A mutation in algN permits trans activation of alginate production by algT in Pseudomonas species.

Conversion of the mucoid phenotype, which results from the production of the exopolysaccharide alginate, is a feature typical of Pseudomonas aeruginosa strains causing chronic pulmonary infections in patients with cystic fibrosis. In this study, we further characterized a recombinant plasmid, called pJF15, that contains DNA from the 65- to 70-min region of the chromosome of mucoid P. aeruginosa FRD1 and has loci involved in alginate conversion. Plasmid pJF15 complements algT mutations in trans and confers the mucoid phenotype in cis following gene replacement. However, the phenotype of nonmucoid P. aeruginosa carrying pJF15 is unchanged. Here we report the identification of a locus immediately downstream of algT, called algN, that may be a negative regulator that blocks algT from activating alginate production. Inactivation of algN by transposon Tn501 insertion allowed algT to stimulate alginate production in trans. The DNA sequence of this region identified an open reading frame that predicts an algN gene product of 33 kDa, but no homology was found to other proteins in a sequence data base. Clones of algT in which algN was deleted caused the activation of alginate biosynthesis in transconjugants of several P. aeruginosa strains. DNA containing algT was shown to hybridize to the genomes of several Pseudomonas species, including P. putida, P. stutzeri, and P. fluorescens. Transconjugants of these species carrying algT DNA (with a deletion of algN) from pJF15 showed a mucoid phenotype and increased production of uronic acid-containing polymers that resembled alginate.     

Cloning of genes from mucoid Pseudomonas aeruginosa which control spontaneous conversion to the alginate production phenotype.

Strains of Pseudomonas aeruginosa causing chronic pulmonary infections in patients with cystic fibrosis are known to convert to a mucoid form in vivo characterized by the production of the exopolysaccharide alginate. The alginate production trait is not stable, and mucoid strains frequently convert back to the nonmucoid form in vitro. The DNA involved in these spontaneous alginate conversions, referred to as algS, was shown here to map near hisI and pru markers on the chromosome of strain FRD, an isolate from a cystic fibrosis patient. Although cloning algS+ by trans-complementation was not possible, a clone (pJF5) was isolated that caused algS mutants to convert to the Alg+ phenotype at detectable frequencies (approximately 0.1%) in vitro. Gene replacement with transposon-marked pJF5 followed by mapping studies showed that pJF5 contained DNA transducibly close to algS in the chromosome. Another clone was identified called pJF15 which did contain algS+ from mucoid P. aeruginosa. The plasmid-borne algS+ locus could not complement spontaneous algS mutations in trans, but its cis-acting activity was readily observed after gene replacement with the algS mutant chromosome by using an adjacent transposon as the selectable marker. pJF15 also contained a trans-active gene called algT+ in addition to the cis-active gene algS+. The algT gene was localized on pJF15 by using deletion mapping and transposon mutagenesis. By using gene replacement, algT::Tn501 mutants of P. aeruginosa were constructed which were shown to be complemented in trans by pJF15. Both algS and algT were located on a DNA fragment approximately 3 kilobases in size. The algS gene may be a genetic switch which regulates the process of alginate conversion.     

Use of a gene replacement cosmid vector for cloning alginate conversion genes from mucoid and nonmucoid Pseudomonas aeruginosa strains: algS controls expression of algT.

Pseudomonas aeruginosa can convert to a mucoid colony morphology by a genetic mechanism called alginate conversion; this results in the production of copious amounts of the exopolysaccharide alginate. The mucoid phenotype of P. aeruginosa is commonly associated with its ability to cause chronic pulmonary tract infections in patients with cystic fibrosis. In this study we isolated the cis-acting locus involved in alginate conversion, called algS, from both mucoid and nonmucoid isogenic strains. We then examined the role of algS in the control of algT, a trans-active gene required for alginate production in P. aeruginosa. We used a new cosmid cloning vector, called pEMR2, that permitted both the cloning of large DNA fragments and their subsequent gene replacement in P. aeruginosa. To verify the predicted properties of this vector, we isolated and tested a pEMR2 hisI+ clone. Using cloned algS-containing DNA and a method for gene replacement, we constructed isogenic strains of P. aeruginosa that had Tn501 adjacent to algS on the chromosome. Two pEMR2 clone banks containing genomic fragments from isogenic algS(On) (exhibiting the alginate production phenotype) and algS(Off) (exhibiting the non-alginate production phenotype) strains were constructed, and Tn501 served as an adjacent marker to select for clones containing the respective algS allele. The pEMR2 algS(On) and pEMR2 algS(Off) clones were shown to contain the indicated algS allele by gene replacement with the chromosome of strains that carried the opposite allele. To test whether algS controls the expression of the adjacent algT gene, we constructed a pLAFR1 algS(Off)T clone and showed it to be unable to complement an algT::Tn501 mutation in trans. In contrast, a pLAFR1 algS(On)T clone did complement algT::Tn501 in trans. Thus, algS appears to control the activation of algT expression, bringing about alginate conversion.     

A complex multilevel attack on Pseudomonas aeruginosa algT/U expression and algT/U activity results in the loss of alginate production

Infection by the opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. This is mainly due to the genotypic and phenotypic changes of the bacteria that cause conversion from a typical nonmucoid to a mucoid form in the CF lung. Mucoid conversion is indicative of overproduction of a capsule-like polysaccharide called alginate. The alginate-overproducing (Alg(+)) mucoid phenotype seen in the CF isolates is extremely unstable. Low oxygen tension growth of mucoid variants readily selects for nonmucoid variants. The switching off mechanism has been mapped to the algT/U locus, and the molecular basis for this conversion was partially attributed to mutations in the algT/U gene itself. To further characterize molecular changes resulting in the unstable phenotype, an isogenic PAO1 derivative that is constitutively Alg(+) due to the replacement of the mucA with mucA22 (PDO300) was used. The mucA22 allele is common in mucoid CF isolates. Thirty-four spontaneous nonmucoid variants, or sap (suppressor of alginate production) mutants, of PDO300 were isolated under low oxygen tension. About 40% of the sap mutants were rescued by a plasmid carrying algT/U (Group A). The remaining sap mutants were not (Group B). The members of Group B fall into two subsets: one similar to PAO1, and another comparable to PDO300. Sequence analysis of the algT/U and mucA genes in Group A shows that mucA22 is intact, whereas algT/U contains mutations. Genetic complementation and sequencing of one Group B sap mutant, sap22, revealed that the nonmucoid phenotype was due to the presence of a mutation in PA3257. PA3257 encodes a putative periplasmic protease. Mutation of PA3257 resulted in decreased algT/U expression. Thus, inhibition of algT/U is a primary mechanism for alginate synthesis suppression.     

Adaptations of Pseudomonas aeruginosa to the cystic fibrosis lung environment can include deregulation of zwf, encoding glucose-6-phosphate dehydrogenase

Cystic fibrosis (CF) patients are highly susceptible to chronic pulmonary disease caused by mucoid Pseudomonas aeruginosa strains that overproduce the exopolysaccharide alginate. We showed here that a mutation in zwf, encoding glucose-6-phosphate dehydrogenase (G6PDH), leads to a approximately 90% reduction in alginate production in the mucoid, CF isolate, P. aeruginosa FRD1. The main regulator of alginate, sigma-22 encoded by algT (algU), plays a small but demonstrable role in the induction of zwf expression in P. aeruginosa. However, G6PDH activity and zwf expression were higher in FRD1 strains than in PAO1 strains. In PAO1, zwf expression and G6PDH activity are known to be subject to catabolite repression by succinate. In contrast, FRD1 zwf expression and G6PDH activity were shown to be refractory to such catabolite repression. This was apparently not due to a defect in the catabolite repression control (Crc) protein. Such relaxed control of zwf was found to be common among several examined CF isolates but was not seen in other strains of clinical and environmental origin. Two sets of clonal isolates from individual CF patient indicated that the resident P. aeruginosa strain underwent an adaptive change that deregulated zwf expression. We hypothesized that high-level, unregulated G6PDH activity provided a survival advantage to P. aeruginosa within the lung environment. Interestingly, zwf expression in P. aeruginosa was shown to be required for its resistance to human sputum. This study illustrates that adaptation to the CF pulmonary environment by P. aeruginosa can include altered regulation of basic metabolic activities, including carbon catabolism.     

Impact of Alginate Conditioning Film on Deposition Kinetics of Motile and Nonmotile Pseudomonas aeruginosa Strains

The initial deposition of bacteria in most aquatic systems is affected by the presence of a conditioning film adsorbed at the liquid-solid interface. Due to the inherent complexity of such films, their impact on bacterial deposition remains poorly defined. The aim of this study was to gain a better understanding of the effect of a conditioning film on the deposition of motile and nonmotile Pseudomonas aeruginosa cells in a radial stagnation point flow system. A well-defined alginate film was used as a model conditioning film because of its polysaccharide and polyelectrolyte nature. Deposition experiments under favorable (nonrepulsive) conditions demonstrated the importance of swimming motility for cell transport towards the substrate. The impact of the flagella of motile cells on deposition is dependent on the presence of the conditioning film. We showed that on a clean substrate surface, electrostatic repulsion governs bacterial deposition and the presence of flagella increases cell deposition. However, our results suggest that steric interactions between flagella and extended polyelectrolytes of the conditioning film hinder cell deposition. At a high ionic strength (100 mM), active swimming motility and changes in alginate film structure suppressed the steric barrier and allowed conditions favorable for deposition. We demonstrated that bacterial deposition is highly influenced by cell motility and the structure of the conditioning film, which are both dependent on ionic strength.     

Impact of alginate conditioning film on deposition kinetics of motile and nonmotile Pseudomonas aeruginosa strains

The initial deposition of bacteria in most aquatic systems is affected by the presence of a conditioning film adsorbed at the liquid-solid interface. Due to the inherent complexity of such films, their impact on bacterial deposition remains poorly defined. The aim of this study was to gain a better understanding of the effect of a conditioning film on the deposition of motile and nonmotile Pseudomonas aeruginosa cells in a radial stagnation point flow system. A well-defined alginate film was used as a model conditioning film because of its polysaccharide and polyelectrolyte nature. Deposition experiments under favorable (nonrepulsive) conditions demonstrated the importance of swimming motility for cell transport towards the substrate. The impact of the flagella of motile cells on deposition is dependent on the presence of the conditioning film. We showed that on a clean substrate surface, electrostatic repulsion governs bacterial deposition and the presence of flagella increases cell deposition. However, our results suggest that steric interactions between flagella and extended polyelectrolytes of the conditioning film hinder cell deposition. At a high ionic strength (100 mM), active swimming motility and changes in alginate film structure suppressed the steric barrier and allowed conditions favorable for deposition. We demonstrated that bacterial deposition is highly influenced by cell motility and the structure of the conditioning film, which are both dependent on ionic strength.     

Use of cell wall stress to characterize σ22 (AlgT/U) activation by regulated proteolysis and its regulon in Pseudomonas aeruginosa

MucA sequesters extracytoplasmic function (ECF) σ²² ( algT/U encoded) from target promoters including P algD for alginate biosynthesis. We have shown that cell wall stress (e.g. d -cycloserine) is a potent inducer of the algD operon. Here we showed that MucB, encoded by the algT-mucABCD operon, interacts with MucA in the sigma–sequestration complex. We hypothesized that AlgW protease (a DegS homologue) is activated by cell wall stress to cleave MucA and release σ²². When strain PAO1 was exposed to d -cycloserine, MucA was degraded within just 10 min, and σ²² was activated. However, in an algW mutant, MucA was stable with no increased σ²² activity. Studies on a yaeL mutant, defective in an RseP/YaeL homologue, suggest that YaeL protease cleaves MucA only after cleavage by AlgW. A defect in mucD , encoding a periplasmic HtrA/DegP homologue, caused MucA instability, suggesting MucD degrades cell wall stress signals. Overall, these data indicate that cell wall stress signals release σ²² by regulated intramembrane proteolysis (RIP). Microarray analyses identified genes of the early and late cell wall stress stimulon, which included genes for alginate production. The subset of genes in the σ²² regulon was then determined, which included gene products predicted to contribute to recovery from cell wall stress.     

A spider web strategy of type IV pili‐mediated migration to build a fibre‐like Psl polysaccharide matrix in Pseudomonas aeruginosa biofilms

Bacterial motilities participate in biofilm development. However, it is unknown how/if bacterial motility affects formation of the biofilm matrix. Psl polysaccharide is a key biofilm matrix component of Pseudomonas aeruginosa. Here we report that type IV pili (T4P)‐mediated bacterial migration leads to the formation of a fibre‐like Psl matrix. Deletion of T4P in wild type and flagella‐deficient strains results in loss of the Psl‐fibres and reduction of biofilm biomass in flow cell biofilms as well as pellicles at air‐liquid interface. Bacteria lacking T4P‐driven twitching motility including those that still express surface T4P are unable to form the Psl‐fibres. Formation of a Psl‐fibre matrix is critical for efficient biofilm formation, yet does not require flagella and polysaccharide Pel or alginate. The Psl‐fibres are likely formed by Psl released from bacteria during T4P‐mediated migration, a strategy similar to spider web formation. Starvation can couple Psl release and T4P‐driven twitching motility. Furthermore, a radial‐pattern Psl‐fibre matrix is present in the middle of biofilms, a nutrient‐deprived region. These imply a plausible model for how bacteria respond to nutrient‐limited local environment to build a polysaccharide‐fibre matrix by T4P‐dependent bacterial migration strategy. This strategy may have general significance for bacterial survival in natural and clinical settings.     

Proteomics-based identification of outer-membrane proteins responsible for import of macromolecules in Sphingomonas sp. A1: alginate-binding flagellin on the cell surface

A nonmotile gram-negative bacterium, Sphingomonas sp. A1, directly incorporates macromolecules such as alginate through a "super-channel" consisting of a pit formed on the cell surface, alginate-binding proteins in the periplasm, and an ATP-binding cassette transporter in the inner membrane. Here, we demonstrate the proteomics-based identification of cell-surface proteins involved in the formation of the pit and/or import of alginate. Cell-surface proteins were prepared from the outer membrane released as vesicles during the conversion of intact cells to spheroplasts. Seven proteins (p1-p7) with acidic isoelectric points were inducibly expressed in the outer membrane of strain A1 cells grown on alginate and showed significant identity with bacterial cell-surface proteins (p1-p4, TonB-dependent outer-membrane transporter; p5 and p6, flagellin; and p7, lipoprotein). Each mutant with a disruption of the p1-p4 or p6 gene showed significant growth retardation in the alginate medium. Flagellin homologues (p5 and p6) were further analyzed because strain A1 forms no flagellum. p5 was found to be uniformly distributed on the cell surface by immunogold-labeling electron microscopy and to exhibit alginate binding with a nanomolar dissociation constant by a surface plasmon resonance sensor. The cell surface of the p6 gene disruptant differed from that of the wild-type strain A1 in that pit formation was incomplete and cell-surface structures shifted from pleats to networks. These results suggest that, distinct from bacterial flagellins constituting a helical filament of flagella, strain A1 cell-surface flagellin homologues function as receptors for alginate and/or regulators of cell-surface structures.     

The archaeal flagellum: a different kind of prokaryotic motility structure

The archaeal flagellum is a unique motility apparatus distinct in composition and likely in assembly from the bacterial flagellum. Gene families comprised of multiple flagellin genes co-transcribed with a number of conserved, archaeal-specific accessory genes have been identified in several archaea. However, no homologues of any bacterial genes involved in flagella structure have yet been identified in any archaeon, including those archaea in which the complete genome sequence has been published. Archaeal flagellins possess a highly conserved hydrophobic N-terminal sequence that is similar to that of type IV pilins and clearly unlike that of bacterial flagellins. Also unlike bacterial flagellins but similar to type IV pilins, archaeal flagellins are initially synthesized with a short leader peptide that is cleaved by a membrane-located peptidase. With recent advances in genetic transfer systems in archaea, knockouts have been reported in several genes involved in flagellation in different archaea. In addition, techniques to isolate flagella with attached hook and anchoring structures have been developed. Analysis of these preparations is under way to identify minor structural components of archaeal flagella. This and the continued isolation and characterization of flagella mutants should lead to significant advances in our knowledge of the composition and assembly of archaeal flagella.     

Crystal structure of a novel bacterial cell-surface flagellin binding to a polysaccharide

Bacterial flagellins are generally self-assembled into extracellular flagella for cell motility. However, the flagellin homologue p5 is found on the cell surface of Sphingomonas sp. A1 (strain A1) and binds tightly to the alginate polysaccharide. To assimilate alginate, strain A1 forms a mouthlike pit on the cell surface and concentrates the polymer in the pit. p5 is a candidate receptor that recognizes extracellular alginate and controls pit formation. To improve our understanding of the structure and function of p5, we determined the crystal structure of truncated p5 (p5DeltaN53C45) at 2.0 A resolution. This, to our knowledge, is the first structure of flagellin_IN motif-containing flagellin. p5DeltaN53C45 consists of two domains: an alpha-domain rich in alpha-helices that forms the N- and C-terminal regions and a beta-domain rich in beta-strands that constitutes the central region. The alpha-domain is structurally similar to the D1 domain of Salmonella typhimurium flagellin, while the beta-domain is structurally similar to the finger domain of the bacteriophage T4 baseplate protein that is important for intermolecular interactions between baseplate and a long or short tail fiber. Results from the deletion mutant analysis suggest that residues 20-40 and 353-363 are responsible for alginate binding. Truncated N- and C-terminal regions are thought to constitute alpha-helices extending from the alpha-domain. On the basis of the size and surface charge, the cleft in extended alpha-helices is proposed as an alginate binding site of p5. Structural similarity in the beta-domain suggests that the beta-domain is involved in the proper localization and/or orientation of p5 on the cell surface.     

Alginate Hydrogel Protects Encapsulated Hepatic HuH-7 Cells against Hepatitis C Virus and Other Viral Infections

Cell microencapsulation in alginate hydrogel has shown interesting applications in regenerative medicine and the biomedical field through implantation of encapsulated tissue or for bioartificial organ development. Although alginate solution is known to have low antiviral activity, the same property regarding alginate gel has not yet been studied. The aim of this work is to investigate the potential protective effect of alginate encapsulation against hepatitis C virus (HCV) infection for a hepatic cell line (HuH-7) normally permissive to the virus. Our results showed that alginate hydrogel protects HuH-7 cells against HCV when the supernatant was loaded with HCV. In addition, alginate hydrogel blocked HCV particle release out of the beads when the HuH-7 cells were previously infected and encapsulated. There was evidence of interaction between the molecules of alginate hydrogel and HCV, which was dose- and incubation time-dependent. The protective efficiency of alginate hydrogel towards HCV infection was confirmed against a variety of viruses, whether or not they were enveloped. This promising interaction between an alginate matrix and viruses, whose chemical mechanisms are discussed, is of great interest for further medical therapeutic applications based on tissue engineering.     

Superchannel of bacteria: biological significance and new horizons

Sphingomonas species A1 is a newly identified pit-forming bacterium that directly incorporates a macromolecule (alginate) into its cytoplasm through a pit-dependent transport system, which we termed a superchannel. A pit is a novel, high-dimensional organ acquired through the fluidity and reconstitution of cell surface molecules, including flagellin, and through cooperation with the transport machinery in the cells, which confers upon bacterial cells a more efficient way to secure and assimilate macromolecules. The analysis of the superchannel changes general ideas regarding the fluidity and function of the cell surface, evolution and origin of cell-surface organs, including flagella, transport, and assimilation systems of macromolecules, and the divergence and energetics of metabolism.