O operon pst de Escherichia coli é formado pelos genes pstS, pstC, pstA, pstB e phoU. Os quatro primeiros genes codificam proteínas que compõem um sistema de transporte do tipo ABC denominado Pst. O sistema Pst, junto com a proteína PhoU participa da repressão dos genes do regulon PHO. A transcrição dos genes do operon pst é induzida pela carência de fosfato inorgânico (Pi). Neste trabalho, o padrão de transcrição do operon pst foi analisado. A existência de um transcrito primário instável foi comprovada através de uma nova técnica de RT-PCR. O papel da RNase E na degradação do mRNA de pst foi demonstrado. A análise das seqüências intergênicas do operon revelou a importância da seqüência REP (Repetitive Extragenic Palindrome) localizada na região intergênica entre pstS e pstC na estabilização da mensagem de pstS. As seqüências localizadas a 5\' dos genes pstC, pstB e phoU também foram analisadas e demonstraram uma atividade promotora fraca, que resulta na síntese de transcritos dos genes distais de pst. / The pst operon of Escherichia coli consists of the genes pstS, pstC, pstA, pstB and phoU. The four proximal genes of the operon encode the proteins of the ABC-type Pi phosphate (Pi) transporter Pst.The Pst system, together with the PhoU protein, also acts as a negative regulator of the PHO regulon. Transcription of the pst genes is induced by Pi starvation. The present study describes the transcription pattern of the pst operon. The existence of an unstable primary transcript was confirmed by using an improved RT-PCR protocol. The role of RNase E in pst transcript decay was demonstrated.Analysis of the operon intergenic regions revealed the role of a REP sequence (Repetitive Extragenic Palindrome) located between pstS and pstC in pst mRNA stability. The regions upstream of pstC, pstB and phoU displayed promoter activity. Transcription from these internal promoters resulted in a small amount of mRNAs corresponding to the pst distal genes.

Xylella fastidiosa é uma bactéria Gram-negativa, limitada ao xilema das plantas e o agente causador de diversas doenças em importantes plantações como citros, videiras, mirta, amêndoa, arbustos e café. Em citros, X. fastidiosa causa a Clorose Variegada dos Citros (CVC) ou “amarelinho". Nove enzimas (GumB, C, D, E, F, H, J, K e M) estão envolvidas nas etapas biossintéticas de um polissacarídeo extracelular (EPS), chamado de goma fastidiana, um dos mecanismos envolvidos na patogênese da bactéria. Essas enzimas catalisam reações de adição de açúcares, polimerização e exportação do EPS através da membrana da bactéria. No presente trabalho, ferramentas de bioinformática foram utilizadas para o estudo e entendimento da biossíntese da goma fastidiana. As nove enzimas foram estudadas quanto ao seu conteúdo de estrutura secundária, análise de hidrofobicidade e das regiões transmembrânicas, classificação quanto as suas funções. A construção de modelos estruturais para as enzimas Gums através de comparação por homologia seqüencial mostrou ser um processo impossível, devido a falta de moléculas homólogas com estruturas tridimensionais conhecidas. Por outro lado, métodos de reconhecimento de enovelamento mostraram bons resultados e comparações entre as estruturas secundárias das enzimas Gums foram calculadas com a utilização dos programas GenThreader e THREADER 3.3. Modelos tridimensionais para as enzimas GumB, GumK, GumM, GumJ e GumC foram construídos com o programa MODELLER 6.0a e validados com o programa Procheck e VERIFY 3D. Para construção do modelo da GumH (enzima que catalisa a adição da GDP-manose em um lipídio carreador poliprenol), o GenThreader encontrou similaridades quando comparada a MurG (E.coli), 2-epimerase (E. coli), GtfB (Amycolatopsis orientalis) e beta-GT de fago T4. Todos os modelos são bastante semelhantes e compostos por dois domínios (alfa/beta), ambos similares ao motivo de ligação de nucleotídeos Rossmann fold e separados por uma fenda profunda, que, provavelmente, forma o sítio de ligação da GDP-manose. Estudos da interação entre proteína e substrato foram obtidos com a utilização do programa FLO. O alinhamento seqüencial da GumH com outras onze glicosiltransferases mostrou regiões bastante conservadas, incluindo o motivo EX7E presente no sítio de ligação do substrato na proteína. Considerações a respeito das interações do substrato GDP-manose com a enzima GumH e do mecanismo da reação foram feitas. Essas análises enfatizam o modelo obtido para a GumH, que representa a primeira estrutura proposta para as enzimas envolvidas na síntese da goma fastidiana. / Xylella fastidiosa is a xylem-dwelling, insect-transmitted gamma-protobacterium that causes pathogenicity in citrus plants and many others important crops such as grapevine, periwinkle, almond, oleander and coffee. In citrus plants, X. fastidiosa causes citrus variegated chlorosis (CVC) or “amarelinho". Nine enzymes (GumB, C, D, E, F, H, J, K and M) are involved in the biosynthetic pathway of an exopolysaccharide (EPS) called fastidian gum which could be involved in the pathogenicity of the bacterium. These enzymes catalyses sugars addition reactions, polymerization and discharge of the EPS through the bacteria’s membrane. We have used bioinformatic tools to study these enzymes and to understand the gum biosynthesis. The nine enzymes were studied regarding to its secondary structure content, analysis of hidrophobicity and transmembrane regions, and yet function classification. The construction of structural models using sequential homology was shown to be impossible, due to the necessity of homologues molecules whose three-dimensional structures are known. On the other hand, pairwises comparisons of secondary structures showed good results and were realized with GenThreader and THREADER 3.3 programs. Three-dimensional structures to GumB, GumK, GumM, GumJ and GumC enzymes were constructed using MODELLER 6.0a and validated with Procheck and VERIFY 3D programs. To construct the model of GumH (enzyme that catalyse the addiction of a GDP-mannose on a polyprenol phosphate carrier), GenThreader found folding similarities when compared to MurG and UDP-Acetylglucosamine 2-Epimerase (from E. coli), GtfB (from Amycolatopsis orientalis) and beta-GT (from T4 phage). The models are very similar consisting of two alpha/beta open sheet domains, both alike in topology to the Rossmann nucleotide-binding folds, and separated by a deep cleft which probably forms the GDP-mannose binding site. Studies of the interaction between enzyme and docked substrate were carried out using the FLO program. The sequence alignment between GumH and another eleven glycosiltransferases showed several preserved regions including the EX7E motif present on the substrate binding site. The interactions between enzyme-GDP-mannose substrate and the mechanism of the reaction were studied. These analyses emphasize the three-dimensional model constructed for GumH that represents the first structural information for enzymes involved in fastidian gum synthesis.

Xylella fastidiosa é uma bactéria Gram-negativa, limitada ao xilema das plantas e o agente causador de diversas doenças em importantes plantações como citros, videiras, mirta, amêndoa, arbustos e café. Em citros, X. fastidiosa causa a Clorose Variegada dos Citros (CVC) ou “amarelinho”. Nove enzimas (GumB, C, D, E, F, H, J, K e M) estão envolvidas nas etapas biossintéticas de um polissacarídeo extracelular (EPS), chamado de goma fastidiana, um dos mecanismos envolvidos na patogênese da bactéria. Essas enzimas catalisam reações de adição de açúcares, polimerização e exportação do EPS através da membrana da bactéria. No presente trabalho, ferramentas de bioinformática foram utilizadas para o estudo e entendimento da biossíntese da goma fastidiana. As nove enzimas foram estudadas quanto ao seu conteúdo de estrutura secundária, análise de hidrofobicidade e das regiões transmembrânicas, classificação quanto as suas funções. A construção de modelos estruturais para as enzimas Gums através de comparação por homologia seqüencial mostrou ser um processo impossível, devido a falta de moléculas homólogas com estruturas tridimensionais conhecidas. Por outro lado, métodos de reconhecimento de enovelamento mostraram bons resultados e comparações entre as estruturas secundárias das enzimas Gums foram calculadas com a utilização dos programas GenThreader e THREADER 3.3. Modelos tridimensionais para as enzimas GumB, GumK, GumM, GumJ e GumC foram construídos com o programa MODELLER 6.0a e validados com o programa Procheck e VERIFY 3D. Para construção do modelo da GumH (enzima que catalisa a adição da GDP-manose em um lipídio carreador poliprenol), o GenThreader encontrou similaridades quando comparada a MurG (E.coli), 2-epimerase (E. coli), GtfB (Amycolatopsis orientalis) e beta-GT de fago T4. Todos os modelos são bastante semelhantes e compostos por dois domínios (alfa/beta), ambos similares ao motivo de ligação de nucleotídeos Rossmann fold e separados por uma fenda profunda, que, provavelmente, forma o sítio de ligação da GDP-manose. Estudos da interação entre proteína e substrato foram obtidos com a utilização do programa FLO. O alinhamento seqüencial da GumH com outras onze glicosiltransferases mostrou regiões bastante conservadas, incluindo o motivo EX7E presente no sítio de ligação do substrato na proteína. Considerações a respeito das interações do substrato GDP-manose com a enzima GumH e do mecanismo da reação foram feitas. Essas análises enfatizam o modelo obtido para a GumH, que representa a primeira estrutura proposta para as enzimas envolvidas na síntese da goma fastidiana. / Xylella fastidiosa is a xylem-dwelling, insect-transmitted gamma-protobacterium that causes pathogenicity in citrus plants and many others important crops such as grapevine, periwinkle, almond, oleander and coffee. In citrus plants, X. fastidiosa causes citrus variegated chlorosis (CVC) or “amarelinho”. Nine enzymes (GumB, C, D, E, F, H, J, K and M) are involved in the biosynthetic pathway of an exopolysaccharide (EPS) called fastidian gum which could be involved in the pathogenicity of the bacterium. These enzymes catalyses sugars addition reactions, polymerization and discharge of the EPS through the bacteria’s membrane. We have used bioinformatic tools to study these enzymes and to understand the gum biosynthesis. The nine enzymes were studied regarding to its secondary structure content, analysis of hidrophobicity and transmembrane regions, and yet function classification. The construction of structural models using sequential homology was shown to be impossible, due to the necessity of homologues molecules whose three-dimensional structures are known. On the other hand, pairwises comparisons of secondary structures showed good results and were realized with GenThreader and THREADER 3.3 programs. Three-dimensional structures to GumB, GumK, GumM, GumJ and GumC enzymes were constructed using MODELLER 6.0a and validated with Procheck and VERIFY 3D programs. To construct the model of GumH (enzyme that catalyse the addiction of a GDP-mannose on a polyprenol phosphate carrier), GenThreader found folding similarities when compared to MurG and UDP-Acetylglucosamine 2-Epimerase (from E. coli), GtfB (from Amycolatopsis orientalis) and beta-GT (from T4 phage). The models are very similar consisting of two alpha/beta open sheet domains, both alike in topology to the Rossmann nucleotide-binding folds, and separated by a deep cleft which probably forms the GDP-mannose binding site. Studies of the interaction between enzyme and docked substrate were carried out using the FLO program. The sequence alignment between GumH and another eleven glycosiltransferases showed several preserved regions including the EX7E motif present on the substrate binding site. The interactions between enzyme-GDP-mannose substrate and the mechanism of the reaction were studied. These analyses emphasize the three-dimensional model constructed for GumH that represents the first structural information for enzymes involved in fastidian gum synthesis.

O operon pst de Escherichia coli é formado pelos genes pstS, pstC, pstA, pstB e phoU. Os quatro primeiros genes codificam proteínas que compõem um sistema de transporte do tipo ABC denominado Pst. O sistema Pst, junto com a proteína PhoU participa da repressão dos genes do regulon PHO. A transcrição dos genes do operon pst é induzida pela carência de fosfato inorgânico (Pi). Neste trabalho, o padrão de transcrição do operon pst foi analisado. A existência de um transcrito primário instável foi comprovada através de uma nova técnica de RT-PCR. O papel da RNase E na degradação do mRNA de pst foi demonstrado. A análise das seqüências intergênicas do operon revelou a importância da seqüência REP (Repetitive Extragenic Palindrome) localizada na região intergênica entre pstS e pstC na estabilização da mensagem de pstS. As seqüências localizadas a 5\' dos genes pstC, pstB e phoU também foram analisadas e demonstraram uma atividade promotora fraca, que resulta na síntese de transcritos dos genes distais de pst. / The pst operon of Escherichia coli consists of the genes pstS, pstC, pstA, pstB and phoU. The four proximal genes of the operon encode the proteins of the ABC-type Pi phosphate (Pi) transporter Pst.The Pst system, together with the PhoU protein, also acts as a negative regulator of the PHO regulon. Transcription of the pst genes is induced by Pi starvation. The present study describes the transcription pattern of the pst operon. The existence of an unstable primary transcript was confirmed by using an improved RT-PCR protocol. The role of RNase E in pst transcript decay was demonstrated.Analysis of the operon intergenic regions revealed the role of a REP sequence (Repetitive Extragenic Palindrome) located between pstS and pstC in pst mRNA stability. The regions upstream of pstC, pstB and phoU displayed promoter activity. Transcription from these internal promoters resulted in a small amount of mRNAs corresponding to the pst distal genes.

L'objet de cette thèse de bioinformatique est de mieux comprendre l’ensemble des systèmes de régulation génique chez les bactéries. La disponibilité de centaines de génomes complets chez les bactéries ouvre la voie aux approches de génomique comparative et donc à l’étude de l’évolution des réseaux transcriptionnels bactériens. Dans un premier temps, nous avons implémenté et validé plusieurs méthodes de prédiction d’opérons sur base des génomes bactériens séquencés. Suite à cette étude, nous avons décidé d’utiliser un algorithme qui se base simplement sur un seuil sur la distance intergénique, à savoir la distance en paires de bases entre deux gènes adjacents. Notre évaluation sur base d’opérons annotés chez Escherichia coli et Bacillus subtilis nous permet de définir un seuil optimal de 55pb pour lequel nous obtenons respectivement 78 et 79% de précision. Deuxièmement, l’identification des motifs de régulation transcriptionnelle, tels les sites de liaison des facteurs de transcription, donne des indications de l’organisation de la régulation. Nous avons développé une méthode de recherche d’empreintes phylogénétiques qui consiste à découvrir des paires de mots espacés (dyades) statistiquement sur-représentées en amont de gènes orthologues bactériens. Notre méthode est particulièrement adaptée à la recherche de motifs chez les bactéries puisqu’elle profite d’une part des centaines de génomes bactériens séquencés et d’autre part les facteurs de transcription bactériens présentent des domaines Hélice-Tour-Hélice qui reconnaissent spécifiquement des dyades. Une évaluation systématique sur 368 gènes de E.coli a permis d’évaluer les performances de notre méthode et de tester l’influence de plus de 40 combinaisons de paramètres concernant le niveau taxonomique, l’inférence d’opérons, le filtrage des dyades spécifiques de E.coli, le choix des modèles de fond pour le calcul du score de significativité, et enfin un seuil sur ce score. L’analyse détaillée pour un cas d’étude, l’autorégulation du facteur de transcription LexA, a montré que notre approche permet d’étudier l’évolution des sites d’auto-régulation dans plusieurs branches taxonomiques des bactéries. Nous avons ensuite appliqué la détection d’empreintes phylogénétiques à chaque gène de E.coli, et utilisé les motifs détectés comme significatifs afin de prédire les gènes co-régulés. Au centre de cette dernière stratégie, est définie une matrice de scores de significativité pour chaque mot détecté par gène chez l’organisme de référence. Plusieurs métriques ont été définies pour la comparaison de paires de profils de scores de sorte que des paires de gènes ayant des motifs détectés significativement en commun peuvent être regroupées. Ainsi, l’ensemble des nos méthodes nous permet de reconstruire des réseaux de co-régulation uniquement à partir de séquences génomiques, et nous ouvre la voie à l’étude de l’organisation et de l’évolution de la régulation transcriptionnelle pour des génomes dont on ne connaît rien. The purpose of my thesis is to study the evolution of regulation within bacterial genomes by using a cross-genomic comparative approach. Nowadays, numerous genomes have been sequenced facilitating in silico analysis in order to detect groups of functionally related genes and to predict the mechanism of their relative regulation. In this project, we combined prediction of operons and regulons in order to reconstruct the transcriptional regulatory network for a bacterial genome. We have implemented three methods in order to predict operons from a bacterial genome and evaluated them on hundreds of annotated operons of Escherichia coli and Bacillus subtilis. It turns out that a simple distance-based threshold method gives good results with about 80% of accuracy. The principle of this method is to classify pairs of adjacent genes as “within operon” or “transcription unit border”, respectively, by using a threshold on their intergenic distance: two adjacent genes are predicted to be within an operon if their intergenic distance is smaller than 55bp. In the second part of my thesis, I evaluated the performances of a phylogenetic footprinting approach based on the detection of over-represented spaced motifs. This method is particularly suitable for (but not restricted to) Bacteria, since such motifs are typically bound by factors containing a Helix-Turn-Helix domain. We evaluated footprint discovery in 368 E.coli K12 genes with annotated sites, under 40 different combinations of parameters (taxonomical level, background model, organism-specific filtering, operon inference, significance threshold). Motifs are assessed both at the level of correctness and significance. The footprint discovery method proposed here shows excellent results with E. coli and can readily be extended to predict cis-acting regulatory signals and propose testable hypotheses in bacterial genomes for which nothing is known about regulation. Moreover, the predictive power of the strategy, and its capability to track the evolutionary divergence of cis-regulatory motifs was illustrated with the example of LexA auto-regulation, for which our predictions are remarkably consistent with the binding sites characterized in different taxonomical groups. A next challenge was to identify groups of co-regulated genes (regulons), by regrouping genes with similar motifs, in order to address the challenging domain of the evolution of transcriptional regulatory networks. We tested different metrics to detect putative pairs of co-regulated genes. The comparison between predicted and annotated co-regulation networks shows a high positive predictive value, since a good fraction of the predicted associations correspond to annotated co-regulations, and a low sensitivity, which may be due to the consequence of highly connected transcription factors (global regulator). A regulon-per-regulon analysis indeed shows that the sensitivity is very weak for these transcription factors, but can be quite good for specific transcription factors. The originality of this global strategy is to be able to infer a potential network from the sole analysis of genome sequences, and without any prior knowledge about the regulation in the considered organism.

In the nature, lots of organism apply different kinds of lights such as flourscence or luminoscence for some purposes such as defence or hunting. Firefly luciferase and Bacterial luciferase are the most famous ones which have been used to design Biosensors or Bioreporters in recent decades. Their applications are so extensive from detecting pollutions in the environment to medical and treatment usages. To design Copper Bioreporter, copper resistance promoter from COP operon which plays an important role in Pseudomonas syringae and pGL3 plasmid which has luciferase gene were utilized. To achieve that target, sequences of promoter were synthesized and inserted to pCR2.1 vector, then suitable primers with considering restriction sites were designed to get high concentration of DNA. After digestion of pGL3 and interested gene by Nhe I and Sac I enzymes, ligation was performed, and then recombinat plasmids were transferred to E. coli BL-21 as a host cell. Finallay, luciferase assay of designed bioreporter was performed by Luminometer in presence of different concentration of CuSO4. The result was maginificant that confirmed design of Copper Bioreporter.

Chez Bacillus subtilis l’expression de l’opéron mtl pour l’utilisation du mannitol est contrôlé par MtlR. MtlR est un activateur transcriptionnel qui appartient à la famille des régulateurs DeoR composé d’un domaine HTH suivi de deux PRDs, un domaine EIIBGat et un domaine EIIAMtl-like.Le mécanisme général de la régulation de l’activité de MtlR est basé sur sa phosphorylation par des composants du PTS. La phosphorylation sur la Cystéine 419 du domaine EIIBGat par P~EIIAMtl a un effet négatif majeur sur l’activité de MtlR. Par conséquent, dans un mutant mtlF où EIIAMtl est délétée MtlR est constitutivement actif.Dans cette étude nous avons mis en évidence un nouveau phénomène de régulation de MtlR impliquant la protéine du PTS, EIIBMtl.Nous avons observé que lorsque on déléte l’opéron mtl ou EIIBMtl et EIIAMtl, l’activité constitutive de MtlR dans un mutant mtlF déjà observée est abolie d’où notre hypothèse que EIIBMtl aura un effet sur l’activité de MtlR. Par des expériences de double hybride nous avons montré une interaction directe, spécifique et bidirectionnelle entre les deux protéines EIIBMtl et EIIBGatEIIAMtl-like de MtlR. D’une manière comparable à la cellule où EIIBMtl est fusionnée à la protéine EIICMtl nous avons démontré que seulement la forme EIIBMtl fusionné à la perméase EIICMtl est capable d’activer MtlR mais nous avons également démontré que ce n’est pas EIICMtl qui est essentielle à l’interaction entre EIIBMtl et MtlR mais c’est le voisinage de la membrane qui est essentielle pour l’établissement de cette interaction et l’activation de MtlRUn modèle de régulation de l’activité du régulateur MtlR est proposé. Dans ce modèle l’induction de l’opéron mtl via l’activation de MtlR requiert la phosphorylation de PRDII de MtlR par P~His-HPr, la déphosphorylation de EIIBGat de MtlR par EIIAMtl et la présence de la forme non-phosphorylée de l’EIIBMtl qui est dominante en présence du substrat inducteur, le mannitol. Ainsi, l’EIIBMtl non-phosphorylée séquestre MtlR déphosphorylé sur sa cystéine 419 à la membrane, l’active et induit l’expression de l’opéron mtl. / The Bacillus subtilis mtl operon encodes the enzymes necessary for mannitol utilization. Its expression is controlled by MtlR, a transcriptional activator belonging to the DeoR family. MtlR contains a HTH domain followed by two PTS regulation domains (PRDs), an EIIBGat domain and an EIIAMtl-like domain.The general mechanism of the regulation of MtlR activity is based on its phosphorylation by components of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). The phosphorylation of EIIBGat on cysteine 419 by P~EIIAMtl has a major negative effect on the activity of MtlR. The absence of EIIAMtl in a mtlF mutant therefore leads to constitutively active MtlR.In this study a new mechanism of MtlR regulation based on the interaction of the PTS component EIIBMtl with MtlR is presented.We observed that the deletion of the entire mtlAFD operon or of mtlF and only the 3’-part of mtlA (encoding the EIIBMtl domain) abolishes the constitutive MtlR activity of the mtlF mutant, suggesting that MtlR activity depends on functional EIIBMtl. By carrying out yeast two-hybrid experiments we could establish a direct, specific and bidirectional interaction between EIIBMtl and the EIIBGatEIIAMtl-like part of MtlR.Complementation of the above mutants was possible with entire MtlA, but not with the EIIBMtl domain. EIIBMtl is normally fused to the membrane protein EIICMtl; we therefore fused EIIBMtl to another membrane, which indeed restored MtlR function in the absence of EIICMtl. The EIICMtl domain is therefore not essential for the interaction between EIIBMtl and MtlR; it is rather the vicinity of the membrane which is required for the activation of MtlR.A regulation model of MtlR activity is proposed. In this model, the MtlR-mediated induction of the mtlAFD operon requires the phosphorylation of PRDII by P~His-HPr and the dephosphorylation of EIIBGat by EIIAMtl. The presence of unphosphorylated EIIBMtl, which prevails when the inducer mannitol is present, is also required. Under these conditions unphosphorylated EIIBMtl sequesters MtlR dephosphoryled on cysteine 419, but phosphorylated at His-342, to the membrane thereby activating the transcription activator, which leads to increased expression of the mtlAFD operon.

The initial aim of this project was to uncover novel cellulolytic organisms or enzymes from the diverse microbial source, activated sludge. Two isolation methods were used; either directly inoculating the sludge material onto filter paper as a carbon source, or using the Evolver� technology as an enrichment device. In both cases, as expected, cellulase activity was evident, however attributing this activity to one species was difficult in either case. This highlighted the complex interrelationships that existed between the many microorganisms present as the cellulosic carbon sources were degraded. In one instance, a Cellvibrio sp. was isolated. This genus of bacteria is known to possess both types of cellulase activity (exo- and endo- acting) and was therefore likely to contribute to the degradation of the cellulose. However, the isolate, once purified, did not display significant cellulolytic ability as compared to the unpurified consortium of microorganisms. Therefore, in each case, microorganisms responsible for the cellulolytic activity were not uncovered. It was suspected that the microorganisms responsible for some of the cellulolytic activity were protists. During the isolation of microorganisms, an Aeromonas sp. bearing the novel phenotype (for this genus) of CMCase activity was isolated. This activity was at first suspected to contribute to the degradation of the filter paper that was seen during isolation. However, tests with the pure isolate suggested that the Aeromonas sp. CMCase was not used for cellulose catabolism. Ironically, the enzyme may instead function in the production of a cellulose-like exopolysaccharide by the bacterium. Part of a cellulose synthase operon was found in the genome of the Aeromonas sp. isolate, including a gene coding for an endoglucanase that gives a predicted molecular weight enzyme similar to the 39 kDa CMCase purified from the bacterium. The CMCase enzyme, operating as part of of a synthetic operon is expected to be important in terms of the biofilm forming ability of this Aeromonas strain. Such capabilities of the bacterium were investigated here, including observing motility behaviour of the organism on agar surfaces. Studying the biofilm forming ability of this genus in general will be important in understanding how the fish and human pathogens persist in aquatic environments

Background: The loss of photosynthesis has occurred often in eukaryotic evolution, even more than its acquisition, which occurred at least nine times independently and which generated the evolution of the supergroups Archaeplastida, Rhizaria, Chromalveolata and Excavata. This secondary loss of autotrophic capability is essential to explain the evolution of eukaryotes and the high diversity of protists, which has been severely underestimated until recently. However, the ecological and evolutionary scenarios behind this evolutionary ‘‘step back’’ are still largely unknown. Methodology/Principal Findings: Using a dynamic model of heterotrophic and mixotrophic flagellates and two types of prey, large bacteria and ultramicrobacteria, we examine the influence of DOC concentration, mixotroph’s photosynthetic growth rate, and external limitations of photosynthesis on the coexistence of both types of flagellates. Our key premises are: large bacteria grow faster than small ones at high DOC concentrations, and vice versa; and heterotrophic flagellates are more efficient than the mixotrophs grazing small bacteria (both empirically supported). We show that differential efficiency in bacteria grazing, which strongly depends on cell size, is a key factor to explain the loss of photosynthesis in mixotrophs (which combine photosynthesis and bacterivory) leading to purely heterotrophic lineages. Further, we show in what conditions an heterotroph mutant can coexist, or even out-compete, its mixotrophic ancestor, suggesting that bacterivory and cell size reduction may have been major triggers for the diversification of eukaryotes. Conclusions/Significance: Our results suggest that, provided the mixotroph’s photosynthetic advantage is not too large, the (small) heterotroph will also dominate in nutrient-poor environments and will readily invade a community of mixotrophs and bacteria, due to its higher efficiency exploiting the ultramicrobacteria. As carbon-limited conditions were presumably widespread throughout Earth history, such a scenario may explain the numerous transitions from phototrophy to mixotrophy and further to heterotrophy within virtually all major algal lineages. We challenge prevailing concepts that affiliated the evolution of phagotrophy with eutrophic or strongly light-limited environments only.

No description available.