L'utilisation de virus de bactéries ou bactériophages pourrait être un complément efficace à l'antibiothérapie. Mon travail a porté sur la caractérisation de bactériophages dirigés contre l'espèce Pseudomonas aeruginosa, pathogène opportuniste responsable d'infections des voies respiratoires des patients atteints de mucoviscidose.J'ai tout d'abord déterminé la sensibilité des souches mucoviscidosiques au Pyophage (un cocktail de phages thérapeutiques Géorgien) et identifié six phages lytiques de quatre genres différents. Environ 15% des souches sont résistantes au Pyophage. Ensuite, en utilisant les souches cliniques multi-résistantes aux phages comme bactérie d'enrichissement, 32 phages ont été obtenus à partir des eaux usées de France et Côte d'Ivoire. Tous les phages analysés sont caudés et distribués au sein de dix genres parmi lesquels six exclusivement lytiques. J'ai identifié des souches bactériennes qui demeurent insensibles à tous les phages. J'ai montré que le système CRISPRs-Cas n'est pas associé à la résistance des souches aux phages lytiques.

La fibrose kystique est une maladie autosomale récessive multisystémique se manifestant principalement au niveau du système respiratoire et digestif. En raison d’une altération de la protéine régulatrice CFTR, l’équilibre ionique chez les cellules de l’hôte est déficient. Il en résulte, entre autres, une sécrétion et une accumulation d’un mucus pulmonaire très consistant. L’évacuation du mucus étant très fastidieuse chez les patients atteints de fibrose kystique, l’élimination des particules et bactéries des voies respiratoires est inadéquate. Cet aspect de la maladie participe grandement à sa progression, car l’établissement d’infections bactériennes chroniques contribue principalement à l’effet délétère sur la santé respiratoire des patients atteints de fibrose kystique. La colonisation du mucus pulmonaire par les bactéries pathogènes est le sujet de nombreuses recherches extensives dans le contexte de la fibrose kystique. La présence d’une diversité microbienne dans les voies respiratoires est connue depuis longtemps, mais avec les techniques récentes d’analyse moléculaire, il a été constaté que celle-ci peut présenter une très grande richesse qui était préalablement non détectée par les méthodes d’isolement classiques. Les interactions et comportements adoptés par les populations polymicrobiennes du microbiome FK dans la progression de la maladie sont maintenant de grand intérêt. La virulence des principaux pathogènes en FK a préalablement et exhaustivement été décrite de façon spécifique. Parmi les plus notoires, comme Staphylococcus aureus, Pseudomonas aeruginosa et Burkholderia cepacia, certains utilisent des systèmes de communication cellulaire élaborés («quorum sensing») dans le but de réguler leur virulence. De nouvelles voies de recherche sont construites sur la suggestion qu’une population polymicrobienne peut être perçue comme un seul agent infectieux. Notre étude a été conduite afin de documenter les possibles réponses interespèces entre S. aureus et P. aeruginosa suite à l’échange de molécules sécrétées dans le cadre du «quorum sensing». Pour ce faire, nous avons caractérisé un éventail d’interactions existantes entre des isolats cliniques des deux pathogènes d’intérêt. Nous avons également développé un modèle nous permettant de mieux comprendre l’impact de la coexistence sur leur pathogenèse. Inévitablement, l’étude vise à définir l’instance de la présence simultanée des deux bactéries dans l’établissement d’infections chroniques chez les patients atteints de fibrose kystique ainsi que sur leur santé respiratoire. Cette étude tente ultimement de contribuer à l’effort visant à comprendre l’importance des interactions polymicrobiennes dans un contexte infectieux dans le but de trouver de nouveaux indices quant à l’élaboration de stratégies thérapeutiques.

Light is the primary source of energy in most of earth's ecosystems . In freshwater ecosystems the major interacting factors that determine the abundance and species composition of planktonic phototrophs, the primary utilizers of light, are nutrients, temperature and light. With increasing eutrophication and declining geographical latitude, nutrient availability becomes in excess of the organisms' requirements, water temperature is more favourable for growth, and community structure depends to a greater extent on light availability. This study focuses on the population dynamics of the bloom-forming cyanobacterium Microcystis aeruginosa Kutz. emend. Elenkin in subtropical Hartbeespoort Dam, South Africa. The objectives of the study were: to investigate the annual cycle, and the factors leading to the dominance and success of the cyanobacterium in this hypertrophic, warm monomictic lake, where light availability is the major factor limiting phytoplankton growth rates; to study the surface blooms and ultimately hyperscums that this species forms; and to assess the ecological significance of hyperscums. A 4. 5-years field study of phytoplankton abundance and species composition in relation to changes in the physical environment, was undertaken. The hypothesis was that M. aeruginosa dominated the phytoplankton population (> 80 % by volume) up to 10 months of every year because it maintained itself within shallow diurnal mixed layers and was thus ensured access to light. It was shown that wind speeds over Hartbeespoort Dam were strong enough to mix the epilimnion (7 - 18 m depth) through Langmuir circulations only 12 % of the time. At other times solar heating led to the formation of shallow ( < 2 m) diurnal mixed layers (Z[1]) that were usually shallower than the euphotic zone (Zeu; x = 3.5 m), while the seasonal mixed layer (zrn) was always deeper than Zeu. From the correspondence between vertical gradients of chlorophyll a concentrations and density gradients, when M. aeruginosa was dominant, it was implied that this species maintained the bulk of its population within Z[1]. Under the same mixing conditions non-buoyant species sank into dark layers. These data point out the importance of distinguishing between Zrn and Z[1], and show the profound effect that the daily pattern of Z[1], as opposed to the seasonal pattern of Zrn can have on phytoplankton species composition Adaptation to strong light intensities at the surface was implicated from low cellular chlorophyll a content (0.132 μg per 10[6] cells) and high I[k ](up to 1230 μE m⁻² S¯¹). Ensured access to light, the postmaximum summer populations persisted throughout autumn and winter, despite suboptimal winter temperatures, by sustaining low losses. Sedimentation caused a sharp decline of the population at the end of winter each year and a short ( 2-3 months) successional episode follCMed, rut by late spring M. aeruginosa. was again dominant. The mixing regime in Hartbeespoort Dam and the buoyancy mechanism of M. aeruginosa led to frequent formation of surface bloons and ultimately hyperscums. Hyperscums were defined as thick (decimeters), crusted, buoyant cyanobacterial mats, in which the organisms are so densely packed that free water is not evident. In Hartbeespoort Dam in winter M. aeruginosa formed hyperscums that measured up to 0.75 m in thickness, covered more than a hectare, contained up to 2 tonnes of chlorophyll a, and persisted for 2 - 3 monnths. Hyperscum formation was shown to depend upon the coincidence of the following preconditions: a large, pre-existing standing crop of positively buoyant cyanobacteria; turbulent mixing that is too weak to overcome the tendency of the cells to float, over prolonged periods (weeks); lake morphometry with wind-protected sites on lee shores; and high incident solar radiation. The infrequent occurrence of hyperscums can be attributed to the rare co-occurrence of these conditions. Colonies in the hyperscum were arranged in a steep vertical gradient, where colony compaction increased exponentially with decreasing distance form the surface. This structure was caused by evaporative dehydration at the surface, and by the buoyancy regulation mechanism of M. aeruginosa., which results with cells being unable to lose boyancy when deprived access to light from above. The mean chlorophyll a concentration and water content were 3.0 g 1¯¹ and 14 % at the surface crust, 1.0 g 1¯¹ and 77 % at a few mm depth, and 0.3 g 1¯¹ and 94 % at 10 cm depth, where M. aeruginosa cell concentration exceeded 109 ml¯¹. A consequence of the high cell and pigment concentrations was that light penetrated only 3 mm or less, below which anaerobic, highly reduced conditions developed. Nutrient concentrations in hyperscum interstitial water, collected by dialysis, increased dramatically with time (phosphate: 30-fold over 3 months; ammonia: 260-fold). Volatile fatty acids, intermediate metabolites in anaerobic decomposition processes, were present. Gas bubbles trapped within the hyperscum contained methane (28 %) , and CO[2] (19 %), the major end products of anaerobic decomposition, and no oxygen. The structure and function of M. aeruginosa in hyperscum was examined in relation to the vertical position of colonies and the duration of exposure to hyperscum condition. Colonies and cells collected from 10 em depth in the hyperscum were similar in their morphology (light and fluorescent microscopy) and ultrastructure (transmission and scanning electron microscopy) to those of colonies from surface blooms in the main basin of the lake. With declining depth over the uppermost 10 mm of the hyperscum cells appeared increasingly dehydrated, decomposed and' colonized by bacteria. studies employing microelectrode techniques demonstrated that photosynthetic activity of colonies at the surface of a newly accumulated hyperscum rapidly photoinhibited, substrate-limited, and then ceased within hours of exposure to light intensities > 625 μE m⁻² S¯¹. Photooxidative death followed. The dead cells dehydrated to form the dry crust, from underneath. and space was thus created for colonies rising Cells collected from 10 cm depth retained their photosynthetic capacity ([14]C-uptake experiments) throughout the hyperscum season, although a considerable decline in this capacity was noted over the last (third) month. Altogether the data indicated that spatial separation developed within the hyperscum, between a zone at the surface of lethal physical conditions, a zone beneath the surface of stressful and probably lethal chemical conditions, and a deeper zone of more moderate conditions, which nevertheless, deteriorated after 2 - 3 months. A conceptual model describing the fate of a colony entering a hyperscum was then proposed. According to this model, a colony that arrives below a hyperscum and is not carried away by currents, becomes over-buoyant in the dark and floats into the bottom of the hyperscum. With time it migrates towards, due to its own positive buoyancy, the buoyancy of colonies rising from underneath, and the collapse of cells at the top. It survives in the dark, anaerobic environment by maintaining low levels of basal metabolism while utilizing stored reserves. Depending on weather conditions, the colony mayor may not remain within the hyperscum long enough to reach the zone of decomposition near the surface, where it would die. With the aging of the hyperscum and the accumulation of trapped decomposition products, the zone of decomposition expands. Thus, a hyperscum is essentially a site of a continuous cycle of death and dehydration at the surface and upward migration of colonies from below to replace those that died, although not all colonies entering the hyperscum necessarily reach the lethal zone. The formation of hyperscums was shown to have no major influence on the annual cycle of M. aeruginosa in Hartbeespoort Dam. The seasonality of increase and decline of the planktonic population was similar from year to year, irrespective of whether or not hyperscums formed. The phenomenon of hyperscums demonnstrated that, as Reynolds and Walsby (1975) claimed, thick cyanobacterial water-blooms do form incidentally and have no vital function in the biology of the organism. water temperature did have a major effect on the annual cycle of this species in Hartbeespoort Dam. In temperate lakes the low water temperatures in autumn and winter (<10° C) cause M. aeruginosa to lose its ability to regain buoyancy in the dark, and consequently it sinks to bottom sediments. The higher ( > l2°C) minimum winter temperature in Hartbeespoort Dam leads to the maintenance of a relatively large residual planktonic population throughout the winter. Unlike the case in temperate lakes, the long-term survival of M. aeruginosa in warm-water lakes probably does not depend on winter benthic stocks for the provision of an inoculum for the following growth season. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1987.

The widespread use of β-lactams has undermined their effectiveness as chemotherapeutic agents by fueling the evolution and dissemination of multiple resistance mechanisms, including: (1) production of hydrolytic β-lactamase enzymes that inactivate β-­lactams, (2) expression of PBPs with low-affinity for β-­lactams and (3) overexpression of multidrug efflux pumps which actively expunge β-­lactams and other toxic substances. The overall goal of this thesis is the structural characterization of bacterial proteins involved in regulating β-lactam resistance. The notorious resistance of Staphylococcus aureus primarily stems from the production of β-lactamases and PBP2a, a low-affinity PBP which confers broad-spectrum β-­lactam resistance in methicillin-resistant S. aureus (MRSA) strains. Expression of these resistance determinants is controlled by a β-­lactam-inducible transmembrane receptor (BlaR1/MecR1) and repressor (BlaI/MecI). This dissertation presents the crystal structure of the BlaR1 sensor domain (BlaRs) from S. aureus, determined in its apo form and acylated with penicillin G. These structures reveal that acylation by β-lactams is not accompanied by a BlaRs conformational change. It is also shown that mutation of the BlaR1 L2 loop prevents induction of β-­lactamase expression in vivo, supporting that the L2 loop plays an important role in signal transduction. The intrinsic resistance of Pseudomonas aeruginosa to a variety of antibiotics (including β-lactams) is exacerbated in mutant strains that overexpress multidrug efflux pumps such as MexAB-OprM. Production of MexAB-OprM is controlled by the MarR family repressor, MexR, and several hyper-resistant strains of P. aeruginosa appear to involve mutations in either MexR or additional regulatory factors upstream of MexR. The allosteric effectors of MarR proteins are typically small lipophenolic compounds. This dissertation confirms that MexR is uniquely modulated by the 53 residue protein, ArmR. Electromobility gel shift assays and isothermal titration calorimetry demonstrate that a direct MexR-ArmR interaction is responsible for neutralizing the affinity of MexR for its DNA operator. The allosteric conformational change induced by ArmR-binding was assessed by determining the crystal structure of MexR double mutant Q106L/A110L (MexRLL) in complex with ArmR residues 29-53 (ArmRC). This structure shows that ArmR induces a dramatic conformational change which repositions the MexR DNA-binding lobes into an orientation that is incompatible with binding DNA.

Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.

In nature bacteria predominantly live on surfaces, in matrix-encased communities called biofilms. Biofilm formation displays dynamic developmental patterns resembling those of multicellular organisms. Using cooperative traits such as cell-cell signaling, bacteria in biofilms form complex architectures, known as microcolonies, in which cells become highly differentiated from their planktonic counterparts. Microcolonies are generally highly tolerant to bactericides, rendering biofilms extremely difficult to eradicate. The aim of this study was to investigate the last, and least understood stage of biofilm development, which involves the coordinated dispersal of single cells that revert to a free-swimming planktonic phenotype and escape from the biofilm. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. In the model organism Pseudomonas aeruginosa, reproducible patterns of cell death and dispersal can occur within biofilm structures, leaving behind empty or hollow microcolonies. These events were previously linked with the appearance of oxidative and/or nitrosative stress in mature microcolonies. Here, the involvement of reactive oxygen and nitrogen intermediates in biofilm development and dispersal processes was investigated in both mono- and mixed-species biofilms. By using specific fluorescent dyes and P. aeruginosa mutant strains, nitric oxide (NO), a by-product of anaerobic respiration and an important messenger molecule in biological systems, was found to play a major role in P. aeruginosa biofilm dispersal. Further, the results demonstrated that exposure to physiological, non-toxic concentrations of NO (in the low nanomolar range) causes biofilm dispersal in P. aeruginosa and restores its vulnerability to conventional antimicrobials. By using microarray techniques, NO was shown to induce global changes in genetic expression, including enhanced metabolic activity and motility and decreased adhesion and virulence in P. aeruginosa biofilms. The regulatory pathway implicated c-di-GMP, a newly discovered messenger molecule involved in the transition from sessility to motility in many bacterial species. NO-mediated dispersal was also observed in other single- and multi-species biofilms of clinically and industrially relevant organisms. Hence, the combined exposure to NO and bactericides was identified as a potential novel strategy for the removal of microbial communities, providing a low cost and environmentally safe solution to biofilm control.

Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.

Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.

Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.

Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.