Shifting paradigms in Pseudomonas aeruginosa biofilm research.

Biofilms formed by Pseudomonas aeruginosa have long been recognized as a challenge in clinical settings. Cystic fibrosis, endocarditis, device-related infections, and ventilator-associated pneumonia are some of the diseases that are considerably complicated by the formation of bacterial biofilms, which are resistant to most current antimicrobial therapies. Due to intense research efforts, our understanding of the molecular events involved in P. aeruginosa biofilm formation, maintenance, and antimicrobial resistance has advanced significantly. Over the years, several dogmas regarding these multicellular structures have emerged. However, more recent data reveal a remarkable complexity of P. aeruginosa biofilms and force investigators to continually re-evaluate previous findings. This chapter provides examples in which paradigms regarding P. aeruginosa biofilms have been challenged, reflecting the need to critically re-assess what is emerging in this rapidly growing field. In this process, several avenues of research have been opened that will ultimately provide the foundation for the development of preventative measures and therapeutic strategies to successfully treat P. aeruginosa biofilm infections.

Loss of Pseudomonas aeruginosa PhpA aminopeptidase activity results in increased algD transcription.

Inactivation of Pseudomonas aeruginosa phpA, encoding a putative leucine aminopeptidase, results in increased transcription of algD. The homologous protein in Escherichia coli, PepA, is multifunctional, possessing independent aminopeptidase and DNA-binding activities. Here we provide in vitro evidence that PhpA is an aminopeptidase and show that this activity is the relevant property with regard to algD expression. This regulation occurred at the previously mapped algD transcription initiation site and was not due to activation of an alternative promoter.

Negative control of flagellum synthesis in Pseudomonas aeruginosa is modulated by the alternative sigma factor AlgT (AlgU).

Many respiratory isolates of Pseudomonas aeruginosa from cystic fibrosis patients are mucoid (alginate producing) yet lack flagella. It was hypothesized that an alginate regulator inhibits flagellar gene expression. Mutations in algB, algR, and algT resulted in nonmucoid derivatives, yet algT mutants expressed flagella. AlgT-dependent control of flagellum synthesis occurred through inhibition of fliC but not rpoN transcription.

Pseudomonas aeruginosa AlgZ, a ribbon-helix-helix DNA-binding protein, is essential for alginate synthesis and algD transcriptional activation.

The Pseudomonas aeruginosa algD gene is the first gene of an operon encoding most of the enzymes necessary for biosynthesis of the exopolysaccharide alginate. Transcriptional activation of algD results in the high-level synthesis of alginate, an important P. aeruginosa virulence factor with antiphagocytic and adherence properties. Previously, we have identified a protein(s), AlgZ, expressed in mucoid P. aeruginosa CF isolates that specifically bound to sequences located 280 bp upstream of the algD promoter. Mutagenesis of the AlgZ DNA binding site and transcription assays were used to show that AlgZ was an activator of algD transcription. In the current study, the monomeric size of AlgZ was estimated to be between 6 kDa and 15 kDa by electroelution of a protein preparation from an SDS-PAGE gel and analysis of the fractions via protein staining and electrophoretic mobility shift assays. A biochemical enrichment procedure, resulting in a 130-fold enrichment for AlgZ, was devised, the protein identified and a partial amino-terminal sequence obtained. Using the P. aeruginosa Genome Project database, a complete sequence was obtained, and algZ was cloned and expressed in Escherichia coli. Expression of algZ was sufficient for the observed AlgZ DNA binding previously observed from extracts of P. aeruginosa. A protein database search revealed that AlgZ is homologous to the Mnt and Arc repressors of the ribbon-helix-helix family of DNA-binding proteins. An algZ deletion mutant was constructed in the mucoid CF isolate FRD1. The resulting strain was non-mucoid and exhibited no detectable algD transcription. As an indirect role in transcription would probably result in some residual algD transcription, these data suggest that AlgZ is an integral activator of algD and support the hypothesis that both AlgZ and the response regulator AlgR are involved in direct contact with RNA polymerase containing the alternative sigma factor, AlgT. The cloning of algZ is a crucial step in determining the mechanism of algD activation.

Identification of an Escherichia coli pepA homolog and its involvement in suppression of the algB phenotype in mucoid Pseudomonas aeruginosa.

Strains of Pseudomonas aeruginosa isolated from the respiratory tracts of patients with cystic fibrosis often display a mucoid morphology due to high levels of expression of the exopolysaccharide alginate. The response regulator AlgB is required for full transcription of the alginate biosynthetic operon. Repeated attempts to demonstrate a direct interaction between AlgB and the promoter region of algD, the first gene in the alginate operon, have thus far been unsuccessful. The possibility that AlgB exerts its effect on algD indirectly exists. To identify putative genes under the control of AlgB which affect algD transcription, transposon mutagenesis of nonmucoid algB derivatives of the mucoid strain FRD1 was employed. Of approximately 3,000 transposon mutants screened, 6 were found to display phenotypes which were mucoid relative to the phenotype of the parental algB strain. The phenotypes of these mutants ranged from being only slightly mucoid to being indistinguishable from that of the original FRD1 strain. One of the particularly mucoid transposon mutants was chosen for further study. This strain was found to be disrupted in a previously uncharacterized open reading frame with 56% amino acid identity to PepA of Escherichia coli. PepA is classified as a leucine aminopeptidase, and homologs have been detected in a number of bacterial, plant, and animal species. This novel gene has been designated phpA (P. aeruginosa homolog of pepA). The insertional inactivation of phpA was found to correlate with the mucoid phenotype and an increase in algD transcription in the algB strain. Expression of phpA from an ectopic chromosomal locus compensated for the transposon insertion in the native phpA gene, restoring algD transcription to levels similar to those observed in the parental algB strain. While phpA expression did not appear to be under the control of AlgB at the transcriptional level, this study demonstrates that loss of phpA in an algB genetic background had a positive effect on alginate expression and, more specifically, on transcription of the alginate biosynthetic operon.

Phosphorylation-independent activity of the response regulators AlgB and AlgR in promoting alginate biosynthesis in mucoid Pseudomonas aeruginosa.

Overproduction of the capsular polysaccharide alginate appears to confer a selective advantage for Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. The regulators AlgB and AlgR, which are both required as positive activators in alginate overproduction, have homology with the regulator class of two-component environmental responsive proteins which coordinate gene expression through signal transduction mechanisms. Signal transduction in this class of proteins generally occurs via autophosphorylation of the sensor kinase protein and phosphotransfer from the sensor to a conserved aspartate residue, which is present in the amino terminus of the response regulator. Recently, kinB was identified downstream of algB and was shown to encode the cognate histidine protein kinase that efficiently phosphorylates AlgB. However, we show here that a null mutation in kinB in a mucoid cystic fibrosis isolate, P. aeruginosa FRD1, did not block alginate production. The role of the conserved aspartate residue in the phosphorylation of AlgB was examined. The predicted phosphorylation site of AlgB (D59) was mutated to asparagine (N), and a derivative of an AlgB lacking the entire amino-terminal phosphorylation domain (AlgB delta1-145) was constructed. A hexahistidine tag was included at the amino terminus of the wild-type (H-AlgB), H-AlgB delta1-145, and mutant (H-AlgB.59N) AlgB proteins. These derivatives were purified by Ni2+ affinity chromatography and examined for in vitro phosphorylation by the purified sensor kinase protein, KinB. The results indicated that while KinB efficiently phosphorylated H-AlgB, no phosphorylation of H-AlgB delta1-145 or H-AlgB.D59N was apparent. An allelic exchange system was developed to transfer mutant algB alleles onto the chromosome of a P. aeruginosa algB mutant to examine the effect on alginate production. Despite the defect in AlgB phosphorylation, P. aeruginosa strains expressing AlgB.D59N or H-AlgB delta1-145 remained mucoid. The roles of the conserved aspartate residues in the phosphorylation of AlgR were also examined. As seen with AlgB, mutations in the predicted phosphorylation site of AlgR (AlgR.D54N and AlgR.D85N) did not affect alginate production. These results indicate that in vivo phosphorylation of AlgB and AlgR are not required for their roles in alginate production. Thus, the mechanism by which these response regulators activate alginate genes in mucoid P. aeruginosa appears not to be mediated by conventional phosphorylation-dependent signal transduction.

Identification of the histidine protein kinase KinB in Pseudomonas aeruginosa and its phosphorylation of the alginate regulator algB.

The exopolysaccharide alginate is an important virulence factor in chronic lung infections caused by the bacterium Pseudomonas aeruginosa. Two positive activators for alginate synthesis, algB and algR, are members of a superfamily of response regulators of the two-component regulatory system. AlgB belongs to the NtrC subfamily of response regulators and is required for high-level production of alginate. In this study, an open reading frame encoding a polypeptide of 66 kDa, designated kinB, was identified immediately downstream of algB. The sequence of KinB is homologous to the histidine protein kinase members of two-component regulatory systems. Western blot analysis of a P. aeruginosa strain carrying a kinB-lacZ protein fusion and studies of kinB-phoA fusions indicate that KinB localizes to the inner membrane and has a NH2-terminal periplasmic domain. A KinB derivative containing the COOH terminus of KinB was generated and purified. In the presence of [gamma-32P]ATP, the purified COOH-terminal KinB protein was observed to undergo progressive autophosphorylation in vitro. Moreover, the phosphoryl label of KinB could be rapidly transferred to purified AlgB. Substitutions of the residues conserved among histidine protein kinases abolished KinB autophosphorylation. These results provide evidence that kinB encodes the AlgB cognate histidine protein kinase.

Identification and characterization of AlgZ, an AlgT-dependent DNA-binding protein required for Pseudomonas aeruginosa algD transcription.

Transcriptional activation of the Pseudomonas aeruginosa algD gene results in high-level synthesis of the capsular polysaccharide alginate, an important P. aeruginosa virulence factor expressed in cystic fibrosis (CF) patients with chronic pulmonary disease. In this study, electrophoretic mobility-shift assays were used to identify a novel protein (AlgZ), which binds specifically to a sequence located 280 bp upstream of the algD promoter. While AlgZ-binding activity did not require the response regulators AlgB or AlgR, expression of AlgZ was found to be absolutely dependent on the alternative sigma factor AlgT. Electrophoretic mobility-shift assays and copper-phenanthroline footprinting localized AlgZ binding to a 36 bp algD region, which includes several helical repeats. A collection of alginate-producing (mucoid) and non-mucoid P. aeruginosa strains, derived from CF patients, was characterized for AlgZ-binding activity. In all cases, AlgZ binding to algD sequences was observed when extracts derived from mucoid P. aeruginosa CF isolates were examined. However, this binding activity was not present when extracts from non-mucoid P. aeruginosa CF isolates were tested. Oligonucleotide mutagenesis was employed to create an algD allele with a 4 bp mutation in the predicted AlgZ-binding site (algD38) and a heterologous substitution allele (algD40), in which the entire AlgZ-binding site was replaced with a non-specific DNA sequence of identical size. When the algD38 mutation was cloned into an algD-cat transcriptional fusion, this resulted in a 28-fold reduction in algD expression, whereas the algD40 mutation abolished algD transcription, indicating that AlgZ acts as an activator of algD transcription. These results support the hypothesis that activation of algD involves the formation of a high-order looped structure allowing for multivalent contacts between AlgZ, AlgR and RNA polymerase containing the alternative sigma factor AlgT. Characterization of the molecular details of algD activation will provide insights into the control of other prokaryotic and eukaryotic promoters that utilize multiple activators.

Construction and use of a nontoxigenic strain of Pseudomonas aeruginosa for the production of recombinant exotoxin A.

To express recombinant forms of Pseudomonas aeruginosa exotoxin A in high yield, we have developed a nontoxigenic strain of P. aeruginosa derived from the hypertoxigenic strain PA103. The nontoxigenic strain, designated PA103A, was produced by the excision marker rescue technique to replace the toxA structural gene in PA103 with an insertionally inactivated toxA gene. The PA103A strain (ToxA-) was used subsequently as the host strain for the expression and production of several recombinant versions of exotoxin A, and the results were compared with exotoxin A production in other P. aeruginosa and Escherichia coli strains. Use of the PA103A strain transformed with the high-copy-number pRO1614 plasmid bearing various toxA alleles resulted in final purification yields of exotoxin A averaging 23 mg/liter of culture. By comparison, exotoxin A production in other expression systems and host strains yields approximately 1/4 to 1/10 as much toxin.

ToxR (RegA)-mediated in vitro transcription of Pseudomonas aeruginosa toxA.

Exotoxin A (ETA) has been described as a major virulence factor produced by the opportunistic pathogen Pseudomonas aeruginosa. The transcription of the ETA structural gene (toxA) has been shown to be positively regulated by the product of the toxR gene (also called regA). However, the mechanism by which ToxR regulates toxA transcription is still under investigation. We have expressed toxR in Escherichia coli under the control of the T7 promoter and purified the wild-type ToxR protein. We have also produced ToxR as a fusion protein consisting of the first 12 amino acids of the T7 capsid protein attached to the N terminus of the intact ToxR protein. In the present study we have developed and used an in vitro transcription assay in order to investigate the mechanism of ToxR-mediated transcriptional regulation of toxA. Under the conditions of this in vitro assay toxA transcription requires the toxR product in addition to P. aeruginosa RNA polymerase (RNAP). Both the native and the T7::ToxR fusion proteins facilitate initiation of toxA transcription in vitro in the presence of Pseudomonas RNAP. Additional studies using (i) specific enzyme-linked immunosorbent assay; (ii) indirect immunoprecipitation; and (iii) gel-filtration chromatography, indicate that ToxR binds to the purified Pseudomonas RNAP and strengthens the possibility that ToxR may be an alternative sigma factor. Furthermore, the ToxR-mediated transcription of toxA is increased approx. threefold in the presence of crude cytoplasmic extracts from P. aeruginosa ToxR+ or ToxR-RegB- strains, indicating that additional factors play a role in the efficient and optimal transcription of toxA.

Transcriptional analysis of the Pseudomonas aeruginosa genes algR, algB, and algD reveals a hierarchy of alginate gene expression which is modulated by algT.

Strains of Pseudomonas aeruginosa which colonize and infect the lungs of cystic fibrosis patients have a mucoid colony morphology due to the overproduction of the exopolysaccharide alginate. The response regulators AlgB and AlgR are required for the transcription of algD, a tightly regulated gene encoding GDP-mannose dehydrogenase, which is critical for P. aeruginosa alginate biosynthesis. Previous studies indicated that mutations in the algT gene of mucoid FRD1 P. aeruginosa result in nonmucoid derivatives. However, the specific role for algT in alginate gene regulation has not been elucidated. In this study, transcription of algB, algD, and algR was characterized by gene fusion and primer extension analysis. Expression of algR and algD was abolished in P. aeruginosa strains containing algT::Tn501 insertions because of lack of transcription initiation at the algR and algD promoters. An algR mutation was constructed in FRD1, and this resulted in the loss of alginate production and a dramatic decrease in algD transcription. RNA and gene fusion analysis revealed that algB is not required for algR expression, nor is algR necessary for transcription of algB. Thus, with the exception of a requirement for AlgT, the AlgB and AlgR pathways appear to be independent of each other. In gel band mobility shift assays, a protein(s) present in extracts from mucoid and algB and algR mutant P. aeruginosa strains formed a specific complex with algD sequences located immediately upstream of the start of transcription. No binding to these sequences was observed when extracts from algT mutant strains were examined. A model proposed suggests that a hierarchy of alginate gene expression exists in which AlgT is required for transcription of the response regulators algB and algR, which in turn are necessary for algD expression. AlgT or a protein under algT control also binds to sequences located within the algD promoter.

Integration host factor and sequences downstream of the Pseudomonas aeruginosa algD transcription start site are required for expression.

Pseudomonas aeruginosa is an extremely important opportunistic pathogen in immunocompromised individuals. Strains of P. aeruginosa isolated from chronic lung infections in patients with the genetic disease cystic fibrosis have a mucoid colony morphology. This phenotype is due to overproduction of the exopolysaccharide alginate, which is believed to confer a selective advantage on P. aeruginosa in cystic fibrosis lungs. Alginate biosynthesis is controlled by a complex regulatory mechanism. Genes located in the 34-min region of the P. aeruginosa chromosome form an operon which encodes most of the biosynthetic enzymes necessary for alginate production. algD, the first gene in the operon and a critical point for the transcriptional regulation of alginate biosynthesis, is controlled by several trans, cis, and environmental factors. In this study, the involvement of the histone-like protein integration host factor (IHF) in algD expression was examined. Sequences with similarity to consensus IHF-binding sites of Escherichia coli were identified 75 bp upstream (site 1) and 90 bp downstream (site 2) of the start of algD transcription. In gel band mobility shift assays, DNA fragments containing either site bind IHF but site 2 has an approximately 90-fold higher affinity for IHF. Mutations in each of the elements were generated, and they resulted in the reduction or loss of in vitro IHF binding and a three- to fourfold decrease in algD-cat expression. This indicates that IHF binding is necessary for high-level algD transcription. The presence of a high-affinity IHF-binding site located 3' of the algD transcription start site suggested that sequences further downstream of this element are involved in algD expression. When a fragment located downstream of site 2 and upstream of the promoterless cat gene (+110 to +835) was deleted, algD-cat expression was reduced 10-fold supporting the notion that 3' enhancer elements are required for algD transcription. This is the first direct evidence of a 3' element involved in the control of a P. aeruginosa gene. It is postulated that IHF mediates the formation of a higher-order looped structure which is necessary for efficient algD transcription.

Cloning and characterization of the Pseudomonas aeruginosa sodA and sodB genes encoding manganese- and iron-cofactored superoxide dismutase: demonstration of increased manganese superoxide dismutase activity in alginate-producing bacteria.

Pseudomonas aeruginosa is a strict aerobe which is likely exposed to oxygen reduction products including superoxide and hydrogen peroxide during the metabolism of molecular oxygen. To counterbalance the potentially hazardous effects of elevated endogenous levels of superoxide, most aerobic organisms possess one or more superoxide dismutases or compounds capable of scavenging superoxide. We have previously shown that P. aeruginosa possesses both an iron- and a manganese-cofactored superoxide dismutase (D. J. Hassett, L. Charniga, K. A. Bean, D. E. Ohman, and M. S. Cohen, Infect. Immun. 60:328-336, 1992). In this study, the genes encoding manganese (sodA)- and iron (sodB)- cofactored superoxide dismutase were cloned by using a cosmid library of P. aeruginosa FRD which complemented an Escherichia coli (JI132) strain devoid of superoxide dismutase activity. The sodA and sodB genes of P. aeruginosa, when cloned into a high-copy-number vector (pKS-), partially restored the aerobic growth rate defect, characteristic of the Sod- strain, to that of the wild type (AB1157) when grown in Luria broth. The nucleotide sequences of sodA and sodB have open reading frames of 612 and 579 bp that encode dimeric proteins of 22.9 and 21.2 kDa, respectively. These data were also supported by the results of in vitro expression studies. The deduced amino acid sequence of the P. aeruginosa manganese and iron superoxide dismutase revealed approximately 50 and 67% similarity with manganese and iron superoxide dismutases from E. coli, respectively. There was also remarkable similarity with iron and manganese superoxide dismutases from other phyla. The mRNA start site of sodB was mapped to 174 bp upstream of the ATG codon. A likely promoter with similarity to the -10 and -35 consensus sequence of E. coli was observed upstream of the ATG start codon of sodB. Regions sequenced 519 bp upstream of the sodA electrophoresis, sodA gene revealed no such promoter, suggesting an alternative mode of control for sodA. By transverse field electrophoresis, sodA and sodB were mapped to the 71- to 75-min region on the P. aeruginosa PAO1 chromosome. Strikingly, mucoid alginate-producing bacteria generated greater levels of manganese superoxide dismutase than nonmucoid revertants, suggesting that mucoid P. aeruginosa is responding to oxidative stress and/or changes in the redox status of the cell.

Genetic exchange of determinants for capsular polysaccharide biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a.

The production of a capsular polysaccharide (CPS; K antigen) is characteristic of Klebsiella pneumoniae, but CPS structure varies among strains, and many different serotypes are now known. In this study, cps gene clusters encoding the elements of capsular polysaccharide biosynthesis were exchanged by homologous recombination between strains expressing different serotypes. The wild-type K. pneumoniae strains used for genetic exchange were KPA1 (cpsK2), expressing K2 CPS, and KPB1 (cpsK21a), expressing K21a CPS. Plasmid R68.45 was used to mobilize fragments of chromosomal DNA from auxotrophic derivatives of donor strains. Auxotrophic his alleles introduced into recipient strains provided selectable markers to coinherit the adjacent cps gene clusters from donors expressing a heterologous CPS. Each of the capsule-switched recombinants, KPA5 (cpsK21a) and KPB20 (cpsK2), was shown to have a CPS that was immunologically identical to the serotype of the respective donor. The recombinants retained their respective recipient strain background, as evidenced by a genetic marker and demonstration of a distinctive restriction fragment length polymorphism in genomic DNA. KPB1 CPS contained a sequence (mannose-alpha-2-mannose) that binds to a macrophage lectin and may be responsible for their higher susceptibility to macrophage binding and phagocytosis compared with KPA1, whose CPS lacked such sequences. The recombinant strains expressing heterologous cps genes inherited the macrophage-binding phenotype of the donor, thus confirming that relative susceptibility to phagocytosis was determined by the capsule type expressed. KPA1 was highly virulent in a mouse lethality assay, which is a feature typical of K2 strains, whereas KPB1 was not virulent in mice. Recombinant KPA5 retained relatively high virulence in mice, even though it produced the heterologous K21a CPS, which suggests that a virulence factor other than capsule biosynthesis is encoded by the KPA genomic strain background. In contrast, KPB20 gained marginal virulence in the mouse lethality assay through the inheritance and expression of the K2 CPS from the virulent strain. Thus, pathogenesis in K. pneumoniae may be multifactorial. Specific antibody was used to stabilize the CPS on the surface of K. pneumoniae, and the structural organization of the homologous and heterologous capsules was examined by electron microscopy. Recombinant KPB20, expressing heterologous K2 CPS, had a uniform layer of capsule surrounding the organism that was similar to that seen on the surfaces of the parental strains. However, KPA5, expressing the heterologous K21a CPS, was unusual in that the uniform capsular layer was physically separated from the cell wall by approximately 50 nm.(ABSTRACT TRUNCATED AT 400 WORDS)

Involvement of the alginate algT gene and integration host factor in the regulation of the Pseudomonas aeruginosa algB gene.

Strains of Pseudomonas aeruginosa causing pulmonary infection in cystic fibrosis patients are often mucoid because of the synthesis of a capsular polysaccharide called alginate. Regulation of alginate biosynthesis includes the algB gene product (AlgB), which belongs to a class of proteins that control gene transcription in response to environmental stimuli. In this study, a homolog of the DNA-binding-and-bending protein integration host factor (IHF) and the positive regulatory gene algT were shown to be involved in algB expression. An algB-cat gene fusion was constructed on a low-copy-number, broad-host-range plasmid. In alginate-producing (Alg+) P. aeruginosa, levels of chloramphenicol acetyltransferase from algB-cat were twofold higher than in spontaneous Alg- or algT::Tn501 mutant strains, indicating that the mucoid status of the cell influences algB transcription. An algB transcription initiation site was identified 286 nucleotides upstream of translation initiation and revealed an Escherichia coli sigma 70-like promoter. Sequences in the algB promoter region were highly similar to the consensus E. coli IHF binding site. In DNA gel band mobility shift assays, a protein present in extracts from IHF+ E. coli strains and IHF purified from E. coli bound specifically to these algB DNA fragments, while extracts prepared from isogenic IHF- E. coli strains failed to alter the mobility of algB DNA fragments containing the consensus IHF binding site. A protein in cell extracts prepared from P. aeruginosa strains also demonstrated binding to algB fragments containing the IHF binding site, and the position of the complex formed with these extracts was identical to that of the complex formed with purified IHF. Moreover, this binding could be inhibited by anti-IHF antibodies. To test the role of the IHF site in algB regulation, site-specific mutations in the algB IHF site, based on changes which severely affect IHF binding in E. coli, were generated. When either purified E. coli IHF or extracts from P. aeruginosa were used in DNA binding studies, the algB mutant DNAs were severely reduced in IHF binding. Mutations affecting IHF binding at the algB promoter were introduced into the algB-cat plasmid, and all resulted in severely impaired transcriptional activity in Alg- and algT mutant strains of P. aeruginosa. However, these mutations resulted in similar or slightly reduced algB-cat transcription in Alg+ and algB::Tn501 mutant strains. Thus, the algT product plays a positive role in the high-level expression of algB in mucoid cells, whereas as protein present in P.aeruginosa extracts which is likely an IHF homolog plays a positive role in maintaining a basal level of algB expression in nonmucoid strains.

Pseudomonas aeruginosa AlgB, a two-component response regulator of the NtrC family, is required for algD transcription.

Most strains of Pseudomonas aeruginosa isolated from the respiratory tracts of cystic fibrosis patients have a mucoid colony morphology due to the synthesis of an expolysaccharide called alginate. The algB gene product (AlgB) is necessary for the high-level production of alginate in mucoid P. aeruginosa. In this study, AlgB was shown to be involved in the transcription of algD, a gene previously demonstrated to be activated in mucoid P. aeruginosa. In vitro and in vivo expression studies reveal that algB encodes a protein with a molecular size of 49 kDa. The DNA sequence of a 2.2-kb P. aeruginosa fragment containing algB was also determined. The amino-terminal domain of AlgB was found to be conserved with the amino-terminal domains of the response regulator class of two-component regulatory proteins. The central domain of AlgB has sequences highly conserved with those in the NtrC subfamily of transcriptional activators (NtrC, NifA, HydG, DctD, FlbD, TyrR, and PgtA). The central domain of AlgB also contains a potential nucleotide binding site. AlgB is the first NtrC homolog described from P. aeruginosa. At the carboxy terminus of AlgB, a helix-turn-helix motif was observed, suggesting that AlgB is a DNA-binding protein. The strongly conserved NtrC-like central domain of AlgB is not present in AlgR, another alginate response regulator. This study therefore identifies and characterizes the second of at least two unique response regulators used by P. aeruginosa to control alginate gene expression.

Biochemical analysis of CRM 66. A nonfunctional Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa exotoxin A (ETA) is an ADP-ribosyltransferase which inactivates protein synthesis by covalently attaching the ADP-ribose portion of NAD+ onto eucaryotic elongation factor 2 (EF-2). A direct biochemical comparison has been made between ETA and a nonenzymatically active mutant toxin (CRM 66) using highly purified preparations of each protein. The loss of ADP-ribosyltransferase activity and subsequent cytotoxicity have been correlated with the presence of a tyrosine residue in place of a histidine at position 426 in CRM 66. In the native conformation, CRM 66 demonstrated a limited ability (by a factor or at least 100,000) to modify EF-2 covalently and lacked in vitro and in vivo cytotoxicity, yet CRM 66 appeared to be normal with respect to NAD+ binding. Upon activation with urea and dithiothreitol, CRM 66 lost ADP-ribosyltransferase activity entirely yet CRM 66 retained the ability to bind NAD+. Replacement of Tyr-426 with histidine in CRM 66 completely restored cytotoxicity and ADP-ribosyltransferase activity. These results support previous findings from this laboratory (Wozniak, D. J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8880-8884) which suggest that the His-426 residue of ETA is not involved in NAD+ binding but appears to be associated with the interaction between ETA and EF-2.

His-426 of the Pseudomonas aeruginosa exotoxin A is required for ADP-ribosylation of elongation factor II.

Exotoxin A (ETA) is recognized as the most toxic product associated with the opportunistic pathogen Pseudomonas aeruginosa. Identification of the amino acids in the polypeptide sequence that are required for toxin activity is critical for vaccine development. By defining the nucleotide sequence of the structural gene of a mutant that encodes an enzymatically inactive ETA (CRM 66), we identified an essential amino acid (His-426), which is involved in the ADP-ribosyltransferase activity associated with functional ETA. A monoclonal antibody that inhibits ETA enzymatic activity in vitro fails to react with ETA variants that have a His 426----Tyr substitution. Several mono-ADP-ribosylating toxins, including diphtheria and pertussis toxins, within the primary amino acid sequences carry a histidine residue that is conserved in spacing and in location with respect to other critical residues. Analysis of the three-dimensional structure of ETA revealed that His-426 is not associated with the proposed NAD+ binding site. These findings should be useful for the design and construction of toxin vaccines.