The response of aminopeptidases of Phaseolus vulgaris to drought depends on the developmental stage of the leaves.

Aminopeptidases, together with other proteases, execute and regulate the total and specifically limited protein breakdown involved in plant physiology, raising the possibility of their involvement in response to drought. We have identified, in leaves of Phaseolus vulgaris L., five aminopeptidases (E.C.3.4.11) whose levels of activity changed when three week old plants were subjected to drought. First, second and third trifoliate leaves were investigated separately. The aminopeptidases were first identified then isolated using ion exchange chromatography of leaf extracts. Three, named PvAP1, PvAP2 and PvAP4, are metallo aminopeptidases with broad substrate specificity, active against substrates conjugated to alanine and lysine. Two others, PvAP3 and PvAP5, are apparently serine aminopeptidases, the former active against substrates conjugated to phenylalanine and leucine, and the latter characterised by narrow specificity against substrates conjugated to phenylalanine. Their apparent molecular weights range from ∼37 kDa to ∼80 kDa. Levels of activity of individual aminopeptidases in both watered and drought stressed plants are shown to depend on the age of leaves. In watered plants they were generally highest in young, and very low in older, trifoliate leaves, the latter with the exception of PvAP5. Drought initiated an almost general increase of their activities, although to different extents, with the exception of PvAP4 and PvAP5 in young trifoliate leaves. Thus, in such studies it is necessary to investigate the effects of drought separately in leaves of different ages in order to elucidate the different complex and probably specific roles of aminopeptidases in the response of common bean to drought.

The Escherichia coli uropathogenic-specific-protein-associated immunity protein 3 (Imu3) has nucleic acid -binding activity.

BACKGROUND: The Escherichia coli uropathogenic-specific protein (Usp) is a bacteriocin-like genotoxin, active against mammalian cells and associated with E. coli strains that provoke pyelonephritis, prostatitis and bacteraemia. Usp is encoded by a small pathogenicity island with three downstream small open reading frames (Imu1-3) that are believed to provide immunity to the producer. To prevent host suicide, colicins, bacteriocins of E. coli, form tight complexes with their cognate immunity proteins. Colicin - immunity protein complexes are among the strongest protein complexes known. Here, the Usp associated immunity protein 3 (Imu3) was partially characterized to gain insight into its role and mechanism of activity. RESULTS: Isolation and partial characterisation of the Usp-associated immunity protein-3 (Imu3) revealed that, while Usp and Imu3 do not form a high affinity complex, Imu3 exhibits DNA and RNA binding activity. Imu3 was also shown to protect DNA against degradation by colicin E7. CONCLUSIONS: Our data infer that nonspecific DNA binding of the Imu3 immunity protein, prevents suicide of E. coli producing the genotoxin Usp.

Differential inhibition of LINE1 and LINE2 retrotransposition by vertebrate AID/APOBEC proteins.

BACKGROUND: The role of AID/APOBEC proteins in the mammalian immune response against retroviruses and retrotransposons is well established. G to A hypermutations, the hallmark of their cytidine deaminase activity, are present in several mammalian retrotransposons. However, the role of AID/APOBEC proteins in non-mammalian retroelement restriction is not completely understood. RESULTS: Here we provide the first evidence of anti-retroelement activity of a reptilian APOBEC protein. The green anole lizard A1 protein displayed potent DNA mutator activity and inhibited ex vivo retrotransposition of LINE1 and LINE2 ORF1 protein encoding elements, displaying a mechanism of action similar to that of the human A1 protein. In contrast, the human A3 proteins did not require ORF1 protein to inhibit LINE retrotransposition, suggesting a differential mechanism of anti-LINE action of A1 proteins, which emerged in amniotes, and A3 proteins, exclusive to placental mammals. In accordance, genomic analyses demonstrate differential G to A DNA editing of LINE retrotransposons in the lizard genome, which is also the first evidence for G to A DNA editing in non-mammalian genomes. CONCLUSION: Our data suggest that vertebrate APOBEC proteins differentially inhibit the retrotransposition of LINE elements and that the anti-retroelement activity of APOBEC proteins predates mammals.

Escherichia coli uropathogenic-specific protein, Usp, is a bacteriocin-like genotoxin.

BACKGROUND: Bacterial genotoxins provoke DNA damage and carcinogenesis. The Escherichia coli uropathogenic-specific protein gene, usp, and its linked genes, imu1-3, are associated with strains from pyelonephritis, prostatitis, and bacteremia of urinary tract origin. While the Usp C-terminal domain exhibits similarity with DNase-like colicins and pyocins, its role and mechanisms of action, as well as those of the 3 associated proteins, is unknown. METHODS: We isolated Usp and Imu1-3 and examined their activity on plasmid DNA, human umbilical vein endothelial cells, and human embryonic kidney cells (cell line HEK293). The affect of Usp and Imu1-3 was assessed by MTT and Comet assays, infection assays, caspase 3/7 activity, fluorescently labeled actin staining, and Western blotting. RESULTS: Usp possesses DNase activity and, particularly when coapplied with Imu2, exhibits genotoxic activity in mammalian cells. Infection assays demonstrated that E. coli usp(+) imu1-3(+) affects the viability of mammalian cells, induces increased caspase 3/7 activity, and perturbs cell cytoskeleton structure. CONCLUSIONS: Usp is a novel E. coli genotoxin active against mammalian cells. Optimal in vivo activity of Usp requires Imu2. Infection with E. coli usp(+) imu1-3(+) induces a response characteristic of apoptosis.

Characterization of two novel subtilases from common bean (Phaseolus vulgaris L.) and their responses to drought.

Protein breakdown by proteases is basic to the plant response to abiotic stresses such as drought. A large number of genes encoding proteases or putative proteases exist in plants. Only a few of those involved in the response to drought have been characterized, and their regulation is poorly understood. We have identified two new subtilases from leaves of Phaseolus vulgaris L. cultivar Zorin, PvSLP1 and PvSLP2. PvSLP1 was identified at the gene level, using primers based on the gene sequence of the putative drought induced serine protease from Arachis hypogaea L. In P. vulgaris, expression of the PvSLP1 transcript did not change on water withdrawal. PvSLP2 was isolated and characterized at the protein level, together with complete gene and cDNA sequences. The deduced amino acid sequences of both PvSLP1 and PvSLP2 are characteristic of plant subtilases of the S8 family of clan SB. PvSLP2 shows 33% sequence identity to PvSLP1. Expression of the PvSLP2 transcript did not change on withdrawal of water, but its proteolytic activity in leaves increased, depending on the age and position of the leaf. In addition, the level of activity in senescent leaves of well watered plants was higher than in mature or young leaves. These results, together with the fact that PvSLP2 cleaves peptide bonds following an Arg residue, point to regulation of PvSLP2 subtilase activity at translational and/or post-translational levels and suggest a specific role in the response to drought and senescence.

Escherichia coli bacteriocins: antimicrobial efficacy and prevalence among isolates from patients with bacteraemia.

Bacteriocins are antimicrobial peptides generally active against bacteria closely related to the producer. Escherichia coli produces two types of bacteriocins, colicins and microcins. The in vitro efficacy of isolated colicins E1, E6, E7, K and M, was assessed against Escherichia coli strains from patients with bacteraemia of urinary tract origin. Colicin E7 was most effective, as only 13% of the tested strains were resistant. On the other hand, 32%, 33%, 43% and 53% of the tested strains exhibited resistance to colicins E6, K, M and E1. Moreover, the inhibitory activity of individual colicins E1, E6, E7, K and M and combinations of colicins K, M, E7 and E1, E6, E7, K, M were followed in liquid broth for 24 hours. Resistance against individual colicins developed after 9 hours of treatment. On the contrary, resistance development against the combined action of 5 colicins was not observed. One hundred and five E. coli strains from patients with bacteraemia were screened by PCR for the presence of 5 colicins and 7 microcins. Sixty-six percent of the strains encoded at least one bacteriocin, 43% one or more colicins, and 54% one or more microcins. Microcins were found to co-occur with toxins, siderophores, adhesins and with the Toll/Interleukin-1 receptor domain-containing protein involved in suppression of innate immunity, and were significantly more prevalent among strains from non-immunocompromised patients. In addition, microcins were highly prevalent among non-multidrug-resistant strains compared to multidrug-resistant strains. Our results indicate that microcins contribute to virulence of E. coli instigating bacteraemia of urinary tract origin.

Protease inhibitors clitocypin and macrocypin are differentially expressed within basidiomycete fruiting bodies.

Clitocypin and macrocypin are cysteine protease inhibitors of the mycocypin family which is unique to basidiomycetes. We have established that Clitocybe nebularis and Macrolepiota procera each contain genes for both macrocypin and clitocypin. Both are expressed in M. procera but only clitocypin in C. nebularis. Further analysis of mycocypin expression at the mRNA and protein levels in mature fruiting bodies of M. procera revealed that clitocypin is expressed evenly throughout the fruiting body, while the level of expression of macrocypins varies, and, at the protein level, is much higher in the veil fragments and the ring. The expression patterns of various mycocypins were determined in Coprinopsis cinerea, using promoters linked to a reporter gene. The expression profile of the clitocypin promoter was similar to that of the constitutive promoter gpdII from Agaricus bisporus, while that of the macrocypin 4 promoter was limited to the outer edges of the fruiting body throughout development. In addition, the activity of the macrocypin 3 promoter was different, indicating different regulation of expression for different macrocypin genes. The complex, tissue specific expression patterns for mycocypin genes suggest different biological roles for the products, either in regulation of endogenous proteases or in defense against pathogens or predators.

A quantitative technique for determining proteases and their substrate specificities and pH optima in crude enzyme extracts.

A zymography technique based on native polyacrylamide gel electrophoresis (PAGE) has been devised, which enables the substrate specificities, content and pH profiles of proteolytic enzymes to be determined in an unfractionated tissue extract. Enzymes were visualized by exogenous application of small molecule substrates that fluoresce when hydrolyzed. The linearity of response, treatment of background fluorescence, and effects of diffusion of substrate and enzyme were taken into account. Based on these studies, successive application of different substrates on the same gel has enabled the presence and specificity of individual enzymes to be determined. Differences in the concentrations and profiles of enzymes, resulting from environmental factors or ontogeny of the organism, can be assessed from crude extracts on a single gel. The technique was applied to aminopeptidases and peptidases in crude Phaseolus vulgaris leaf extracts. One enzyme active against Ala-AMC (7-amino-4-methylcoumarin), one enzyme active against Z-Arg-AMC, several enzymes active against Leu-AMC, and (for the first time in plants) several enzymes active against Phe-AMC were identified. The technique is very sensitive, and microgram quantities of total protein led to picomoles of liberated AMC, with a linear response over a 32-fold range of concentration. The experimental procedure, including electrophoresis, is rapid, taking approximately 1 h.

Prevalence of ColE1-like plasmids and colicin K production among uropathogenic Escherichia coli strains and quantification of inhibitory activity of colicin K.

Colicin K exhibited pronounced inhibitory activity against uropathogenic Escherichia coli (UPEC) strains. Low prevalence of colicin K production and a relatively high prevalence of ColE1-like plasmids were determined among 215 UPEC strains from Slovenia. Sequencing of the colicin K-encoding pColK-K235 revealed a mosaic structure and the presence of the insertion sequence IS2.