Bacterial chromosomes are highly dynamic, continuously changing with respect to gene content and size via a number of processes, including deletions that result in gene loss. How deletions form and at what rates has been the focus of this thesis. In paper II we investigated how chromosomal location affects chromosomal deletion rates in S. typhimurium. Deletion rates varied more than 100-fold between different chromosomal locations and some large deletions significantly increased the exponential growth rate of the cells. Our results suggest that the chromosome is heterogeneous with respect to deletion rates and that deletions may be genetically fixed as a consequence of natural selection rather than by drift or mutational biases. In paper I we examined in a laboratory setting how rapidly reductive evolution, i.e. gene loss, could occur. Using a serial passage approach, we showed that extensive genome reduction potentially could occur on a very short evolutionary time scale. For most deletions we observed little or no homology at the deletion endpoints, indicating that spontaneous deletions often form through a RecA independent process. In paper III we examined further how large spontaneous deletions form and, unexpectedly, showed that 90% of all spontaneous chromosomal deletions required error-prone translesion DNA polymerases for their formation. We propose that the translesion polymerases stimulate deletion formation by allowing extension of misaligned single-strand DNA ends. In paper IV we investigated how the translesion DNA polymerase Pol IV, RpoS and different types of stresses affect mutation rates in bacteria. Derepression of the LexA regulon caused a small to moderate increase in mutation rates that was fully dependent on functional endonucleases but only partly dependent on translesion DNA polymerases. RpoS levels and growth stresses had only minor effects on mutation rates. Thus, mutation rates appear very robust and are only weakly affected by growth conditions and induction of translesion polymerases and RpoS.

Orientador: Taís Maria Bauab / Resumo: Apesar do grande avanço tecnológico direcionado à indústria farmacêutica, os micro-organismos continuam adquirindo cada vez mais resistência aos fármacos convencionalmente utilizados e, pesquisadores de diversas áreas buscam novas fontes antimicrobianas de origem natural, a exemplo das plantas medicinais. Dentre as infecções fúngicas, destacam-se aquelas causadas por leveduras do gênero Candida sp. a exemplo da a candidíase vulvovaginal (CVV). A planta Astronium urundeuva (Allemao) Engl. apresenta uma série de propriedades biológicas, incluindo a atividade antifúngica. Este trabalho teve como objetivo aprofundar os estudos direcionados a atividade antifúngica dessa planta medicinal de modo a investigar as substâncias responsáveis por esta propriedade biológica através do fracionamento biomonitorado. Dentre os componentes majoritários identificados no extrato (ácido gálico, galato de metila e galato de etila), pode-se observar que as substâncias galato de etila e galato de metila são responsáveis por grande parte da atividade antifúngica contra a espécie C. glabrata enquanto que a atividade contra a espécie C. albicans parece estar relacionada a um efeito sinérgico dos componentes do extrato, incluindo os minoritários. Os resultados do ensaio in vivo confirmaram a atividade antifúngica exibida pelo extrato, mas, além disso, mostrou uma melhor atividade do extrato incorporado na microemulsão (ME), confirmando o efeito fungicida in vitro apresentado por este. Os ensaios de citot... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Despite the great technological advances directed to the pharmaceutical industry, microorganisms continue to acquire more and more resistance to the drugs conventionally used in the therapy, and researchers from several areas keep searching new natural antimicrobial sources, such as medicinal plants. Among fungal infections, we highlight those caused by yeasts from the genus Candida sp., for example vulvovaginal candidiasis (VVC). Astronium urundeuva (Allemao) Engl. plant has various biological properties, including antifungal activity. This work aimed to improve the researches directed to the antifungal activity of this medicinal plant in order to investigate the substances responsible for this biological property using bioassay-guided fractionation technique. Among the major components identified in the extract (gallic acid, methyl gallate and ethyl gallate), we could observe that the substances ethyl gallate and methyl gallate are responsible a large part of the antifungal activity against C. glabrata species whereas the activity against C. albicans species seems to be related to a synergistic effect of the components of the extract, including the minority ones. The in vivo results confirmed the antifungal activity exhibited by the extract, but, in addition, showed a better activity of the extract loaded into a microemulsion (ME), confirming the in vitro fungicidal effect presented by it. The cytotoxicity assays (IC50) performed for VERO, MRC5 and HaCaT cell lines showed t... (Complete abstract click electronic access below) / Doutor

TEM and SHV β-lactamases are the most prevalent β-lactamases among Gram-negative bacteria. The introduction and widespread use of expanded-spectrum antibiotics, particularly third generation cephalosporins, has led to the evolution of bacterial strains expressing extended spectrum β-lactamases (ESBLs). ESBLs emerge by genetic point mutation from non-extended spectrum precursors. It was found that multiple β-lactamase families within single isolates complicate the process of detecting the resistance status of isolate using non-quantitative DNA diagnostic methods. Preliminary phenotypic characterisation of probable β-lactamase enzyme family types present in 100 isolates from the Asia-Pacific and South African locales showed that single isolates frequently contained multiple β-lactamase families. SHV, TEM, AMPC and CTX-M β-lactamase families were detected in these isolates using PCR detection methods. Ninety-eight percent of all isolates tested contained as least one β-lactamase gene, with up to four to β-lactamase gene families found to co-exist in single isolates. Kinetic PCR methods for interrogating the polymorphic sites at blaSHV codons 238 & 240 and blaTEM codons 164, 238, 240 as well as promoter polymorphism were developed. A high proportion of blaSHV 238 and 240 mutant alleles was found to correlate with cefotaxime, ceftazidime and aztreonam resistance levels. In an attempt to understand the molecular basis for the co-existence of multiple blaSHV alleles within single isolates, the blaSHV promoter region was cloned from one ESBL expressing isolate. Experimental results showed that blaSHV can exist downstream of two different promoters within a single isolate. Both promoters have previously been reported, and differ by the presence or absence of IS26, which results in a change in the transcription initiation site. The blaSHV gene copy numbers in cis with the different promoters were measured, and it was found that the copy number of the IS26::blaSHV promoter was positively correlated with resistance levels. Cloning and analysis of PCR products showed that different blaSHV variants existed in cis with promoters in individual isolates. However, mutant genes were more abundant downstream of the IS26 promoter. There were no ESBL+ isolates without this promoter. It was concluded that blaSHV in cis with the IS26 promoter is located on an amplifiable replicon, and the presence of the IS26 insertion may facilitate the acquisition of an ESBL+ phenotype. To further confirm the role of IS26 in resistance acquisition, ESBL negative isolates were subjected to serial passage in vitro evolution experiments and fluctuation assays. Results confirm that the insertion of the IS26 element upstream of blaSHV is positively correlated with the ability to exhibit an ESBL phenotype, when such isolates also contain the critical G238S substitution. It was also found that IS26 can catalyse the duplication and mobilisation of blaSHV within an isolate. Fluctuation experiments have shown that the frequency at which such genomic events occur resulting in ESBL phenotypes is extremely low and requires many generations of selection under sub-lethal conditions. A survey of a geographically diverse set of isolates has shown that IS26-blaSHV was found in all of the bacterial populations surveyed. However, it does not appear to be exclusively associated with SHV-mediated ESBL production.

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