FusC, a member of the M16 protease family acquired by bacteria for iron piracy against plants.

Iron is essential for life. Accessing iron from the environment can be a limiting factor that determines success in a given environmental niche. For bacteria, access of chelated iron from the environment is often mediated by TonB-dependent transporters (TBDTs), which are ß-barrel proteins that form sophisticated channels in the outer membrane. Reports of iron-bearing proteins being used as a source of iron indicate specific protein import reactions across the bacterial outer membrane. The molecular mechanism by which a folded protein can be imported in this way had remained mysterious, as did the evolutionary process that could lead to such a protein import pathway. How does the bacterium evolve the specificity factors that would be required to select and import a protein encoded on another organism's genome? We describe here a model whereby the plant iron-bearing protein ferredoxin can be imported across the outer membrane of the plant pathogen Pectobacterium by means of a Brownian ratchet mechanism, thereby liberating iron into the bacterium to enable its growth in plant tissues. This import pathway is facilitated by FusC, a member of the same protein family as the mitochondrial processing peptidase (MPP). The Brownian ratchet depends on binding sites discovered in crystal structures of FusC that engage a linear segment of the plant protein ferredoxin. Sequence relationships suggest that the bacterial gene encoding FusC has previously unappreciated homologues in plants and that the protein import mechanism employed by the bacterium is an evolutionary echo of the protein import pathway in plant mitochondria and plastids.

Filamentous phages: masters of a microbial sharing economy.

Bacteriophage ("bacteria eaters") or phage is the collective term for viruses that infect bacteria. While most phages are pathogens that kill their bacterial hosts, the filamentous phages of the sub-class Inoviridae live in cooperative relationships with their bacterial hosts, akin to the principal behaviours found in the modern-day sharing economy: peer-to-peer support, to offset any burden. Filamentous phages impose very little burden on bacteria and offset this by providing service to help build better biofilms, or provision of toxins and other factors that increase virulence, or modified behaviours that provide novel motile activity to their bacterial hosts. Past, present and future biotechnology applications have been built on this phage-host cooperativity, including DNA sequencing technology, tools for genetic engineering and molecular analysis of gene expression and protein production, and phage-display technologies for screening protein-ligand and protein-protein interactions. With the explosion of genome and metagenome sequencing surveys around the world, we are coming to realize that our knowledge of filamentous phage diversity remains at a tip-of-the-iceberg stage, promising that new biology and biotechnology are soon to come.

Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity.

The multifunctional enzyme S-adenosylhomocysteine/methylthioadenosine nucleosidase is a key metabolic enzyme in the virulence of Salmonella enterica var Typhimurium.

Key physiological differences between bacterial and mammalian metabolism provide opportunities for the development of novel antimicrobials. We examined the role of the multifunctional enzyme S-adenosylhomocysteine/Methylthioadenosine (SAH/MTA) nucleosidase (Pfs) in the virulence of S. enterica var Typhimurium (S. Typhimurium) in mice, using a defined Pfs deletion mutant (i.e. Δpfs). Pfs was essential for growth of S. Typhimurium in M9 minimal medium, in tissue cultured cells, and in mice. Studies to resolve which of the three known functions of Pfs were key to murine virulence suggested that downstream production of autoinducer-2, spermidine and methylthioribose were non-essential for Salmonella virulence in a highly sensitive murine model. Mass spectrometry revealed the accumulation of SAH in S. Typhimurium Δpfs and complementation of the Pfs mutant with the specific SAH hydrolase from Legionella pneumophila reduced SAH levels, fully restored growth ex vivo and the virulence of S. Typhimurium Δpfs for mice. The data suggest that Pfs may be a legitimate target for antimicrobial development, and that the key role of Pfs in bacterial virulence may be in reducing the toxic accumulation of SAH which, in turn, suppresses an undefined methyltransferase.

An investigation into the Omp85 protein BamK in hypervirulent Klebsiella pneumoniae, and its role in outer membrane biogenesis.

Members of the Omp85 protein superfamily have important roles in Gram-negative bacteria, with the archetypal protein BamA being ubiquitous given its essential function in the assembly of outer membrane proteins. In some bacterial lineages, additional members of the family exist and, in most of these cases, the function of the protein is unknown. We detected one of these Omp85 proteins in the pathogen Klebsiella pneumoniae B5055, and refer to the protein as BamK. Here, we show that bamK is a conserved element in the core genome of Klebsiella, and its expression rescues a loss-of-function ∆bamA mutant. We developed an E. coli model system to measure and compare the specific activity of BamA and BamK in the assembly reaction for the critical substrate LptD, and find that BamK is as efficient as BamA in assembling the native LptDE complex. Comparative structural analysis revealed that the major distinction between BamK and BamA is in the external facing surface of the protein, and we discuss how such changes may contribute to a mechanism for resistance against infection by bacteriophage.

Structure and Membrane Topography of the Vibrio-Type Secretin Complex from the Type 2 Secretion System of Enteropathogenic Escherichia coli.

The ß-barrel assembly machinery (BAM) complex is the core machinery for the assembly of ß-barrel membrane proteins, and inhibition of BAM complex activity is lethal to bacteria. Discovery of integral membrane proteins that are key to pathogenesis and yet do not require assistance from the BAM complex raises the question of how these proteins assemble into bacterial outer membranes. Here, we address this question through a structural analysis of the type 2 secretion system (T2SS) secretin from enteropathogenic Escherichia coli O127:H6 strain E2348/69. Long ß-strands assemble into a barrel extending 17 Å through and beyond the outer membrane, adding insight to how these extensive ß-strands are assembled into the E. coli outer membrane. The substrate docking chamber of this secretin is shown to be sufficient to accommodate the substrate mucinase SteC.IMPORTANCE In order to cause disease, bacterial pathogens inhibit immune responses and induce pathology that will favor their replication and dissemination. In Gram-negative bacteria, these key attributes of pathogenesis depend on structures assembled into or onto the outer membrane. One of these is the T2SS. The Vibrio-type T2SS mediates cholera toxin secretion in Vibrio cholerae, and in Escherichia coli O127:H6 strain E2348/69, the same machinery mediates secretion of the mucinases that enable the pathogen to penetrate intestinal mucus and thereby establish deadly infections.

Assembly of the secretion pores GspD, Wza and CsgG into bacterial outer membranes does not require the Omp85 proteins BamA or TamA.

In Gram-negative bacteria, ß-barrel proteins are integrated into the outer membrane by the ß-barrel assembly machinery, with key components of the machinery being the Omp85 family members BamA and TamA. Recent crystal structures and cryo-electron microscopy show a diverse set of secretion pores in Gram-negative bacteria, with α-helix (Wza and GspD) or ß-strand (CsgG) transmembrane segments in the outer membrane. We developed assays to measure the assembly of three distinct secretion pores that mediate protein (GspD), curli fibre (CsgG) and capsular polysaccharide (Wza) secretion by bacteria and show that depletion of BamA and TamA does not diminish the assembly of Wza, GspD or CsgG. Like the well characterised pilotins for GspD and other secretins, small periplasmic proteins enhance the assembly of the CsgG ß-barrel. We discuss a model for integral protein assembly into the bacterial outer membrane, focusing on the commonalities and differences in the assembly of Wza, GspD and CsgG.

Searching for new strategies against polymicrobial biofilm infections: guanylated polymethacrylates kill mixed fungal/bacterial biofilms.

OBJECTIVES: Biofilm-related human infections have high mortality rates due to drug resistance. Cohabitation of diverse microbes in polymicrobial biofilms is common and these infections present additional challenges for treatment compared with monomicrobial biofilms. Here, we address this therapeutic gap by assessing the potential of a new class of antimicrobial agents, guanylated polymethacrylates, in the treatment of polymicrobial biofilms built by two prominent human pathogens, the fungus Candida albicans and the bacterium Staphylococcus aureus. METHODS: We used imaging and quantitative methods to test the antibiofilm efficacy of guanylated polymethacrylates, a new class of drugs that structurally mimic antimicrobial peptides. We further compared guanylated polymethacrylates with first-line antistaphylococcal and anti-Candida agents used as combinatorial therapy against polymicrobial biofilms. RESULTS: Guanylated polymethacrylates were highly effective as a sole agent, killing both C. albicans and S. aureus when applied to established polymicrobial biofilms. Furthermore, they outperformed multiple combinations of current antimicrobial drugs, with one of the tested compounds killing 99.98% of S. aureus and 82.2% of C. albicans at a concentration of 128 mg/L. The extracellular biofilm matrix provided protection, increasing the MIC of the polymethacrylates by 2-4-fold when added to planktonic assays. Using the C. albicans bgl2ΔΔ mutant, we implicate matrix polysaccharide ß-1,3 glucan in the mechanism of protection. Data for two structurally distinct polymers suggest that this mechanism could be minimized through chemical optimization of the polymer structure. Finally, we demonstrate that a potential application for these polymers is in antimicrobial lock therapy. CONCLUSIONS: Guanylated polymethacrylates are a promising lead for the development of an effective monotherapy against C. albicans/S. aureus polymicrobial biofilms.

Bioengineering of bacteria to assemble custom-made polyester affinity resins.

Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced "target protein." Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains.

In vivo polyester immobilized sortase for tagless protein purification.

BACKGROUND: Laboratory scale recombinant protein production and purification techniques are often complicated, involving multiple chromatography steps and specialized equipment and reagents. Here it was demonstrated that recombinant proteins can be expressed as covalently immobilized to the surface of polyester (polyhydroxyalkanoate, PHA) beads in vivo in Escherichia coli by genetically fusing them to a polyester synthase gene (phaC). The insertion of a self-cleaving module, a modified sortase A (SrtA) from Staphylococcus aureus and its five amino acid recognition sequence between the synthase and the target protein led to a simple protein production and purification method. RESULTS: The generation of hybrid genes encoding tripartite PhaC-SrtA-Target fusion proteins, enabled immobilization of proteins of interest to the surface of PHA beads in vivo. After simple cell lysis and isolation of the PHA beads, the target proteins could be selectively and efficiently released form the beads by activating the sortase with CaCl2 and triglycine. Up to 6 mg/l of soluble proteins at a purity of ~98 % could be isolated in one step with no optimization. This process was used to produce and isolate three proteins: Green fluorescent protein, maltose binding protein and the Mycobacterium tuberculosis vaccine candidate Rv1626. CONCLUSIONS: We have developed a new technique for easy production and purification of recombinant proteins. This technique is capable of producing and purifying high yields of proteins suitable for research application in less than 2 days. No costly or specialized protein chromatography equipment, resins, reagents or expertise are required.

Membrane-anchored MucR mediates nitrate-dependent regulation of alginate production in Pseudomonas aeruginosa.

Alginates exhibit unique material properties suitable for medical and industrial applications. However, if produced by Pseudomonas aeruginosa, it is an important virulence factor in infection of cystic fibrosis patients. The alginate biosynthesis machinery is activated by c-di-GMP imparted by the inner membrane protein, MucR. Here, it was shown that MucR impairs alginate production in response to nitrate in P. aeruginosa. Subsequent site-specific mutagenesis of MucR revealed that the second MHYT sensor motif (MHYT II, amino acids 121-124) of MucR sensor domain was involved in nitrate sensing. We also showed that both c-di-GMP synthesizing and degrading active sites of MucR were important for alginate production. Although nitrate and deletion of MucR impaired alginate promoter activity and global c-di-GMP levels, alginate yields were not directly correlated with alginate promoter activity or c-di-GMP levels, suggesting that nitrate and MucR modulate alginate production at a post-translational level through a localized pool of c-di-GMP. Nitrate increased pel promoter activity in the mucR mutant while in the same mutant the psl promoter activity was independent of nitrate. Nitrate and deletion of mucR did not impact on swarming motility but impaired attachment to solid surfaces. Nitrate and deletion of mucR promoted the formation of biofilms with increased thickness, cell density, and survival. Overall, this study provided insight into the functional role of MucR with respect to nitrate-mediated regulation of alginate biosynthesis.

Genetics and regulation of bacterial alginate production.

A vast range of extracellular polysaccharides are produced by bacteria in order to adapt to and thrive in diverse environmental niches. Many of these polymers have attracted great attention due to their implication in biofilm formation, capsule formation, virulence, or for their potential medical and industrial uses. One important exopolysaccharide, alginate, is produced by various Pseudomonas spp. and Azotobacter vinelandii. Alginate is of particular interest due to its role in the pathogenesis of Pseudomonas aeruginosa lung infection in cystic fibrosis patients. Here, we will discuss the genetic organization and distribution of the genes involved in the biosynthesis of this significant polymer. The complex regulatory networks involved in the production of bacterial alginate will be reviewed, including transcriptional, posttranscriptional and posttranslational forms of regulation.

Structural basis for alginate secretion across the bacterial outer membrane.

Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 Å crystal structure of AlgE, which reveals a monomeric 18-stranded ß-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 (ΔT8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or ΔL2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with ΔT8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

Insights into the assembly of the alginate biosynthesis machinery in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen of particular significance to cystic fibrosis patients. This bacterium produces the exopolysaccharide alginate, which is an indicator of poor prognosis for these patients. The proteins required for alginate polymerization and secretion are encoded by genes organized in a single operon; however, the existence of internal promoters has been reported. It has been proposed that these proteins form a multiprotein complex which extends from the inner to outer membrane. Here, experimental evidence supporting such a multiprotein complex was obtained via mutual stability analysis, pulldown assays, and coimmunoprecipitation. The impact of the absence of single proteins or subunits on this multiprotein complex, i.e., on the stability of potentially interacting proteins, as well as on alginate production was investigated. Deletion of algK in an alginate-overproducing strain, PDO300, interfered with the polymerization of alginate, suggesting that in the absence of AlgK, the polymerase and copolymerase subunits, Alg8 and Alg44, are destabilized. Based on mutual stability analysis, interactions between AlgE (outer membrane), AlgK (periplasm), AlgX (periplasm), Alg44 (inner membrane), Alg8 (inner membrane), and AlgG (periplasm) were proposed. Coimmunoprecipitation using a FLAG-tagged variant of AlgE further demonstrated its interaction with AlgK. Pulldown assays using histidine-tagged AlgK showed that AlgK interacts with AlgX, which in turn was also copurified with histidine-tagged Alg44. Detection of AlgG and AlgE in PAO1 supported the existence of internal promoters controlling expression of the respective genes. Overall experimental evidence was provided for the existence of a multiprotein complex required for alginate polymerization and secretion.

Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation.

Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture.

Pseudomonas aeruginosa is an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslA Δalg8 overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture of P. aeruginosa.

Three-Dimensional Micropatterning Deters Early Bacterial Adherence and Can Eliminate Colonization.

Developing strategies to prevent bacterial infections that do not rely on the use of drugs is regarded globally as an important means to stem the tide of antimicrobial resistance, as argued by the World Health Organization (WHO) (Mendelson, M.; Matsoso, M. P. The World Health Organization Global Action Plan for Antimicrobial Resistance. S. Afr. Med. J. 2015, 105 (5), 325-325. DOI: 10.7196/SAMJ.9644). Given that many antimicrobial-resistant infections are caused by the bacterial colonization of indwelling medical devices such as catheters and ventilators, the use of microengineered surfaces to prevent the initial attachment of microbes to these devices is a promising solution. In this work, it is demonstrated that 3D engineered surfaces can inhibit the initial phases of surface colonization for Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, representing the three most common catheter-associated urinary tract bacterial infections, identified by the WHO as urgent threats. A variety of designs including 11 different topographies and configurations that exhibited random distributions, sharp protrusions, and/or curvilinear shapes with dimensions ranging between 500 nm and 2 µm were tested to better understand the initial stages of surface colonization and how to optimize the design of fabricated surfaces for improved inhibition. These topographies were fabricated in two configurations to obtain either a standard 2D cross section or a 3D engineered topography using a novel UV lithography process enabling cost-efficient high-throughput manufacturing. Evaluating both the number of adhered bacteria and microcolonies formed by all three bacterial pathogens on the different surfaces provides insight into the initial colonization phase of bacterial growth on the various surfaces. The results demonstrate that both initial attachment and subsequent colonization can be significantly reduced on concrete 3D engineered patterns when compared to flat substrates and standard 2D micropatterns. Thus, this technology has great potential to reduce the colonization of bacteria on surfaces in clinical settings without the need for chemical treatments that might enhance antimicrobial resistance.

Membrane topology of outer membrane protein AlgE, which is required for alginate production in Pseudomonas aeruginosa.

The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa secretes a viscous extracellular polysaccharide, called alginate, as a virulence factor during chronic infection of patients with cystic fibrosis. In the present study, it was demonstrated that the outer membrane protein AlgE is required for the production of alginate in P. aeruginosa. An isogenic marker-free algE deletion mutant was constructed. This strain was incapable of producing alginate but did secrete alginate degradation products, indicating that polymerization occurs but that the alginate chain is subsequently degraded during transit through the periplasm. Alginate production was restored by introducing the algE gene. The membrane topology of the outer membrane protein AlgE was assessed by site-specific insertions of FLAG epitopes into predicted extracellular loop regions.

MucR, a novel membrane-associated regulator of alginate biosynthesis in Pseudomonas aeruginosa.

Alginate biosynthesis by Pseudomonas aeruginosa was shown to be regulated by the intracellular second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP), and binding of c-di-GMP to the membrane protein Alg44 was required for alginate production. In this study, PA1727, a c-di-GMP-synthesizing enzyme was functionally analyzed and identified to be involved in regulation of alginate production. Deletion of the PA1727 gene in the mucoid alginate-overproducing P. aeruginosa strain PDO300 resulted in a nonmucoid phenotype and an about 38-fold decrease in alginate production; thus, this gene is designated mucR. The mucoid alginate-overproducing phenotype was restored by introducing the mucR gene into the isogenic DeltamucR mutant. Moreover, transfer of the MucR-encoding plasmid into strain PDO300 led to an about sevenfold increase in alginate production, wrinkly colony morphology, increased pellicle formation, auto-aggregation, and the formation of highly structured biofilms as well as the inhibition of swarming motility. Outer membrane protein profile analysis showed that overproduction of MucR mediates a strong reduction in the copy number of FliC (flagellin), required for flagellum-mediated motility. Translational reporter enzyme fusions with LacZ and PhoA suggested that MucR is located in the cytoplasmic membrane with a cytosolic C terminus. Deletion of the proposed C-terminal GGDEF domain abolished MucR function. MucR was purified and identified using tryptic peptide fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overall, experimental evidence was provided suggesting that MucR specifically regulates alginate biosynthesis by activation of alginate production through generation of a localized c-di-GMP pool in the vicinity of Alg44.

Impact of alginate overproduction on attachment and biofilm architecture of a supermucoid Pseudomonas aeruginosa strain.

The supermucoid Pseudomonas aeruginosa strain PDO300Deltaalg8(pBBR1MCS-5:alg8) showed strongly impaired attachment compared with the respective mucoid or nonmucoid strains and formed a thicker biofilm with large extended mushroom-like microcolonies. Alginate lyase treatment dissolved microcolonies. The data suggested that alginate overproduction impairs attachment but plays a structural role in microcolony formation.