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PubMed2004

Tissue-engineered human nasal septal cartilage using the alginate-recovered-chondrocyte method.

OBJECTIVES: Tissue engineering of nasal septal cartilage has numerous potential applications in craniofacial reconstruction. Chondrocytes suspended in alginate gel have been shown to produce a substantial cell-associated matrix. The objective of this study was to determine whether cartilage tissue could be generated using the alginate-recovered-chondrocyte (ARC) method, in which chondrocytes are cultured in alginate as an intermediate step in tissue fabrication. METHODS: Nasal septal chondrocytes from five patient donors were isolated by enzymatic digestion, then expanded in monolayer culture. At confluency, a portion of those cells were seeded at high density onto a semipermeable membrane and cultured for 14, 21, or 28 days (monolayer group). The remaining cells were suspended in alginate and cultured until a cell-associated matrix was observed (10-17 days). Cells and their associated matrix were released from alginate (ARC group), seeded onto a semipermeable membrane, and cultured as already described. DNA (Hoechst 33258 Assay), glycosaminoglycan (GAG; dimethylmethylene blue assay), and collagen (hydroxyproline assay) were analyzed biochemically. Immunohistochemistry was performed to assess expression of collagens type I and type II. Histochemistry was performed to localize cells accumulating sulfated GAG (Alcian blue stain). RESULTS: The ARC constructs, in contrast to the monolayer constructs, had substantial structural stability and the histologic and gross appearance of cartilaginous tissue. ARC constructs demonstrated significantly greater GAG and collagen accumulation than monolayer constructs (P <.05). Histologic analysis revealed substantial GAG and collagen type II production and only moderate collagen type I production. The composition of the matrix was thus similar to that of native human septal cartilage. CONCLUSIONS: Tissue-engineered human nasal septal cartilage using the ARC method has the histologic and gross appearance of native cartilage and has biochemical composition more like that of native cartilage than monolayer constructs. This is the first report of human nasal septal neocartilage formation without the use of biodegradable scaffolds.

Alginates↗

PubMed1997

Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate.

STUDY DESIGN: Cells from normal rabbit nucleus pulposus (NP) and anulus fibrosus (AF) were cultured in alginate beads for as long as 14 days to allow them to reform a matrix made up of two compartments: the cell-associated matrix (CM) and further removed matrix (FRM). At different time points, the CM and FRM made by each cell population were analyzed using histologic, biochemical, and immunologic assays. OBJECTIVES: To study the metabolism of normal rabbit NP and AF cells in alginate by characterizing the CM and FRM formed by each cell population, and to identify metabolic properties that may shed light on mechanisms at play in disc degeneration. SUMMARY OF BACKGROUND DATA: Little is known about the metabolism of intervertebral disc cells, in part because of the lack of microculture systems appropriate for the study of these cells in vitro. In recent studies from our laboratories, it was suggested that articular chondrocytes cultured in alginate beads remain phenotypically stable and reform a matrix similar to the one they populate in vivo. This culture system appears ideally suited for the study of intervertebral cells available only in limited numbers. METHODS: Rabbit NP and AF cells released from the matrix by sequential enzyme digestion were encapsulated in alginate beads (20,000 cells/bead) and cultured for as long as 14 days. At selected time points, beads were solubilized with calcium chelating agents, and the CM and FRM were isolated. The rate of 35S-sulfate incorporation into proteoglycans, and the contents of various extracellular matrix molecules (total sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, collagen, and pyridinium crosslinks) were measured. RESULTS: Both NP and AF cells remained phenotypically stable in the alginate gel throughout the culture period and reestablished a matrix composed of CM and FRM compartments. The two cell populations exhibited numerous differences in their metabolic activities in vitro. Nucleus pulposus cells synthesized fewer proteoglycan and collagen molecules and were less effective in incorporating these into the CM than AF cells. CONCLUSIONS: Intervertebral disc cells, especially NP cells, are extremely sluggish in reforming a CM, a protective shell rich in proteoglycans and collagen molecules. This may help explain why damage to the NP often is accompanied by progressive degeneration of the disc in vivo.

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PubMed2001

Porcine neonatal pancreatic cell clusters in tissue culture: benefits of serum and immobilization in alginate hydrogel.

Porcine neonatal pancreatic cell clusters (NPCCs) may be a suitable source of insulin producing tissue for transplantation in diabetic patients. The possible beneficial effect of serum on maturation of NPCCs in vitro is difficult to achieve because of cell clumping, which can be avoided by immobilization in alginate hydrogel matrix. Collagenase treated pancreata, cultured for 4 days, formed NPCCs that were embedded in alginate cross-linked with CaCl2 and cultured in modified Ham's F10 medium with 10% fetal calf serum (FCS) for 10 days. NPCCs cultured as suspension in F10+ with 0.5% bovine serum albumin or with 10% FCS were used as control. To prevent the aggregation when cultured with serum, NPCCs were kept as a very diluted suspension. At the beginning and end of the culture, samples were taken for insulin and DNA content and immunostained for beta and non-beta cells. The culture of NPCCs immobilized in alginate resulted with 3-fold increase in insulin content and 9-fold increase in insulin/DNA ratio. Histology revealed evident increase of number of insulin- and other hormone-positive cells compared with the control. Even though 2 weeks in culture resulted in impaired glucose-induced insulin release, the amount of insulin secreted by clusters cultured in the presence of serum was 4-fold higher than in serum-free conditions. After transplantation, NPCCs retrieved from alginate reversed hyperglycemia similarly to NPCCs cultured in standard conditions. In conclusion, this study shows the feasibility of in vitro immobilization of NPCCs in alginate three-dimensional matrix, allowing cell clusters to be cultured at least two times higher density compared with culture in suspension.

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PubMed1992

Heterologous expression of an alginate lyase gene in mucoid and non-mucoid strains of Pseudomonas aeruginosa.

A 1.95 kb DNA fragment containing the aly gene from Klebsiella pneumoniae, which encodes an alginate lyase, has been ligated into the broad-host-range vector pLAFR3. Transfer of the resultant recombinant plasmid, pALY8, into mucoid and non-mucoid strains of Pseudomonas aeruginosa resulted in expression of the alginate lyase. The heterologously expressed alginate lyase, which had the same isoelectric point and substrate specificity as the native enzyme, altered the morphology of mucoid strains. Analysis of the extracellular material from mucoid strains revealed that lyase expression reduced the M(r) and overall yield of alginate produced. The mature form of the recombinant enzyme was the same as that produced extracellularly by Klebsiella pneumoniae; however, most of the alginate lyase was retained intracellularly by P. aeruginosa.

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PubMed1993

A metabolic study of the activity of GDP-mannose dehydrogenase and concentrations of activated intermediates of alginate biosynthesis in Pseudomonas aeruginosa.

GDP-mannose dehydrogenase (GMD) is a key regulatory enzyme and the committal step in alginate biosynthesis. In this study, a metabolic approach has been used to investigate GMD activity in non-mucoid and isogenically related mucoid strains of Pseudomonas aeruginosa. Intracellular concentrations of GDP-mannose and GDP-mannuronate have been quantified using HPLC separation methods, and their concentrations have been related to GMD activity and total alginate production. In all strains of P. aeruginosa tested, GDP-mannose accumulated particularly during the exponential phase of growth in batch culture; the GDP-mannose concentrations in mucoid strains were significantly lower compared with isogenic non-mucoid strains. The product of GMD activity, GDP-mannuronate, was detectable only in mucoid strains, albeit at low but relatively constant levels irrespective of growth phase. The GDP-mannose concentrations in mucoid strains were always significantly greater than those of GDP-mannuronate, indicating that GMD is a rate-limiting enzyme in the biosynthesis of alginate. Significant GMD activity and extracellular alginate production were detected only in mucoid strains. The metabolic data reported here, together with previous genetic studies, provide strong evidence that GMD is the key regulatory enzyme controlling alginate biosynthesis in mucoid strains of P. aeruginosa.

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PubMed1997

Pseudomonas aeruginosa in cystic fibrosis: role of mucC in the regulation of alginate production and stress sensitivity.

Alginate production in Pseudomonas aeruginosa and the associated mucoid phenotype of isolates from cystic fibrosis patients are under the control of the algU mucABCD cluster. This group of genes encodes AlgU, the P. aeruginosa equivalent of the extreme heat shock sigma factor sigma E in Gram-negative bacteria, the AlgU-cognate anti-sigma factor MucA, the periplasmic protein MucB and a serine protease homologue, MucD. While mucA, mucB or mucD act as negative regulators of AlgU, the function of mucC is not known. In this study the role of mucC in P. aeruginosa physiology and alginate production has been addressed. Insertional inactivation of mucC in the wild-type P. aeruginosa strain PAO1 did not cause any overt effects on alginate synthesis. However, it affected growth of P. aeruginosa under conditions of combined elevated temperature and increased ionic strength or osmolarity. Inactivation of mucC in mucA or mucB mutant backgrounds resulted in a mucoid phenotype when the cells were grown under combined stress conditions of elevated temperature and osmolarity. Each of the stress factors tested separately did not cause comparable effects. The combined stress factors were not sufficient to cause phenotypically appreciable enhancement of alginate production in mucA or mucB mutants unless mucC was also inactivated. These findings support a negative regulatory role of mucC in alginate production by P. aeruginosa, indicate additive effects of muc genes in the regulation of mucoidy in this organism and suggest that multiple stress signals and recognition systems participate in the regulation of algU-dependent functions.

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PubMed1989

Isolation of mucoid strains of Pseudomonas aeruginosa from non-cystic-fibrosis patients and characterisation of the structure of their secreted alginate.

When the incubation period of primary isolation plates was extended to 48 h, mucoid strains of Pseudomonas aeruginosa were found in specimens from various infected sites in patients who did not have cystic fibrosis. The 17 mucoid isolates were characterised in terms of mucoid type, pyocin type, and their sensitivity or resistance to seven beta-lactam and two aminoglycoside antibiotics. The carbohydrate, uronic acid (alginate) and protein content of the water-soluble extracellular material of 15 strains was determined. This material was fractionated by ion-exchange chromatography, and the presence of alginate confirmed by the chemical assay of uronic acids and their quantitation by gas-liquid chromatography. Uronic acids were absent from a non-mucoid revertant of one strain. The strains produced alginate with a high content of mannuronic acid and substituted with O-acetyl groups. By proton nuclear magnetic resonance (1H-nmr) analysis the alginate from three strains was shown to lack polyguluronate blocks in its structure. These properties are also found in the alginate of mucoid P. aeruginosa strains from patients with cystic fibrosis.

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PubMed2003

Alginate gene expression by Pseudomonas syringae pv. tomato DC3000 in host and non-host plants.

Pseudomonas syringae produces the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid. Although alginate has been isolated from plants infected by P. syringae, the signals and timing of alginate gene expression in planta have not been described. In this study, an algD : : uidA transcriptional fusion, designated pDCalgDP, was constructed and used to monitor alginate gene expression in host and non-host plants inoculated with P. syringae pv. tomato DC3000. When leaves of susceptible collard plants were spray-inoculated with DC3000(pDCalgDP), algD was activated within 72 h post-inoculation (p.i.) and was associated with the development of water-soaked lesions. In leaves of the susceptible tomato cv. Rio Grande-PtoS, algD activity was lower than in collard and was not associated with water-soaking. The expression of algD was also monitored in leaves of tomato cv. Rio Grande-PtoR, which is resistant to P. syringae pv. tomato DC3000. Within 12 h p.i., a microscopic hypersensitive response (micro-HR) was observed in Rio Grande-PtoR leaves spray-inoculated with P. syringae pv. tomato DC3000(pDCalgDP). As the HR progressed, histochemical staining indicated that individual bacterial cells on the surface of resistant tomato leaves were expressing algD. These results indicate that algD is expressed in both susceptible (e.g. collard, tomato) and resistant (Rio Grande-PtoR) host plants. The expression of algD in an incompatible host-pathogen interaction was further explored by monitoring transcriptional activity in leaves of tobacco, which is not a host for P. syringae pv. tomato. In tobacco inoculated with DC3000(pDCalgDP), an HR was evident within 12 h p.i., and algD expression was evident within 8-12 h p.i. However, when tobacco was inoculated with an hrcC mutant of DC3000, the HR did not occur and algD expression was substantially lower. These results suggest that signals that precede the HR may stimulate alginate gene expression in P. syringae. Histochemical staining with nitro blue tetrazolium indicated that the superoxide anion () is a signal for algD activation in planta. This study indicates that algD is expressed when P. syringae attempts to colonize both susceptible and resistant plant hosts.

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PubMed2006

Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae.

Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation, biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc, which produces levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme, levansucrase, occurred mainly during early exponential growth of both planktonic and sessile cells. Thus, accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.

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PubMed1996

Immobilization of flax protoplasts in agarose and alginate beads. Correlation between ionically bound cell-wall proteins and morphogenetic response.

Linum usitatissimum protoplast-derived colonies that are cultured in auxin-supplemented medium and immobilized in Ca(2+)-alginate matrix form round colonies that develop into polarized, embryo-like structures. On the other hand, protoplast-derived colonies that are immobilized in agarose do not show an organized morphogenetic response, and unique, ionically bound cell-wall protein patterns match this response. Although only slight differences in neosynthesized or total constitutive polypeptides are observed, dramatic changes in ionically bound cell-wall proteins are seen. In protoplasts grown on Ca(2+)-alginate-solidified, auxin-containing medium, several basic polypeptides were strongly induced and were found tightly bound to the cell wall. In contrast, these basic proteins were found only weakly bound to the walls of protoplasts grown on agarose-solidified, auxin-containing medium or on Ca(2+)-alginate-solidified, auxin-free medium, in which they were released into the medium. Our results suggest that plant cells can perceive and respond to the adjacent extracellular matrix, since we show that the growth of flax cells on Ca(2+)-alginate in the presence of auxin-containing medium may promote the binding of specific proteins to the walls. This establishes a direct correlation of an embryo-like morphogenesis with ionically bound cell-wall basic proteins in flax protoplasts grown on Ca(2+)-alginate-solidified, auxin-containing medium.

Alginates↗

PubMed2005

Understanding the control of Pseudomonas aeruginosa alginate synthesis and the prospects for management of chronic infections in cystic fibrosis.

Decades of research have been dedicated to the study of the opportunistic pathogen Pseudomonas aeruginosa, a Gram-negative, environmental bacterium that secretes the exopolysaccharide alginate during chronic lung infection of cystic fibrosis (CF) patients. Although P. aeruginosa utilizes a variety of factors to establish a successful infection in the lungs of CF patients, alginate has stood out as one of the best-studied prognostic indicators of chronic lung infection. While the genetics, biosynthesis and regulation of alginate are well understood, questions still remain concerning its role in biofilm development and its potential as a therapeutic target. The purpose of this review is to provide a brief summary of alginate biosynthesis and regulation, and to highlight recent discoveries in the areas of alginate production, biofilm formation and vaccine design. This information is placed in context with a proposed P. aeruginosa infectious pathway, highlighting avenues for the use of existing therapies as well as the potential for novel agents to reduce or eliminate chronic infections in CF patients.

Alginates↗

PubMed2001

A novel class of amitogenic alginate microcapsules for long-term immunoisolated transplantation.

In the light of results of clinical trials with immunoisolated human parathyroid tissue Ba2+-alginate capsules were developed that meet the requirements for long-term immunoisolated transplantation of (allogeneic and xenogeneic) cells and tissue fragments. Biocompatibility of the capsules was achieved by subjecting high-M alginate extracted from freshly collected brown algae to a simple purification protocol that removes quantitatively mitogenic and cytotoxic impurities without degradation of the alginate polymers. The final ultra-high-viscosity, clinical-grade (UHV/CG) product did not evoke any (significant) foreign body reaction in BB rats or in baboons. Similarly, the very sensitive pERK assay did not reveal any mitogenic impurities. Encapsulated cells also exhibited excellent secretory properties under in vitro conditions. Despite biocompatible material, pericapsular fibrosis is also induced by imperfect capsule surfaces that can favor cell attachment and migration under the release of material traces. This material can interact with free end monomers of the alginate polymers under formation of mitogenic advanced glycation products. Smooth surfaces, and thus topographical biocompatibility of the capsules (visualized by atomic force microscopy), can be generated by appropriate crosslinking of the UHV/CG-alginate with Ba2+ and simultaneous suppression of capsule swelling by incorporation of proteins and/or perfluorocarbons (i.e., medically approved compounds with high oxygen capacity). Perfluorocarbon-loaded alginate capsules allow long-term non-invasive monitoring of the location and the oxygen supply of the transplants by using 19F-MRI. Transplantation studies in rats demonstrated that these capsules were functional over a period of more than two years.

Alginates↗

PubMed2001

Transplantation of alginate microcapsules with proliferating cells in mice: capsular overgrowth and survival of encapsulated cells of mice and human origin.

Alginate microcapsules may be used to encapsulate therapeutic cells and, thereby, to protect them from the host immune system. Both the biomaterial, as well as the therapeutic cells, may give rise to immunological reactions. We have developed methods that are useful in the study of capsule biocompatibility, as well as reactions against the grafts. These imply investigation of the survival of the encapsulated cells as well as fibrotic reactions against the microcapsules. Studies were performed in Balb/c mice with empty alginate-PLL-alginate microcapsules as well as microcapsules containing cells of human or mouse origin. Confocal laser scanning microscopy (CLSM) was used to visualize live and dead cells within the microcapsules and to define some of the cells involved in the fibrotic reaction against the microcapsules. In both grafts, live cells were detected seven days after transplantation. Minor fibrotic reactions were found against empty alginate-PLL-alginate microcapsules and to microcapsules containing mouse cells. An extensive fibrotic reaction was found one week after transplantation against microcapsules containing human cells, and the secretion of therapeutic protein endostatin had ceased. Fibroblasts and macrophages were involved in the fibrotic reaction against the xenograft.

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PubMed1993

Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa.

Reporter gene technology was employed to detect the activity of an alginate promoter of Pseudomonas aeruginosa when the organism was grown as a biofilm on a Teflon mesh substratum and as planktonic cells in liquid medium. Alginate biosynthetic activity was determined with a mucoid cell line derived from a cystic fibrosis isolate and containing an alginate algC promoter fused to a lacZ reporter gene. Reporter activity was demonstrated with chromogenic and fluorogenic substrates for beta-galactosidase. Expression of algC was shown to be upregulated in biofilm cells compared with planktonic cells in liquid medium. Gene up-expression correlated with alginate biosynthesis as measured by Fourier transform infrared spectroscopy, uronic acid accumulation, and alginate-specific enzyme-linked immunosorbent assay. The algC promoter was shown to have maximum activity in planktonic cultures during the late lag and early log phases of the cell growth cycle. During a time course experiment, biofilm algC activity exceeded planktonic activity except during the period immediately following inoculation into fresh medium. In continuous-culture experiments, conversion of lacZ substrate was demonstrated microscopically in individual cells by epifluorescence microscopy.

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PubMed1984

Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate.

Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis commonly produce a capsule-like exopolysaccharide called alginate. The alginate-producing (Alg+) phenotype results in a mucoid colony morphology and is an unstable trait. A mutant of P. aeruginosa FRD (a cystic fibrosis isolate) was obtained which was temperature sensitive for alginate production ( Algts ). At elevated growth temperatures (41 degrees C), no alginate was detected in culture supernatants of the Algts mutant, but yields of alginate increased as the temperature of incubation was reduced. The mutation responsible for the Algts phenotype, alg-50(Ts), has been mapped to a region of the FRD chromosome closely linked to trp-2. The alg-50(Ts) marker did not map near the met-l-linked chromosomal mutations responsible for the instability of the Alg+ phenotype. A broad host range cosmid cloning system based upon derivatives of plasmid RK2 was used to construct a P. aeruginosa clone bank. After transfer of the clone bank to the Algts mutant, hybrid plasmids were obtained which complemented the Algts defect. Deletion mapping of the original 20.3 kilobases of P. aeruginosa DNA cloned showed that a 4.7-kilobase fragment would complement the alg-50(Ts) mutation.

Alginates↗

PubMed1994

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.

Alginates↗

PubMed1994

Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT, encoding a putative alternate sigma factor, and shows evidence for autoregulation.

The mucoid phenotype is common among strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis and is due to overproduction of an exopolysaccharide called alginate. However, the mucoid phenotype is unstable in vitro, especially when the cells are incubated under low oxygen tension. Spontaneous conversion to the nonmucoid form is typically due to mutations (previously called algS) that are closely linked to the alginate regulatory gene algT, located at 68 min on the chromosome. Our sequence analysis of algT showed that its 22-kDa gene product shares homology with several alternate sigma factors in bacteria, suggesting that AlgT (also known as AlgU) interacts directly with RNA polymerase core to activate the promoters of alginate genes. AlgT showed striking sequence similarity (79%) to sigma E of Escherichia coli, an alternate sigma factor involved in high-temperature gene expression. Our analysis of the molecular basis for spontaneous conversion from mucoid to nonmucoid, in the cystic fibrosis isolate FRD, revealed that nonmucoid conversion was often due to one of two distinct missense mutations in algT that occurred at codons 18 and 29. RNase protection assays showed that spontaneous nonmucoid strains with the algT18 and algT29 alleles have a four- to fivefold reduction in the accumulation of algT transcripts compared with the wild-type mucoid strain. Likewise, a plasmid-borne algT-cat transcriptional fusion was about 3-fold less active in the algT18 and algT29 backgrounds compared with the mucoid wild-type strain, and it was 20-fold less active in an algT::Tn501 background. These data indicate that algT is autoregulated. The spontaneous algT missense alleles also caused about fivefold-reduced expression of the adjacent negative regulator, algN (also known as mucB). Transcripts of algN were essentially absent in the algT::Tn501 strain. Thus, algT regulates the algTN cluster, and the two genes may be cotranscribed. A primer extension analysis showed that algT transcription starts 54 bp upstream of the start of translation. Although the algT promoter showed little similarity to promoters recognized by the vegetative sigma factor, it was similar to the algR promoter. This finding suggests that AlgT may function as a sigma factor to activate its own promoter and those of other alginate genes. The primer extension analysis also showed that algT transcripts were readily detectable in the typical nonmucoid strain PAO1, which was in contrast to a weak signal seen in the algT18 mutant of FRD. A plasmid-borne algT gene in PAO1 resulted in both the mucoid phenotype and high levels of algT transcripts, further supporting the hypothesis that AlgT controls its own gene expression and expression of genes of the alginate regulon.

Alginates↗

PubMed1997

Identification of the algZ gene upstream of the response regulator algR and its participation in control of alginate production in Pseudomonas aeruginosa.

Alginate production in mucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients is under direct control by AlgU, the P. aeruginosa equivalent of the extreme heat shock sigma factor sigma(E) in gram-negative bacteria, and AlgR, a response regulator from the superfamily of two-component signal transduction systems. In this report, we describe the identification of the algZ gene, located immediately upstream of algR, which is involved in the control of alginate production. The predicted product of the algZ gene showed similarity to a subset of sensory components from the superfamily of signal transduction systems but lacked several of the highly conserved motifs typical of histidine protein kinases. Inactivation of algZ in the wild-type standard genetic strain PAO1 did not affect its nonmucoid morphology. However, inactivation of algZ in a mucoid mutant P. aeruginosa strain, which had AlgU freed from control by the anti-sigma factor MucA, resulted in increased alginate production under growth conditions which did not permit expression of mucoidy in the parental algZ+ strain. The observed effects were abrogated when algR was inactivated in the algZ::Tc(r) background. These findings indicate that algZ plays a regulatory role in alginate production, possibly interacting with AlgR, and that it may have negative effects on expression of the mucoid phenotype under the conditions tested. The presented results suggest that elements of negative regulation exist at the levels of both the alternative sigma factor AlgU and the transcriptional activator AlgR which, once relieved from that suppression, cooperate to bring about the expression of the alginate system.

Alginates↗