAGCCTGACACAGCGGCAAATGCCAAGAGAGGTATCGCTATGAGTCGTG60 
AAGCCCTGCAGGAAACTCTGTCCGCTGTGATGGATAACGAAGCGGATGAACTCGAGTTGC120 
GGCGGGTGCTCGCAGCTTGCGGCGAGGATGCCGAGCTGCGTTCCACCTGGTCGCGTTACC180 
AGTTGGCGCGGTCCGTCATGCACCGCGAGCCTACCCTGCCGAAGCTGGATATCGCTGCGG240 
CGGTCTCTGCTGCCCTGGCCGACGAGGCCGCTCCGCCGAAAGCGGAGAAGGGACCGTGGG300 
GGATGGTCGGTCGCCTGGCGGTCGCTGCTCGGTGACCCTGGCGGTGCTGGCCGGCGTGCG360 
TCTGTACAACCAGAACGACGCCCTGCCGCAAATGGCGCAACAGGGGACCACCCCGCAGAT420 
CGCCCTGCCTCAGGTGAAAGGCCCGGCCGTGCTGGCCGGCTACAGCGAAGAGCAGGGGGC480 
GCCGCAGGTGATCACCAACTCCTCGTCCAGCGATACCCGCTGGCATGAGCAGCGTCTGCC540 
CGATCTACCTGCGTCAGCACGTGCAACAATCCGCCGTCAGTGGTACAGAGAGCGCGCTGC600 
CCTACGCTCGGGCAGCCAGCCTGGAAAACCGCTGAGGAGAGACATGCGCACCACCTCCCT660 
GTTGCTTTTGCTTGGCAGCCTGATGGCGGTTCCCGCCACTCAGGCTGCCGACGCTTCCGA720 
CTGGCTGAATCGTCTCGCCGAGGCCGATCGCCAGAACAGTTTCCAAGGCACCTTCGTCTA780 
CGAGCGCAATGGCAGCTTCTCCACCCATGAGATCTGGCATCGCGTGGAGAGCGATGGTGC840 
GGTTCGCGAGCGCCTGCTCCAGCTCGACGGCGCGCGCCAGGAAGTGGTCCGGGTCGACGG900 
GCGCACCCAGTGCATCAGCGGCGGCCTTGCCGACCAACTGGCCGATGCCCAGCTGTGGCC960 
GGTGCGCAAGTTCGATCCCTCCCAGCTGGCTTCCTGGTACGACCTGCGCCTGGTCGGGGA1020 
ATCCCGTGTCGCCGGCCGCCCGGCAGTGGTCCTTGCGGTGACTCCGCGCGACCAGCATCG1080 
CTACGGCTTCGAGCTGCACCTGGACCGCGACACCGGCCTGCCGTTGAAGTCGCTGCTGCT1140 
GAACGAGAAGGGGCAGTTGCTCGAGCGCTTCCAGTTCACCCAGTTGAATACCGGCGCGGC1200 
ACCTGCCGAAGACCAGTTGCAGGCGGGCGCCGAATGCCAGGTCGTCGGCCCGGCCAAGGC1260 
CGACGGGGAGAAGACCGTGGCCTGGCGCTCGGAATGGCTGCCGCCAGGTTTCACCCTGAC1320 
CCGCAGTTTCATGCGTCGCAGTCCGGTCACCCCCGATCCGGTCGCCTGCCTGACCTATGG1380 
CGATGGCCTGGCACGATTCTCGGTGTTCATCGAGCCGCTGCACGGTGCCATGGTTGGCGA1440 
CGCGCGCAGCCAGCTCGGCCCGACCGTGGTGGTTTCCAAGCGCCTGCAGACCGATGACGG1500 
CGGCCAGATGGTGACCGTCGTCGGCGAAGTGCCGCTGGGCACCGCCGAGCGGGTGGCGCT1560 
GTCCATCCGGCCCGAGGCCGCCGCCCAGAAATGATCGAGGAGCAGGGGCGAGTGGTGGCG1620 
ACCGAGCCGGGAGCGGTATGGGTCGAGACCGTGCGCCGAGTACCTGCTCGTCCTGCTCGG1680 
CCAATGCCGGTTGCGGCCAGGGGCTGATGCAGCGCCTGGGCGTCGGCGCGGGGCGTGCCC1740 
GGGTGCGCGCGTTGAGCGACCTGAGCCTGCGGGTCGGCGATGCGGTCGTCCTAGGAATTC1800 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
AGCGAAGAGCAGGGGGCGCCGCAGGTGATCA31 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
GAGCAGGGGGCGCCG15 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
AACAGGGGACCACCCCGCAGATCGCC26 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GGGACCACCCCGC13 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 194 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
MetSerArgGluAlaLeuGlnGluThrLeuSerAlaValMetAspAsn 
151015 
GluAlaAspGluLeuGluLeuArgArgValLeuAlaAlaCysGlyGlu 
202530 
AspAlaGluLeuArgSerThrTrpSerArgTyrGlnLeuAlaArgSer 
354045 
ValMetHisArgGluProThrLeuProLysLeuAspIleAlaAlaAla 
505560 
ValSerAlaAlaLeuAlaAspGluAlaAlaProProLysAlaGluLys 
65707580 
GlyProTrpArgMetValGlyArgLeuAlaValAlaAlaSerValThr 
859095 
LeuAlaValLeuAlaGlyValArgLeuTyrAsnGlnAsnAspAlaLeu 
100105110 
ProGlnMetAlaGlnGlnGlyThrThrProGlnIleAlaLeuProGln 
115120125 
ValLysGlyProAlaValLeuAlaGlyTyrSerGluGluGlnGlyAla 
130135140 
ProGlnValIleThrAsnSerSerSerSerAspThrArgTrpHisGlu 
145150155160 
GlnArgLeuProIleTyrLeuArgGlnHisValGlnGlnSerAlaVal 
165170175 
SerGlyThrGluSerAlaLeuProTyrAlaArgAlaAlaSerLeuGlu 
180185190 
AsnArg 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
LeuLeuArgGluAla 
15 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 194 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
MetSerArgGluAlaLeuGlnGluThrLeuSerAlaValMetAspAsn 
151015 
GluAlaAspGluLeuGluLeuArgArgValLeuAlaAlaCysGlyGlu 
202530 
AspAlaGluLeuArgSerThrTrpSerArgTyrGlnLeuAlaArgSer 
354045 
ValMetHisArgGluProThrLeuProLysLeuAspIleAlaAlaAla 
505560 
ValSerAlaAlaLeuAlaAspGluAlaAlaProProLysAlaGluLys 
65707580 
GlyProTrpArgMetValGlyArgLeuAlaValAlaAlaSerValThr 
859095 
LeuAlaValLeuAlaGlyValArgLeuTyrAsnGlnAsnAspAlaLeu 
100105110 
ProGlnMetAlaGlnGlnGlyThrThrProGlnIleAlaLeuProGln 
115120125 
ValLysGlyProAlaValLeuAlaGlyTyrSerGluGluGlnGlyAla 
130135140 
ProGlnValIleThrAsnSerSerSerSerAspThrArgTrpHisGlu 
145150155160 
GlnArgLeuProIleTyrLeuArgGlnHisValGlnGlnSerAlaVal 
165170175 
SerGlyThrGluSerAlaLeuProTyrAlaArgAlaAlaSerLeuGlu 
180185190 
AsnArg 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 316 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
MetArgThrThrSerLeuLeuLeuLeuLeuGlySerLeuMetAlaVal 
151015 
ProAlaThrGlnAlaAlaAspAlaSerAspTrpLeuAsnArgLeuAla 
202530 
GluAlaAspArgGlnAsnSerPheGlnGlyThrPheValTyrGluArg 
354045 
AsnGlySerPheSerThrHisGluIleTrpHisArgValGluSerAsp 
505560 
GlyAlaValArgGluArgLeuLeuGlnLeuAspGlyAlaArgGlnGlu 
65707580 
ValValArgValAspGlyArgThrGlnCysIleSerGlyGlyLeuAla 
859095 
AspGlnLeuAlaAspAlaGlnLeuTrpProValArgLysPheAspPro 
100105110 
SerGlnLeuAlaSerTrpTyrAspLeuArgLeuValGlyGluSerArg 
115120125 
ValAlaGlyArgProAlaValValLeuAlaValThrProArgAspGln 
130135140 
HisArgTyrGlyPheGluLeuHisLeuAspArgAspThrGlyLeuPro 
145150155160 
LeuLysSerLeuLeuLeuAsnGluLysGlyGlnLeuLeuGluArgPhe 
165170175 
GlnPheThrGlnLeuAsnThrGlyAlaAlaProAlaGluAspGlnLeu 
180185190 
GlnAlaGlyAlaGluCysGlnValValGlyProAlaLysAlaAspGly 
195200205 
GluLysThrValAlaTrpArgSerGluTrpLeuProProGlyPheThr 
210215220 
LeuThrArgSerPheMetArgArgSerProValThrProAspProVal 
225230235240 
AlaCysLeuThrTyrGlyAspGlyLeuAlaArgPheSerValPheIle 
245250255 
GluProLeuHisGlyAlaMetValGlyAspAlaArgSerGlnLeuGly 
260265270 
ProThrValValValSerLysArgLeuGlnThrAspAspGlyGlyGln 
275280285 
MetValThrValValGlyGluValProLeuGlyThrAlaGluArgVal 
290295300 
AlaLeuSerIleArgProGluAlaAlaAlaGlnLys 
305310315 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 193 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
MetLeuThrGlnGluGlnAspGlnGlnLeuValGluArgValGlnArg 
151015 
GlyAspLysArgAlaPheAspLeuLeuValLeuLysTyrGlnHisLys 
202530 
IleLeuGlyLeuIleValArgPheValHisAspAlaGlnGluAlaGln 
354045 
AspValAlaGlnGluAlaPheIleLysAlaTyrArgAlaLeuGlyAsn 
505560 
PheArgGlyAspSerAlaPheTyrThrTrpLeuTyrArgIleAlaIle 
65707580 
AsnThrAlaLysAsnHisLeuValAlaArgGlyArgArgProProAsp 
859095 
SerAspValThrAlaGluAspAlaGluPhePheGluGlyAspHisAla 
100105110 
LeuLysAspIleGluSerProGluArgAlaMetLeuArgAspGluIle 
115120125 
GluAlaThrValHisGlnThrIleGlnGlnLeuProGluAspLeuArg 
130135140 
ThrAlaLeuThrLeuArgGluPheGluGlyLeuSerTyrGluAspIle 
145150155160 
AlaThrValMetGlnCysProValGlyThrValArgSerArgIlePhe 
165170175 
ArgAlaArgGluAlaIleAspLysAlaLeuGlnProLeuLeuArgGlu 
180185190 
Ala 
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