Research performed at the Division of Environmental Microbiology has over the last years resulted in the isolation of possible bacterial key-organisms with efficient nutrient removal properties (Comamonas denitrificans, Brachymonas denitrificans, Aeromonas hydrophila). Effective use of these organisms for enhanced nutrient removal in wastewater treatment applications requires the strains to be retained, to proliferate and to maintain biological activity within theprocess. This can be achieved by immobilization of the organisms using an appropriate system.Two putative immobilization systems, agar entrapment and biofilm formation, wereassessed. Surface attached biofilm growth provided better results with respect to cell retention,proliferation and microbial activity than immobilization in agar beads. Thus, biofilm physiology was further characterized using simplified systems of single, dual or multi strain bacterial consortia containing the key-organisms as well as other wastewater treatment isolates. Mechanisms for initial adherence, biofilm formation over time, dynamics and characteristics of extracellular polymeric substances (EPS) and exopolysaccharides, nutrient removal activity as well as the effect of bacterial interactions were investigated. The results showed that all theassessed bacterial strains could form single strain biofilm providing that a suitable nutrientsupply was given. Production of EPS was found to be critical for biofilm development and both EPS and polysaccharide residue composition varied with bacterial strain, culture conditions and biofilm age. Denitrification and phosphorus removal activity of the keyorganisms was maintained in biofilm growth. Co-culturing of two or more strains resulted in both synergistic and antagonistic effects on biofilm formation as well as the microbial activitywithin the biofilm. Bacterial interactions also induced the synthesis of new polysaccharideswhich were not produced in pure strain biofilms.The complexity of single and mixed strain biofilm development and the implications of interactions on biofilm performance were underlined in this study. The data presented can be useful for modeling of biofilm systems, serve as a tool for selection of bacterial strain combinations to use for bioaugmentation/bioremediation or provide a base for further experiment design. / QC 20100622

Made available in DSpace on 2014-06-11T19:23:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-06-02Bitstream added on 2014-06-13T20:30:11Z : No. of bitstreams: 1 bongiovani_ram_me_sjrp.pdf: 2319132 bytes, checksum: e7d65e5bb475e25f1407badd2bab2ddd (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os exopolissacarídeos (EPS) são polímeros produzidos por uma grande variedade de microrganismos e podem possuir propriedades físicas, estruturais e químicas diferentes. Considerando o interesse crescente das indústrias na busca de polissacarídeos que apresentem comportamento reológico compatível com sua aplicação, tanto nos setores de alimentos como farmacêutico, cosmético e têxtil, pesquisas vêm sendo realizadas com a esperança da descoberta de novas moléculas promissoras para tais aplicações. O objetivo deste trabalho foi determinar as características reológicas do exopolissacarídeo (botriosferana) produzido pelo Botryosphaeria rhodina MAMB-05 em três fontes de carbono: glucose (EPSGLU), sacarose (EPSSAC) e frutose (EPSFRU). A análise da composição monossacarídica, após hidrólise ácida, mostrou que todos os polímeros são constituídos exclusivamente por glucose e a cromatografia de filtração em gel indicou baixo grau de polidispersividade. Experimentos com o corante Congo Red evidenciaram a presença de tripla hélice nos três polímeros produzidos pelo fungo B. rhodina MAMB-05. Os três polissacarídeos comportaram-se como fluidos não-Newtoniano com características de soluções pseudoplásticas e tixotrópicas e com diminuição da viscosidade quando em presença de NaCl. As soluções aquosas de EPSGLU, EPSSAC e EPSFRU apresentaram comportamento viscoelástico, com características de gel forte em 5 e 25°C, não sofrendo mudanças no comportamento, mesmo em presença de NaCl, a 25°C. As soluções polissacarídeos mantiveram as características de géis fortes, com propriedades termoestáveis e termorresistentes, entre 5 a 95°C de temperatura. Esses resultados permitiram concluir que os EPS produzidos pelo fungo B. rhodina MAMB-05 possuem propriedades químicas e físicas, passíveis de utilização nas indústrias de alimentos, farmacêutica, cosmética e biomédica. / Exopolysaccharides (EPS) are polymers produced by a great variety of microorganisms and can possess different physical and chemical properties, and structural features. Considering the growing interest in commercial applications of polysaccharides that show rheological compatible behavior within the application, as in the food, pharmaceutical, cosmetic and textile sectors, research have been developed in attempts to discover new promising biomolecules for such applications. The objective of the work presented was to determine the rheological characteristics of the exopolysaccharide botryosphaeran produced by Botryosphaeria rhodina MAMB-05 on three different sole carbon sources: glucose (EPSGLU), sucrose (EPSSUC) and fructose (EPSFRU). The analysis of monosaccharide composition, after acid hydrolysis, showed glucose as the only component and the gel permeation chromatography indicated that the EPSs have an excellent purity grade. All three biopolymers produced by B. rhodina MAMB-05 were found to exist in the triple helix conformational state. The three exopolysaccharides showed non-Newtonian behavior, with pseudoplastic and thixotropic characteristics, and showed a slight decrease in viscosity in presence of NaCl. The three biopolymer gels showed viscoelastic behavior, with characteristics of strong gelling at 5 ºC and 25 ºC, without suffering behavioral changes, even in the presence of NaCl at 25 ºC. The polysaccharide solutions kept the strong gel characteristics possessing thermo-stable and thermo-resistant properties when evaluating the influence of temperature (5 – 95 ºC) over the viscoelastic behavior of solutions. The results concluded that the EPS’s produced by fungus B. rhodina MAMB-05 have physical and chemical properties amenable for use in the food, pharmaceutical, cosmetic and biomedical industries.

Made available in DSpace on 2014-06-11T19:23:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-21Bitstream added on 2014-06-13T18:50:36Z : No. of bitstreams: 1 arandaselverio_g_me_sjrp.pdf: 1103053 bytes, checksum: 82b446306de04f7e3cee32bff6293b57 (MD5) / Exopolissacarídeos (EPS) são polímeros produzidos por uma grande variedade de microrganismos e possuem diferentes propriedades estruturais, físicas e químicas. A investigação das propriedades reológicas de suas soluções é importante devido ao crescente interesse na aplicação comercial de polissacarídeos, principalmente na indústria de alimentos. Assim, o objetivo deste trabalho foi investigar as características reológicas dos exopolissacarídeos R1, R2, R3 e R4 produzidos por diferentes linhagens de Rhizobium e Mesorhizobium. Análises quantitativas mostraram que o teor de ácido urônico encontrado em R3 (8,4 %) foi maior que em R1 (2,4 %), R2 (1,7 %) e R4 (0,8 %). A cromatografia de filtração em gel mostrou que R2 e R3 são mais homogêneos e menos polidispersos. Hidrólise ácida total e análise por HPAEC/PAD mostrou glucose como constituinte básico majoritário dos EPS, além de galactose e manose. Todos os polissacarídeos apresentaram comportamento não-Newtoniano, com características de soluções pseudoplásticas nas concentrações de 2, 5 e 10 g/L. O modelo reológico de Ostwald-de-Waele (Lei da Potência) foi utilizado para representar os dados experimentais de tensão de cisalhamento versus taxa de deformação. Os EPS R1, R2 e R4 demonstraram pequeno aumento na viscosidade em presença de NaCl, e apresentaram comportamento viscoelástico de gel, sendo R1 o que apresenta características de gel mais forte. O EPS R3 manteve-se como o menos viscoso, tanto em meio aquoso quanto em solução salina, provavelmente devido ao maior percentual de ácidos urônicos em sua estrutura. Além disso, R3 exibiu comportamento de solução diluída a baixas concentrações, e viscoelástico de gel fraco em concentrações mais elevadas. A análise da influência da temperatura sobre o comportamento viscoelástico das soluções polissacarídicas mostrou... / Exopolysaccharides (EPS) are polymers produced by a great variety of microorganisms and possess different structural, physical and chemical properties. Investigation of rheological properties of these solutions is important due to an increasing interest in polysaccharides commercial applications, mainly in food sectors. The objective of this work was investigate rheological characteristics of exopolysaccharides R1, R2, R3 and R4 produced by different Rhizobium and Mesorhizobium strains. Quantitative analysis showed that uronic acid component found in R3 (8,4 %) was higher than R1 (2,4 %), R2 (1,7 %) and R4 (0,8 %). Gel filtration chromatography indicated that EPS R2 and R3 are more homogeneous and less polidisperse. Acid hydrolysis and HPAEC/PAD analysis revealed that glucose was the main monosaccharide, beyond galactose and mannose. All exopolysaccharides had non-Newtonian behavior, with pseudoplastic characteristics at concentrations of 2, 5 and 10 g/L. The Ostwald-de-Waele (Power Law) was the rheological model used to represent the experimental data of the shear stress versus shear rate. EPS R1, R2 and R4 demonstrated a slight increase in viscosity in presence of NaCl, and viscoelastic behavior, R1 had strong gelling characteristics. The EPS R3 was less viscous, in water solutions and presence of salt, probably due to a high percentual of uronic acids on its structure. Moreover, R3 exhibited diluted solution behavior at low concentration, and viscoelastic weak gelling at high concentrations. Analysis of temperature influence over polysaccharide solutions viscoelastic behavior showed that EPS R1, R2 e R4 had strong gelling characteristics, at concentrations of 5 g/L.

Given the concern for environmental preservation, by cause of human activities and its profound impacts, research works have led to a shift from conventional said agriculture using agrochemicals and fertilizers to agriculture that pursues ways less harmful to the environment management. The objective was to prospect and also isolate the bacteria with proper characteristics for growth benefits from the rhizosphere sugarcane soil and its subsequent use in production of microbial inoculants. As to reach this purpose, 24 soil samples from sugarcane mills in the state of Alagoas were collected and isolated for qualification and quantification of soluble phosphorus, production of exopolysaccharides (EPS), verification of biofilm formation, production of indole acetic acid (IAA), cytokinins and gibberellins production, rooting induction and sugarcane budding. The used methodology for phosphate solubilization was performed according to the recommendations of the authors referenced in the bibliography. According to the results obtained for phosphate solubilizing growth medium (Nbrip) showed that 17% had high capacity and inorganic phosphate solubilizing growth medium capacity Reached 67%. The others results stayed between low or absence of solubilization. Of the isolates tested, 75 % Were able to grow in medium with low water activity Aw 0919 at 30 °C and the appearance of colonies was Observed five days after inoculation. Results for EPS production at room temperature showed that 4% of the isolates surveyed achieved a hight production, followed by 42% which has achieved average production and the remaining 54% of low production. Analysing biofilm production capacity in TSB medium in absorbance of 540 nm were observed that 4% of the isolates had a higher frequency for biofilm formation, 83% achieved average production and 13% of isolates with low frequency output. Larger quantity for production of IAA in the presence precursor L-tryptophan was observed at the end of 72 hours despite significant differences among the isolates, except for isolate 7 which was considered high 51.62194 μg.mL-1 and isolated 6 to lower production 14.98113 μg.mL-1, all other isolates were classified as high producers. Among the isolates tested 25% were able to synthesize cytokines and 83.33% synthesized gibberellin compared to the control. All isolates, compared in control, increased in size which suggested the action of one or both hormones in question at the same time. The sprouting of inoculated buds compared in control to those without inoculation, the RB72454 variety of sugarcane, in end of the evaluation, in the presence of isolated samples 4, 16, 21 and 24 demonstrated that only 25% of the buds had sprouted and grew roots, not differing significantly from the control. As the result inoculation significantly increased a sprouting rate up to 83.33% of the isolates, and the best results obtained were in samples 7, 12, 19 and 20 (94.5, 100, 95 and 94.5% respectively) in both the number of sprouted buds as for speed emission of shoots and roots. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Tendo em vista a preocupação com a preservação ambiental, devido à constatação de que as atividades antrópicas podem causar profundos impactos, os trabalhos de pesquisa têm levado a uma mudança da agricultura dita convencional, que utiliza produtos agroquímicos e fertilizantes, para uma agricultura que busca formas de manejo menos nocivos ao ambiente. Objetivou-se a prospecção e o isolamento de bactérias com a características para promoção de crescimento a partir do solo rizosfera de cana – de – açúcar para serem utilizados na produção de inoculantes microbianos. Foram coletadas 24 amostras de solos provenientes de usinas sucroalcooleiras do Estado de Alagoas, de onde foram retirados os isolados para qualificação e quantificação de fósforo solúvel, produção de Exopolissacarídeos (EPS) e a verificação da formação de biofilme, produção de ácido indolacético (AIA), Produção de citocininas e giberelinas, Indução de enraizamento e brotação de gemas de cana-de-açúcar. A metodologia utilizada para solubilização de fosfato foi realizada de acordo com as recomendações dos autores. Os resultados obtidos para solubilização de fosfato em meio Nbrip demonstraram que 17% apresentaram alta capacidade de solubilização de fosfato inorgânico e 67% alcançaram média capacidade. Os demais estão entre baixa e ausência de solubilização. Dos isolados testados, 75% foram capazes de crescer em meio com atividade de água reduzida 0,919 Aw, a 30°C, o aparecimento de colônias foi observado cinco dias após a inoculação.Os resultados para produção de EPS em temperatura ambiente demonstraram que 4% dos isolados pesquisados atingiram uma ótima produção, seguido de 42% que obtiveram média produção e os demais, 54%. Quanto à capacidade de produção de biofilme em meio TSB em absorbância de 540 nm, 4% dos isolados obtiveram maior freqüência para formação de biofilme, 83% atingiram média produção e 13% dos isolados com baixa frequência de produção. As maiores quantidades para produção de AIA, na presença do precursor L-triptofano foi evidenciada ao final de 72 horas, apesar das diferenças significativas entre os isolados estudados, exceto o isolado sete que foi considerado elevado 51,62194 μg.mL-1 e o isolado seis com a produção mais baixa 14,98113 μg.mL-1, os demais isolados foram classificados altos produtores. Dentre os isolados testados 25% foram capazes de sintetizar citocininas e 83,33% sintetizaram giberelinas comparados ao controle. Todos os isolados, comparados a testemunha, aumentaram em tamanho, sugerindo a ação de um ou dos dois hormônios em questão. Na brotação das gemas inoculadas comparados ao controle sem inoculação da variedade RB72454 de cana-de-açúcar, ao final da avaliação, na presença dos isolados 4, 16, 21 e 24, apenas 25% das gemas haviam brotado e emitido raízes, não diferindo significativamente do controle. A inoculação aumentou significativamente a velocidade de brotação para 83,33% dos isolados, sendo os melhores resultados obtidos para 7, 12, 19 e 20 (94.5, 100, 95 e 94.5% respectivamente) tanto para o número de gemas brotadas como para velocidade de emissão de brotos e raizes.

Philosophiae Doctor - PhD / The World Health Organisation predicts that by the year 2050, 10 million people could die annually as a result of infections caused by multidrug resistant bacteria. Individuals with compromised immune systems, caused by underlying disease such as HIV, MTB and COVID-19, are at a greater risk. Antibacterial resistance is a global concern that demands the discovery of novel drugs. Natural products, used since ancient times to treat diseases, are the most successful source of new drug candidates with bioactivities including antibiotic, antifungal, anticancer, antiviral, immunosuppressive, anti-inflammatory and biofilm inhibition. Marine bioprospecting has contributed significantly to the discovery of novel bioactive NPs with unique structures and biological activities, superior to that of compounds from terrestrial origin. Marine invertebrate symbionts are particularly promising sources of marine NPs as the competition between microorganisms associated with invertebrates for space and nutrients is the driving force behind the production of antibiotics, which also constitute pharmaceutically relevant natural products.

Philosophiae Doctor - PhD / bioassay guided isolation approach was then used to isolate the high molecular weight antibacterial compound (50kDa-100kDa) from T. actiniarum fermentations. With common protein isolation, purification and detection methods failing to provide insight into the nature of the antibacterial compound, we hypothesized that the active agent is not proteinaceous in nature and may be a high molecular weight exopolysaccharide. Extraction and antibacterial screening of the exopolysaccharide fraction from T. actiniarum showed antibacterial activity as well as lytic activity when subjected to a zymography assay using Pseudomonas putida whole cells as a substrate. Additionally, the biosynthetic pathways for the production of poly-β-1, 6-N-acetyl-glucosamine (PNAG), an exopolysaccharide involved in biofilm formation and chondroitin sulfate, a known and industrially important glycosaminoglycan with antibacterial and anti-inflammatory activity was identified and the mechanism may be novel. Genome mining identified a variety of novel secondary metabolite gene clusters which could potentially encode other novel bioactivities. Therefore a bioassay guided isolation, focused on the small (<3kDa) molecules, was pursued. Secondary metabolites were extracted, fractionated and screened for biofilm inhibition, antibacterial and anticancer activity and activity was observed in all assays. Active fractions were dereplicated by UHPLC-QToF-MS and compounds of interest were isolated using mass guided preparative HPLC. The purity of the isolated compounds was assessed using UHPLC-QToF-MS and NMR and the structure of the target compounds elucidated. Structures that could be determined were the bile acids cholic acid and 3-oxo cholic acid and although not responsible for the observed activities, this is the first report of bile acid production for this genus. This is the first study investigating the bioactive potential of the strain and the first demonstrating that T. actiniarum is a promising source of potentially novel pharmaceutically relevant natural products depicted through both culture-dependent and culture-independent approaches.

No description available.

Les EPS bactériens peuvent servir d' agents texturants dans l' industrie alimentaire et certaines propriétés santé leur sont proposées. Les séquences des gènes des opérons responsables de la biosynthèse des EPS de Lactobacillus rhamnosus RW-9595M, ATCC 9595, R et RW-6541M montrent très peu de différences, bien que la quantité d'EPS produite par ces souches soit différente (543, 108, 212 et 179 mg d'EPS/l, respectivement). Le but de ce projet est d'établir des liens entre la production d'EPS de ces souches et l'expression de différents gènes, lors de la croissance sur glucose. La technique de rétrotranscriptase-PCR quantitative (qR T -PCR) a été utilisée pour mesurer l'expression de gènes connus et impliqués dans la biosynthèse des EPS. Les 12 gènes ciblés sont associés à la synthèse des sucres précurseurs, à la polymérisation des unités répétitives ou sont des régulateurs de la transcription. La souche RW-9595M montre une surexpression de la des 12 gènes cibles en phase exponentielle de croissance, par rapport à la phase stationnaire, comparativement à la surexpression de seulement trois ou quatre gènes, dans les mêmes conditions, pour les souches ATCC 9595, R et RW-6541M. Ces résultats suggèrent que la quantité totale d'EPS produite par la souche RW-9595M (la plus productrice) est le reflet d'une plus grande activité trancriptionnelle de cette souche lors de la croissance logarithmique, ayant des effets potentiels sur l'activité métabolique durant cette phase. Les souches R et RW-6541M (productions d'EPS moyennes) ne montrent aucune différence d'expression significative des gènes ciblés entre elles. Aussi, dans les conditions utilisées, la souche ATCC 9595 semble moins efficace dans la production de biomasse, ce qui est réflété par sa faible production d'EPS. Afin de découvrir si d'autres différences transcriptionnelles entre les souches sont à l'origine de leurs productions d'EPS différentes, une méthode d'affichage différentiel (cDNA-AFLP), visant la comparaison de l'ensemble des transcriptomes, a également été explorée. Sur trois gènes ainsi ciblés, un seul a montré une différence réelle d'expression entre les souches RW-6541M et RW-9595M. En somme, il apparaît que les différences de production d'EPS entre les souches de L. rhamnosus seraient plutôt causées par des différences globales de transcription, et ainsi peut-être par d'autres différences de métabolisme, que par des différences d'expression des gènes des opérons EPS spécifiquement.

Un procédé de fermentation basé sur une coculture des souches probiotiques Lactobacillus rhamnosus R et Lactobacillus rhamnosus RW9595M a été utilisé pour coproduire des exopolysaccharides (EPS) de poids moléculaires modulés. Dans cette étude, les deux souches ont été cultivées séparément et en coculture. Les souches ont été cultivées durant 72 h, à 37 °C, sous contrôle de pH à 6.0, et avec une agitation de 100 r.p.m. Deux milieux de culture ont été testés, le milieu minimum basai supplémenté (BMM-S) avec 1.1% (v/v) de tryptone et le perméat de lactosérum supplémenté (PLS) avec 1.1% (v/v) de tryptone, 0.5 mg /L de MgS04, 0.05 mg /L de MnS04 et 0.1% de Tween 80. La croissance cellulaire des souches et le poids moléculaire ont été mesurés au cours de la fermentation par dénombrement sur gélose et par HPLC-SEC, respectivement. Lorsque les souches ont été cocultivées dans le BMM-S en proportion de 1 :1, 991 ±30 mg/L d'EPS avec des poids moléculaires de 1.0 ± 0.21 x 106, 1.6 ± 0.16 x 106 Da, et 1.9 ± 0.18 x 105 Da, ont été obtenus. Dans le milieu PLS, les souches ont été cocultivées en proportion de 9:1 (R/RW9595M), 657 ± 90 mg/L d'EPS avec des poids moléculaires de 5.59 ± 0.82 x 104Da, ont été obtenus. La croissance cellulaire individuelle a été mesurée par qPCR-TaqMan pour les essais dans le milieu PLS. La concentration cellulaire maximale pour chaque souche était de 2.05 x 109 UFC/mL pour Lb. rhamnosus R, et de 3.67 x 108 UFC/mL pour Lb. rhamnosus RW9595M. Ce procédé est simple et économique pour la culture des probiotiques à haute valeur ajoutée en fabrications alimentaires.

Les souches Lactobacillus rhamnosus RW-9595M et ATCC 9595 possèdent 17 cadres de lecture ouverts (ORF) identiques à 99 % identifiés comme gènes de biosynthèse putatifs des EPS. Par contre, les quantités produites diffèrent avec 543 mg l-1 pour RW-9595M et 108 mg l-1 pour ATCC 9595. La phosphorylation réversible a été proposée comme mécanisme pour réguler la polymérisation des EPS chez les bactéries lactiques. Parmi les protéines participant à la polymérisation, on propose la tyrosine kinase, la tyrosine phosphatase et la co-polymérase. Cette étude cherche à démontrer l’impact de la phosphorylation de ces protéines, surtout la co-polymérase (Wzd), sur le complexe de ces protéines chez L. rhamnosus. Afin de tester l’effet de la phosphorylation sur leurs interactions, Wzd (co-polymérase) et Wze (kinase) ont été exprimées chez L. lactis ou chez E. coli. Chez L. lactis ssp. cremoris, certaines combinaisons de gènes peuvent être associées à la production d'EPS in vivo. Chez E. coli, l'expression des gènes peut être contrôlée, permettant d'exprimer et de purifier des protéines dans le but d'effectuer l'étude de leurs interactions in vitro. Les clones de la souche L. lactis ssp. cremoris MG1363 (naturellement non productrice d’EPS) transformée avec l’opéron EPS de RW-9595M ou ATCC 9595 produisent respectivement 326 et 302 mg l-1, mais avec des rendements inférieurs à la production chez la souche d'origine RW-9595M. Lors des essais in vitro, Wzd et Wze ne s’autophosphorylent pas lorsqu'elles sont seules, mais ensemble elles forment un complexe. Le complexe entre ces deux protéines non phosphorylées permet la phosphorylation de Wzd par Wze en présence d’ATP. Wze est libérée par la déstabilisation de cette interaction suivant la phosphorylation. L’existence de Wzd phosphorylée et de l’ATP conduit à l’auto-phosphorylation de Wze par une interaction transitoire. De plus, l’activité de Wzd est modulée par la phosphorylation des tyrosines en permettant l’autophosphorylation de Wze. Or, la phosphorylation de Wzd peut être une étape de phosphorylation réversible pour la polymérisation des EPS. Cette étude contribue à la compréhension de la relation entre les caractéristiques et les fonctions biologiques des polymères d'EPS. / Lactobacillus rhamnosus RW-9595M and ATCC 9595 have 99 % identical 17 open reading frames (ORFs) identified as putative genes coding for EPS biosynthesis and both strains produce very different EPS amounts: 543 mg l-1 (RW-9595M) and 108 mg l-1 (ATCC 9595). Reversible phosphorylation has been proposed as a mechanism to regulate the polymerization of EPS in lactic acid bacteria and three proteins, tyrosine kinase, tyrosine phosphatase and co-polymerase are proposed to be responsible for this regulation. The aim of this project was to demonstrate the impact of the phosphorylation of these proteins, especially the co-polymerase (Wzd), on the protein complex for EPS polymerization in L. rhamnosus. To test the effect of phosphorylation on their interactions, Wzd and Wze were expressed in L. lactis subsp. cremoris and E. coli. In L. lactis subsp. cremoris, the presence of certain combinations of genes can be associated with EPS production yields. In E. coli, gene expression can be controled and the proteins purified in order to study their interactions and phosphorylation state in vitro. Lactococcus lactis subsp. cremoris MG1363, a non EPS-producing strain, was transformed with two recombinant plasmids coding for the genes required for EPS synthesis. These transformants with operons from either RW-9595M or ATCC 9595 produce 326 and 302 mg l-1 respectively, with lower yields than RW-9595M. The proteins encoded by wzd and wze are respectively the co-polymerase and the kinase which theoretically participate in determining the chain length of the EPS. These two proteins do not autophosphorylate when they are alone, but together form a complex. The non-phosphorylated complex of two proteins allows the phosphorylation of Wzd by Wze, in the presence of ATP. This phosphorylation destabilizes the protein interaction with Wze. The transient interaction with phosphorylated Wzd leads to autophosphorylation of Wze in the presence of ATP. In addition, the activity of Wzd is modulated by tyrosine phosphorylation allowing autophosphorylation of Wze. Thus, the phosphorylation of Wzd may be an additional step of reversible phosphorylation for polymerization of EPS. This study may help advance our understanding of the relationship between the characteristics and biological functions of these polymers.