RESUMO
BACKGROUND: The cyclic-di-GMP (c-di-GMP) second messenger exemplifies a signaling system that regulates many bacterial behaviors of key importance; among them, c-di-GMP controls the transition between motile and sessile life-styles in bacteria. Cellular c-di-GMP levels in bacteria are regulated by the opposite enzymatic activities of diguanylate cyclases and phosphodiesterases, which are proteins that have GGDEF and EAL domains, respectively. Azospirillum is a genus of plant-growth-promoting bacteria, and members of this genus have beneficial effects in many agronomically and ecologically essential plants. These bacteria also inhabit aquatic ecosystems, and have been isolated from humus-reducing habitats. Bioinformatic and structural approaches were used to identify genes predicted to encode GG[D/E]EF, EAL and GG[D/E]EF-EAL domain proteins from nine genome sequences. RESULTS: The analyzed sequences revealed that the genomes of A. humicireducens SgZ-5T, A. lipoferum 4B, Azospirillum sp. B510, A. thiophilum BV-ST, A. halopraeferens DSM3675, A. oryzae A2P, and A. brasilense Sp7, Sp245 and Az39 encode for 29 to 41 of these predicted proteins. Notably, only 15 proteins were conserved in all nine genomes: eight GGDEF, three EAL and four GGDEF-EAL hybrid domain proteins, all of which corresponded to core genes in the genomes. The predicted proteins exhibited variable lengths, architectures and sensor domains. In addition, the predicted cellular localizations showed that some of the proteins to contain transmembrane domains, suggesting that these proteins are anchored to the membrane. Therefore, as reported in other soil bacteria, the Azospirillum genomes encode a large number of proteins that are likely involved in c-di-GMP metabolism. In addition, the data obtained here strongly suggest host specificity and environment specific adaptation. CONCLUSIONS: Bacteria of the Azospirillum genus cope with diverse environmental conditions to survive in soil and aquatic habitats and, in certain cases, to colonize and benefit their host plant. Gaining information on the structures of proteins involved in c-di-GMP metabolism in Azospirillum appears to be an important step in determining the c-di-GMP signaling pathways, involved in the transition of a motile cell towards a biofilm life-style, as an example of microbial genome plasticity under diverse in situ environments.
Assuntos
Azospirillum/genética , Azospirillum/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Domínios Proteicos , Transdução de Sinais , Adaptação Biológica , Azospirillum/enzimologia , Biofilmes/crescimento & desenvolvimento , Biologia Computacional , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Diester Fosfórico Hidrolases/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Conformação Proteica , Sistemas do Segundo Mensageiro/genéticaRESUMO
It was found that Azospirillum brasilensis strain Sp7 is able to produce extracellular proteolytic enzymes. The enzymes were active within a broad range of pH values, with two peaks of activity being located in the acid and alkaline pH areas; required calcium ions; and exhibited substrate specificity with respect to azogelatin. Zymography allowed at least four proteolytic enzymes with molecular weights of 32, 45, 52, and 174 kDa to be detected in A. brasilense Sp7 culture liquid. It was shown that the lectin from A. brasilense Sp7 can inhibit proteolytic enzymes.
Assuntos
Azospirillum/enzimologia , Lectinas/fisiologia , Peptídeo Hidrolases/metabolismo , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Peptídeo Hidrolases/química , Inibidores de Proteases/farmacologia , Especificidade por SubstratoRESUMO
CD spectroscopic study of the secondary structure of partly adenylylated glutamine synthetase (GS) of the bacterium Azospirillum brasilense showed both the native and cation-free (EDTA-treated) enzyme to be highly structured (58 and 49% as alpha-helices, 10 and 20% as beta-structure, respectively). Mg(2+), Mn(2+), or Co(2+), when added to the native GS, had little effect on its CD spectrum, whereas their effects on the cation-free GS were more pronounced. Emission ((57)Co) Mössbauer spectroscopic (EMS) study of (57)Co(2+)-doped cation-free GS in frozen solution and in the dried state gave similar spectra and Mössbauer parameters for the corresponding spectral components, reflecting the ability of the Co(2+)-enzyme complex to retain its properties upon drying. The EMS data show that (a) A. brasilense GS has 2 cation-binding sites per active center and (b) one site has a higher affinity to Co(2+) than the other, in line with the data on other bacterial GSs.
Assuntos
Azospirillum brasilense/enzimologia , Glutamato-Amônia Ligase/química , Azospirillum/enzimologia , Cátions , Dicroísmo Circular , Cobalto/química , Magnésio/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Espectrofotometria , TemperaturaRESUMO
In this work, we report the detection of aromatic amino acid aminotransferase (AAT) activity from cell-free crude extracts of nine strains of N(2)-fixing bacteria from three genera. Using tyrosine as substrate, AAT activity ranged in specific activity from 0.084 to 0.404 micromol min(-1)mg(-1). When analyzed under non-denaturating PAGE conditions; and using tryptophan, phenylalanine, tyrosine, and histidine as substrates Pseudomonas stutzeri A15 showed three isoforms with molecular mass of 46, 68 and 86 kDa, respectively; Azospirillum strains displayed two isoforms which molecular mass ranged from 44 to 66 kDa and Gluconacetobacter strains revealed one enzyme, which molecular mass was estimated to be much more higher than those of Azospirillum and P. stutzeri strains. After SDS-PAGE, some AAT activity was lost, indicating a differential stability of proteins. All the strains tested produced IAA, especially with tryptophan as precursor. Azospirillum strains produced the highest concentrations of IAA (16.5-38 microg IAA/mg protein), whereas Gluconacetobacter and P. stutzeri strains produced lower concentrations of IAA ranging from 1 to 2.9 microg/mg protein in culture medium supplemented with tryptophan. The IAA production may enable bacteria promote a growth-promoting effect in plants, in addition to their nitrogen fixing ability.
Assuntos
Azospirillum/enzimologia , Gluconacetobacter/enzimologia , Ácidos Indolacéticos/metabolismo , Pseudomonas stutzeri/enzimologia , Transaminases/metabolismo , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Histidina/metabolismo , Ácidos Indolacéticos/farmacologia , Peso Molecular , Fixação de Nitrogênio , Fenilalanina/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Especificidade por Substrato , Transaminases/biossíntese , Triptofano/metabolismo , Tirosina/metabolismoRESUMO
Azospirillum lipoferum RG20, a nitrogen-fixing bacterium found in all kind of soils, was found to be naturally resistant to penicillins and cephalosporins. 6-beta-Bromopenicillanic acid, an irreversible inhibitor of serine-beta-lactamases, completely abolished this resistance. A beta-lactamase was purified 518-fold from a cell-free extract of A. lipoferum RG20. A single band on SDS-PAGE (apparent molecular mass 31000 Da) and on isoelectric focussing (pI9.35) was observed with the purified protein. The enzyme hydrolysed benzylpenicillin, ampicillin, cephalothin and cephaloridine with comparable k(cat) values and catalytic efficiencies. However, carbenicillin and cefotaxime were hydrolysed with significantly lower kinetic parameters and oxacillin was hydrolysed at a rate 100 times slower. The purified beta-lactamase was inhibited by clavulanic acid and sulbactam but not by EDTA or aztreonam. Its substrate and inhibitor profiles are consistent with those of the broad-spectrum beta-lactamases inhibited by clavulanic acid (group 2b of the Bush-Jacoby-Medeiros scheme). The effect of pH on k(cat) and K(m) values for benzylpenicillin hydrolysis was studied. The dependence of k(cat) on pH suggests that the enzyme-substrate (ES) complex must be in at least three protonation states: two with k(cat) values equal to 2800 and 1450 s(-1) and a third inactive one [pK(1(ES)) 4.7 and pK(2(ES)) 7.9]. Similarly, the dependence of k(cat)/K(m) on pH can be explained by postulating that the enzyme free form can be at least in three different protonation states: two of them with k(cat)/K(m) values equal to 2.7 x 10(6) and 3.7 x 10(8) M(-1) s(-1) and a third one unable to productively bind substrate. Interestingly, the dependence of k(cat)/K(m) on pH is consistent with positive cooperativity for proton binding to the enzyme free form [pK(1(E)) 8.5 and pK(2(E)) 7.2].