A trealase solúvel foi purificada a partir do intestino de S. frugiperda. Os pKas dos grupos catalíticos determinados por inativação química são similares aos pKas determinados por analise cinética, indicando que a enzima tem um grupo carboxila que atua como um nucleófilo e um grupo guanidina que atua como doador de prótons. Dietil pirocarbonato não afeta a enzima, exceto na presença de MalfaGlu (inibidor competitivo). A modificação com DPC diminui a atividade enzimática da trealase e muda o valor do pKa do resíduo de Arg, indicando que o resíduo de His modula o pKa do doador de prótons. Trealase tem dois subsítios para a ligação de glicose e baseado na proteção por MalfaGlu durante a modificação química é possível inferir que o subsítio que liga MalfaGlu contém o grupo carboxila, e o outro subsítio possui o resíduo de Arg que atua como grupo catalítico e o resíduo de His. Usando diferentes estratégias nós obtivemos uma seqüência parcial do cDNA que aparentemente codifica para trealase (denominada trealase 1) e clonamos e expressamos a enzima denominada trealase 2. A trealase 2 foi expressa em Bl21 DE3 e purificada, sendo que suas propriedades são similares a enzima solúvel. O cDNA da trealase 1 provavelmente codifica para a trealase de membrana encontrada no intestino de S. frugiperda. A trealase 2 tem 587 aminoácidos, um pepitideo sinal com 23 aminoácidos e seis possíveis sítios para glicosilação. A enzima apresenta alta identidade e similaridade (61% e 76%, respectivamente) com a trealase digestiva de B. mori. Foi determinada a atividade de trealase presente na carcaça, Túbulos de Malpighi, corpo gorduroso, intestino e hemolinfa de Tenebrio molitor, Musca domestica, Spodoptera frugiperda e Diatraea saccharalis na presença e na ausência de ß-glicosideos tóxicos produzidos por plantas. Os glicosídeos usados foram amigdalina, prunasina, florizina e o aglicone mandelonitrila. E a atividade das trealases de T. molitor e S. frugiperda foi determinada também na presença de esculina. Prunasina é o melhor inibidor das trealases de T. molitor, já para as trealases (ligadas a membrana) de D. saccharalis o melhor inibidor é florizina e esculina é o melhor inibidor das trealases de S. frugiperda. Nós alimentamos S. frugiperda com uma dieta contendo 0,1% de esculina e sua presença nós tecidos foi detectada por fluorescência. Esculina foi encontrada no corpo gorduroso, Túbulos de Malpighi e hemolinfa e não foi encontrada na carcaça. A maior quantidade de esculina foi registrada na hemolinfa (0,2 mM) e as larvas alimentadas com uma dieta contendo esculina são 40% menor que as larvas alimentadas numa dieta controle. A inibição das trealases pode ser um dos fatores que leva a diminuição de peso das larvas experimentais. As larvas de S. frugiperda criadas numa dieta com 0,1% de amigdalina apresenta em alguns tecidos um aumento na atividade especifica de trealase o que não é observado quando as larvas são alimentadas com uma dieta com 0,1% de esculina. O aumento na atividade especifica de trealase pode ser uma das razões pela qual o desenvolvimento de S. frugiperda não é afetado pela amigdalina presente na dieta. / A soluble trehalase was purified from Spodoptera frugiperda midgut. The pKas of the catalitical groups determined by chemical inactivation agrees with the ones determined by kinetical analysis, indicating that the enzyme has a carboxyl group that acts as a nucleophile and a guanidine group that is the proton donor. Diethyl pyrocarbonate (DPC) does not affect to the enzyme, except in the presence of MalphaGlu (a competitive inhibitor). DPC modification decreases trehalase activity and changes the pKa value of the catalytical Arg residue, indicating that pKa of the proton donor His residue modulates. Trehalase has two subsites for glucose binding and based on the protection by MalphaGlu against chemical modification it is possible to infer that the subsite that binds MalphaGlu contains the catalytic carboxyl, whereas the other has the catalytical Arg residue and the His residue. Using different strategies we succeeded in obtaining a partial sequence of a cDNA that apparently codes for trehalase (called trehalase 1) and in molecular cloning and expressing the enzyme named trehalase 2. Trehalase 2, expressed in Bl21 DE3 cells was purified and its properties are similar to the soluble enzyme. Trehalase 1 cDNA probably codes for a membrane-bound trehalase found in S. frugiperda midgut. Trehalase 2 has 587 amino acids, a signal peptide with 23 amino acids and six possible sites for glycosilation. The enzyme present higher identity and similarity (61% and 76%, respectively) to digestive trehalase of Bombyx mori. Trehalase from body wall, Malpighian tubules, fat body, midgut and haemolymph from Tenebrio molitor, Musca. domestica, Spodoptera frugiperda and Diatraea saccharalis were assayed with and without the presence of toxic glucosides produced by plants. The glucosides used were amygdalin, prunasin, phlorizin and the aglycone mandelonitrile. In addition, T. molitor and S. frugiperda trehalases were assayed with esculin. Prunasin is the best inhibitor in T. molitor and M. domestica, phlorizin in D. saccharalis (only membrane-bound activity) and esculin in S. frugiperda. We fed S. frugiperda with a diet containing 0.1 % esculin and followed its fate by fluorescence. Esculin is recovered from fat body, Malpighian tubules and haemolymph. No esculin was found in body wall. The majority of esculin was recovered in haemolymph (0.2 mM) and larvae fed on esculin-containing diet weigh 40 % less than control ones. Trehalase inhibition by esculin may account for at least part of the observed decrease in larval weight. S. frugiperda larvae reared in 0.1% amygdalin-containing diet present higher trehalase activities in several tissues than the larvae reared in 0.1% esculin-containing diet. Higher trehalase activity should be the reason why S. frugiperda development is affected by esculin, but is not impaired by amygdalin present in the diet.

A trealase solúvel foi purificada a partir do intestino de S. frugiperda. Os pKas dos grupos catalíticos determinados por inativação química são similares aos pKas determinados por analise cinética, indicando que a enzima tem um grupo carboxila que atua como um nucleófilo e um grupo guanidina que atua como doador de prótons. Dietil pirocarbonato não afeta a enzima, exceto na presença de MalfaGlu (inibidor competitivo). A modificação com DPC diminui a atividade enzimática da trealase e muda o valor do pKa do resíduo de Arg, indicando que o resíduo de His modula o pKa do doador de prótons. Trealase tem dois subsítios para a ligação de glicose e baseado na proteção por MalfaGlu durante a modificação química é possível inferir que o subsítio que liga MalfaGlu contém o grupo carboxila, e o outro subsítio possui o resíduo de Arg que atua como grupo catalítico e o resíduo de His. Usando diferentes estratégias nós obtivemos uma seqüência parcial do cDNA que aparentemente codifica para trealase (denominada trealase 1) e clonamos e expressamos a enzima denominada trealase 2. A trealase 2 foi expressa em Bl21 DE3 e purificada, sendo que suas propriedades são similares a enzima solúvel. O cDNA da trealase 1 provavelmente codifica para a trealase de membrana encontrada no intestino de S. frugiperda. A trealase 2 tem 587 aminoácidos, um pepitideo sinal com 23 aminoácidos e seis possíveis sítios para glicosilação. A enzima apresenta alta identidade e similaridade (61% e 76%, respectivamente) com a trealase digestiva de B. mori. Foi determinada a atividade de trealase presente na carcaça, Túbulos de Malpighi, corpo gorduroso, intestino e hemolinfa de Tenebrio molitor, Musca domestica, Spodoptera frugiperda e Diatraea saccharalis na presença e na ausência de ß-glicosideos tóxicos produzidos por plantas. Os glicosídeos usados foram amigdalina, prunasina, florizina e o aglicone mandelonitrila. E a atividade das trealases de T. molitor e S. frugiperda foi determinada também na presença de esculina. Prunasina é o melhor inibidor das trealases de T. molitor, já para as trealases (ligadas a membrana) de D. saccharalis o melhor inibidor é florizina e esculina é o melhor inibidor das trealases de S. frugiperda. Nós alimentamos S. frugiperda com uma dieta contendo 0,1% de esculina e sua presença nós tecidos foi detectada por fluorescência. Esculina foi encontrada no corpo gorduroso, Túbulos de Malpighi e hemolinfa e não foi encontrada na carcaça. A maior quantidade de esculina foi registrada na hemolinfa (0,2 mM) e as larvas alimentadas com uma dieta contendo esculina são 40% menor que as larvas alimentadas numa dieta controle. A inibição das trealases pode ser um dos fatores que leva a diminuição de peso das larvas experimentais. As larvas de S. frugiperda criadas numa dieta com 0,1% de amigdalina apresenta em alguns tecidos um aumento na atividade especifica de trealase o que não é observado quando as larvas são alimentadas com uma dieta com 0,1% de esculina. O aumento na atividade especifica de trealase pode ser uma das razões pela qual o desenvolvimento de S. frugiperda não é afetado pela amigdalina presente na dieta. / A soluble trehalase was purified from Spodoptera frugiperda midgut. The pKas of the catalitical groups determined by chemical inactivation agrees with the ones determined by kinetical analysis, indicating that the enzyme has a carboxyl group that acts as a nucleophile and a guanidine group that is the proton donor. Diethyl pyrocarbonate (DPC) does not affect to the enzyme, except in the presence of MalphaGlu (a competitive inhibitor). DPC modification decreases trehalase activity and changes the pKa value of the catalytical Arg residue, indicating that pKa of the proton donor His residue modulates. Trehalase has two subsites for glucose binding and based on the protection by MalphaGlu against chemical modification it is possible to infer that the subsite that binds MalphaGlu contains the catalytic carboxyl, whereas the other has the catalytical Arg residue and the His residue. Using different strategies we succeeded in obtaining a partial sequence of a cDNA that apparently codes for trehalase (called trehalase 1) and in molecular cloning and expressing the enzyme named trehalase 2. Trehalase 2, expressed in Bl21 DE3 cells was purified and its properties are similar to the soluble enzyme. Trehalase 1 cDNA probably codes for a membrane-bound trehalase found in S. frugiperda midgut. Trehalase 2 has 587 amino acids, a signal peptide with 23 amino acids and six possible sites for glycosilation. The enzyme present higher identity and similarity (61% and 76%, respectively) to digestive trehalase of Bombyx mori. Trehalase from body wall, Malpighian tubules, fat body, midgut and haemolymph from Tenebrio molitor, Musca. domestica, Spodoptera frugiperda and Diatraea saccharalis were assayed with and without the presence of toxic glucosides produced by plants. The glucosides used were amygdalin, prunasin, phlorizin and the aglycone mandelonitrile. In addition, T. molitor and S. frugiperda trehalases were assayed with esculin. Prunasin is the best inhibitor in T. molitor and M. domestica, phlorizin in D. saccharalis (only membrane-bound activity) and esculin in S. frugiperda. We fed S. frugiperda with a diet containing 0.1 % esculin and followed its fate by fluorescence. Esculin is recovered from fat body, Malpighian tubules and haemolymph. No esculin was found in body wall. The majority of esculin was recovered in haemolymph (0.2 mM) and larvae fed on esculin-containing diet weigh 40 % less than control ones. Trehalase inhibition by esculin may account for at least part of the observed decrease in larval weight. S. frugiperda larvae reared in 0.1% amygdalin-containing diet present higher trehalase activities in several tissues than the larvae reared in 0.1% esculin-containing diet. Higher trehalase activity should be the reason why S. frugiperda development is affected by esculin, but is not impaired by amygdalin present in the diet.

The aromatic beta-glucosides of plant origin, salicin and arbutin, serve as carbon sources for the sustenance of bacteria when ‘preferred’ sugars are absent in the environment. In the family Enterobacteriaceae, there are varied patterns for utilization of these beta-glucosides, wherein, in some members the ability to utilize salicin or arbutin is cryptic while in others it is completely absent. Escherichia coli harbors silent or cryptic genetic systems for the utilization of arbutin and salicin, which are activated by spontaneous mutation(s). Of these systems, the bgl operon of E.coli has been used as a paradigm for silent genes and extensive studies have been carried out to understand its silencing and activating mechanisms. Mutational activation of the wild type bgl operon in E.coli leads to the acquisition of the ability to utilize both arbutin and salicin. Preliminary studies have shown that aromatic beta-glucoside utilization in Shigella sonnei, which is evolutionarily related to E.coli, shows a two-step activation process wherein the wild type strain first becomes Arb+, which subsequently mutates to Sal+. The genetic systems responsible for beta-glucoside utilization, including the bgl operon, are conserved in S.sonnei to a large extent. A major difference is that the bglB gene encoding the phosphor-β-glucosidase B is insertionally inactivated in S.sonnei. As a result, activation of the bgl operon in the first stage leads to expression of the permease, BglF, which along with the phosphor-β-glucosidase A expressed from an unlinked constitutive gene, bglA, confers an Arb+phenotype. Salicin is not a substrate for the enzyme BglA and therefore a second mutational event is required for the acquisition of the Sal+ phenotype. Interestingly, the insertion within bglB is retained in AK102, the Sal+ second step mutant of S.sonnei. Therefore, the locus involved in conferring salicin utilization ability is unknown. However, S.sonnei is not amenable to routine genetic echniques and an E.coli bglB model was generated by creating an insertion in the bglB gene to identify the locus involved in conferring the Sal+ phenotype. Like S.sonnei, this E.coli strain, SD-1.3, also showed a two-step activation process for the utilization of salicin. Utilization of salicin in the Sal+ second step mutant of SD-1.3 could require activation of other silent genetic systems such as the asc operon and the chb operon or mutation in loci such as bglB or bglA. Linkage analysis by P1 transduction showed that activation of the asc operon is required for conferring a Sal+ phenotype in the second step mutant. The asc operon comprises of two genes, ascF encoding a PTS permease and ascB encoding a phosphor-β-glucosidaseB.The Precise mechanism of activation of the asc operon is not known but, it has been speculated that AscG, encoded by an upstream gene, acts as a repressor. Results presented in this thesis show that BglF is responsible for the transport of salicin and AscB provides the phosphor-β-glucosidase B in the Sal+ second step mutant of the E.coli strain SD-1.3. Analysis of the expression of the ascFB operon by measuring the transcripts as well as the activity of phosphor-β-glucosidase B showed that it is enhanced in the Sal+ second step mutant of SD-1.3 in the presence of the inducer. The expression of the ascFB operon is also increased constitutively when ascG is replaced by an antibiotic cassette in the parent strain SD-1.3 and the Arb+ first step mutant, indicating that AscG acts as a repressor for the asc operon. Moreover, inactivation of ascG in the parent leads to utilization of salicin in a single step by the activation of the bgl operon to provide the transport function, indicating that the inactivation of ascG is sufficient to activate the expression of ascB. Similarly, loss of AscG–mediated repression of the asc operon confers salicin utilization ability to the Arb+ first step mutant of SD-1.3. Interestingly, measurement of phosphor-β-glucosidase B activity in a Sal+ second step mutant derivative deleted for ascG showed a constitutive increase in the expression of the ascFB operon. Thus, AscG mediates the induction of the asc operon in response to salicin. In order to study the mechanism of activation of the asc operon, the ascB gene was cloned from the Arb+ first step mutant and the Sal+ second step mutant of SD-1.3 in a low copy number vector. Both these constructs were able to confer a Sal+ phenotype to the Arb+ first step mutant indicating absence of any genetic change in ascB in the Sal+ second step mutant. This was also confirmed by sequencing of ascB gene from the strains that showed no changes in the nucleotide sequence. Absence of any insertions within ascG showed that activation of the ascoperon is not achieved through disruption of ascG in the Sal+ second step mutants analyzed. AscG belongs to the GalR family of repressors in which some members require a mutation to enable the binding of sugar to mediate induction. Nucleotide sequence analysis showed that there was no change in the ascG gene in the Sal+ mutants analyzed. However, when the upstream regulatory region of the ascFB operon was analyzed a mutation was found in the -10 sequence of the putative promoter of the ascFB genes. This change leads to a stronger promoter as it brings the -10 sequence closer to the consensus sequence. Therefore, salicin utilization is achieved in the Sal+ second step mutant analyzed by an increase in expression of the asc operon by a promoter-up mutation. The negative effect of binding of AscG on expression of the ascFB operon is relieved in presence of the inducer, salicin. The possible role of the asc operon in salicin utilization in S.sonnei was tested by replacing the ascB gene by anantibiotic cassette in AK102, the Sal+ second step mutant of S. sonnei. This did not lead to loss of salicin utilization. By gene targeting approach it was also found that none of the phosphor-β-glucosidases known in E.coli are involved in degradation of salicin in AK102. A search of the S. sonnei genome database indicated the presence of two putative phosphor-β-glucosidases encoded by glvG and SSO1595. Replacement of glvG gene by anantibiotic cassette in AK102 did not lead to loss of salicin utilization. However, a similar replacement of SSO1595 in AK102 resulted in a Sal+ phenotype indicating that SSO1595 provides the phosphor-β-glucosidase in the Sal+ second step mutant of S. sonnei. A homolog of this enzyme is not present in E.coliorinany of the other members of the Shigella genus. Transcription alanalysis as well as measurement of phosphor-β-glucosidase B activity showed that expression of SSO1595 is enhanced constitutively in AK102. To study the mechanism of mutational activation for achieving salicin utilization in S. sonnei, SSO1595 was cloned from AK101, theArb+ first step mutant and AK102, the Sal+ second step mutant in a low copy numbe rvector. Both these constructs were able to confer a Sal+ phenotype to AK101 indicating an absence of genetic change in SSO1595 in AK102. This was also confirmed by sequencing of SSO1595 gene from the strains. Analysis of the upstream regulatory region of SSO1595 in AK102 indicated a deletion of around 1.0kbp sequence. This was also confirmed by nucleotide sequencing of the region. By primer extension analysis it was found that a new transcriptional start site is generated upstream to the deletion in the Sal+ second stepmutant of S.sonnei. Acquisition of the Sal+ phenotype in AK102 is therefore the resultof the SSO1595 gene being brought under a new promoter as a result of a DNA rearrangement. Overall, this study suggests that a high degree of similarity at the genomic level between organisms does not always ensure similarity in genetic mechanisms as two distinct pathways are responsible for conferring utilization of salicinin S. sonnei and E.coli.

The genetic systems involved in the utilisation of aromatic β-glucosides in E. coli consist of the bgl, asc, and chb operons and the locus bglA encoding phospho-β-glucosidase A. The bgl and asc operons are known as cryptic or silent systems since their expression is not sufficient for utilisation of these sugars in wild type strains of E. coli. Their transcriptional activation by different classes of mutations confers a Bgl+ phenotype to the mutant. The maintenance of cryptic genes without accumulating deleterious mutation in spite of being silent is an evolutionary puzzle. Several observations have suggested the possibility that these genes may be expressed under specific physiological conditions conferring a fitness advantage to the organism. The main aim of this study was to investigate the possible role of aromatic β-glucoside catabolic systems of E. coli in combating nutrient stress and microaerobic growth conditions. The results presented in Chapter 2 address the evolution of aromatic β-glucoside catabolic systems when exposed to a novel β-glucoside as the sole substrate. The results indicate that the bgl opeon, the primary system involved in the utilisation of the aromatic β-glucosides arbutin and salicin, is also involved in esculin utilisation. In the absence of bglB encoding the enzyme phospho-β-glucosidase B, activation of the silent asc operon enables esculin utilisation. The bglA gene encoding phospho-β-glucosidase A specific for arbutin, can undergo successive mutations to evolve the ability to hydrolyse esculin and salicin sequentially when bglB and ascB are absent. The Esc+ and Sal+ mutants retain their arbutin+ phenotype, indicating that the mutations enhance the promiscuity of the enzyme. Sequencing data indicate that the first step Esc+ mutant carries a four base insertion within the promoter of the bglA gene that results in enhanced transcription of bglA. RT-PCR studies confirm that both the steady-state levels as well as the half-life of the bglA mRNA are enhanced in the mutant. This is further corroborated by the observation that overexpression of wild type bglA in the parent strain using a multicopy plasmid confers an Esc+ phenotype. The second step Sal+ mutant carries a point mutation within bglA ORF, a thymine to guanine transversion at position 583 (T583G) of the bglA gene, resulting in an amino acid change from cysteine to glycine at position 195 (C195G) of the BglA ORF close to the active site. Presence of a plasmid carrying the T583G mutation, introduced by site-directed mutagenesis, results in a Sal+ phenotype, confirming the role of the transversion in conferring the Sal+ phenotype. Based on docking studies, the positioning of salicin into the substrate binding site of the mutant BglA enzyme is different compared to wild type BglA due to the loss of stearic hindrance for the binding of salicin when C195 is replaced by the smaller amino acid glycine in the mutant protein. These observations indicate that under conditions of nutrient deprivation, exposure to novel substrates can result in the evolution of new metabolic capabilities by the sequential modification of a pre-existing genetic system. In the case of one novel substrate, the mutation results in the overexpression of the hydrolytic enzyme, while in the case of the second substrate, a mutation close to its active site increases its substrate specificity. Results presented in Chapter 3 specifically deal with the involvement of the bgl operon under low levels of oxygen. Earlier observations have shown that there is a 22 fold enhancement in the expression of the bgl operon under anaerobic condition. The present results provide evidence that bgl expression has a physiological role under low levels of oxygen and in addition suggest a possible mechanism for the overexpression of the bgl operon that involves the ArcAB two component system known to mediate regulation under microaerobic and static conditions. Transcription studies using a lacZ reporter fused to the wild type bgl promoter show that there is enhanced transcription from the bgl promoter under microaerobic and static conditions in the presence of arcA encoding the response regulator compared to that in its absence. The positive effect of arcA on the expression of the bgl operon is dispensable in the absence of H-NS since presence or absence of arcA does not change the expression of the bgl operon in an hns-null background, implying that the involvement of ArcA is via antagonizing H-NS. Competition experiments indicate that there is growth advantage associated with the activated allele of the bgl operon under low levels of oxygen since Bgl+ strains carrying the activated allele of the bgl operon as well as strains expressing BglG constitutively can out-compete wild-type strains. Presence of the wild type arcA allele results in a strong growth advantage compared to its absence under static conditions but not aerobic condition. The bgl operon seems to be one of the possible downstream targets of ArcA under static condition since absence of the bgl operon results in a modest reduction of the growth advantage (GASP) phenotype conferred by arcA. The up-regulation of the bgl operon is likely to enable the cells to scavenge available nutrients from their niche more efficiently. These experiments also show that the GASP phenotype associated with BglG constitutive strains under static conditions involves downstream genes that are different from oppA known to be one of the downstream targets during aerobic growth. It is possible that under low level of oxygen, the bgl operon is regulating a different set of downstream genes involving a different mechanism. In summary, the results of this investigation show that the aromatic β-glucoside catabolic systems in E. coli play a role in the generation of new metabolic capabilities via mutations in pre-existing genetic systems as well as through changes in gene expression patterns. The mechanisms outlined in this study are likely to be of broader significance applicable to microbial evolution under stress in general.