Characterisation of plasmids conferring ampicillin resistance in South African isolates of haemophilus ducreyi

A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science (medicine).' Johannesburg 1996Fifty-two strains of Haemophilus ducreyi from various geographic regions of southern Africa (Botswana, Lesotho, Namibia, Gauteng, Natal and Transkei) isolated between 1988 to 1994 were tested for susceptibilities to five antimicrobial agents and characterized according to their plasmid content and ampicillin- resistance genes.IT201

Functional genomic analysis of Haemophilus influenzae and application to the study of competence and transformation.

During the progression of this study, hundreds of additional bacterial genomes were sequenced, techniques have evolved, and novel approaches were developed to aid the field of functional and comparative genomics. Some of these techniques were used in this study to identify three novel competence-regulated operons in H. influenzae. The techniques included the use of advanced computer programs and algorithms to assist in predicting protein functions and to facilitate a comparative genomic analysis of H. influenzae with other species of the Pasteurellaceae family. Quantitative PCR was employed to examine the expression of putative transformation-related genes. Finally, PCR-mediated mutagenesis was used in a directed approach to generate mutations in the newly discovered competence-regulated operons to assess their involvement in uptake and transformation of exogenous DNA.The publication of the complete genomic sequence of Haemophilus influenzae Rd KW20 in 1995 was a truly monumental event in molecular biology. For the first time, all of the potential genes of an independent-living organism were known and awaiting functional characterization. This event required the development of fundamentally different methodologies to elucidate gene functions, with systematic global approaches becoming much more feasible. This study describes the development of a transposon-based mutagenesis strategy to facilitate a high-throughput functional analysis of the H. influenzae genome. Mutants created using this strategy were screened in a highly-parallel assay to identify genes mediating transformation in this organism. Additionally, analysis of the transposon insertion sites generated during this study identified a previously unrecognized Tn5 insertion bias

Unveiling electrotransformation of Moraxella catarrhalis as a process of natural transformation

The human respiratory tract pathogen Moraxella catarrhalis is a naturally competent microorganism. However, electrotransformation has long been used to introduce foreign DNA into this organism. This study demonstrated that electrotransformants obtained with linear or circular nonreplicating plasmid DNA originated exclusively from natural transformation processes taking place during the recovery phase after the application of current. Only replicating plasmid DNA could be introduced into M. catarrhalis by electrotransformation, in a type IV pilus-independent manner. Electrotransformation with homologous genomic DNA indicated that restriction of double-stranded DNA was independent of type III restriction-methylation systems. Nontransformability of M. catarrhalis by electrotransformation was observed using double- as well as single-stranded DNA. In addition, the study showed that natural competence is a very constant feature of M. catarrhali

Natural transformation and recombination in Helicobacter pylori

Bacteriën kennen geen geslachtelijke voortplanting, ze hebben altijd één “ouder” in plaats van twee. Ze kunnen dus tijdens de voortplanting niet kruisen. Om toch erfelijke eigenschappen te kunnen uitwisselen hebben ze andere methoden. De maagbacterie Helicobacter pylori kan dit bijvoorbeeld doen door DNA dat in de omgeving aanwezig is (“vrij DNA”) op te nemen, een proces dat transformatie heet. Dit vrije DNA kan afkomstig zijn van een soortgenoot met net iets andere eigenschappen, en deze eigenschappen gaan dan over op de transformerende bacterie. Het doel van dit promotie onderzoek was ten eerste het in kaart brengen van het systeem dat het DNA over de celwand van H. pylori heen kan transporteren, en ten tweede het effect van transformatie op de soort H. pylori te onderzoeken. In hoofdstuk twee worden daarom twee H. pylori-stammen beschreven die langere tijd samen in één maag hebben geleefd. Onderzoek aan deze stammen maakte duidelijk dat er verschillende stukken DNA van de ene op de andere stam waren overgegaan. Het grootste stuk DNA dat in één keer van de ene naar de andere stam is gegaan moet tenminste 1.700 baseparen lang geweest zijn, voldoende om minimaal één intact gen over te dragen. In hoofdstuk 3 wordt in het genoom van H. pylori gezocht naar genen die homologie vertonen met (=lijken op) genen die in andere bacteriesoorten een rol spelen bij transformatie. Dit levert een aantal genen op. Proeven met H. pylori stammen (“mutanten”) waarin deze genen kapot gemaakt zijn, levert één mutant op die niet meer kan transformeren. Hiermee is aangetoond dat het betreffende gen, dprA genaamd, noodzakelijk is voor H. pylori om te transformeren. Onderzoek aan genomen van andere bacteriën maakt duidelijk dat vrijwel iedere bacteriesoort een soortgelijk gen heeft, maar wat het gen precies doet is onbekend. In hoofdstuk 4 wordt de rol van de dprA genfamilie in meer detail onderzocht. Hierbij wordt ten eerste bewezen dat een dprA van de bacteriesoort Escherichia coli de rol van dprA in de soort Haemophilus influenzae kan overnemen, en dat dprA genen in verschillende soorten dus eenzelfde functie hebben. Ondanks verdere proeven aan dprA blijft de exacte functie van dprA onbekend. In hoofdstuk 5 wordt opnieuw gezocht naar genen die nodig zijn voor transformatie. In tegenstelling tot hoofdstuk 3 niet door te kijken naar genen die lijken op bekende ‘transformatiegenen’ uit andere bacteriesoorten, maar door willekeurig genen in H. pylori bacteriën uit te schakelen en te kijken welke uitschakeling leidt tot een mutant die niet meer in staat is tot transformatie. Dit levert opnieuw een transformatiegen op: comH. In tegenstelling tot dprA hebben andere bacteriesoorten geen op comH gelijkende genen: comH is uniek voor H. pylori. Het gen codeert waarschijnlijk voor een eiwit dat aan de buitenzijde of in de celwand van de bacterie is gelegen. Dit suggereert dat het betrokken is bij het proces van DNA transport door de celwand. In hoofdstuk 6 wordt het fenomeen ‘palindrome avoidance’ onderzocht. Palindrome avoidance betekent dat palindromische DNA-sequenties in bacteriën minder vaak voorkomen dan je zou verwachten op grond van kansberekening. De reden hiervoor is onbekend, maar één van de mogelijke verklaringen is gelegen in enzymen die DNA kapotknippen: deze knippen meestal in één specifieke sequentie en dat is vrijwel altijd een palindromische sequentie. Omdat het knippen van DNA potentiëel dodelijk is voor de cel, zou dat een reden kunnen zijn waarom palindromen erg weinig voorkomen. Om deze hypothese te testen zijn twee H. pylori stammen, die verschillende DNA-knippende eiwitten bevatten, onderzocht op palindrome avoidance. Er was echter geen correlatie tussen de aanwezigheid van enerzijds een DNA-knippend enzym en anderzijds de palindrome avoidance van het palindroom dat hierdoor geknipt wordt. De hypothese is dus waarschijnlijk onjuist in H. pylori.Vandenbroucke-Grauls, C.M.J.E. [Promotor]Bitter, W. [Copromotor]Kusters, J.G. [Copromotor

Characterization of genes conferring v factor independence in haemophilus parainfluenzae and haemophilus ducreyi

A thesis submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements of Doctor of Philosophy Johannesburg 1994Haemophilus influenzae and Haemophilus parainfluenzae are obligate human parasites that form part of the flora of the mucous membranes. They are normally present in the mouth and upper respiratory tract of healthy individuals. H. influenzae is known as a major pathogen in children while H. parainfluenzae is an opportunistic pathogen which is also found in the urogenital tract. H. ducreyi, however, has only been isolated from genital ulcers in patients suffering from chancroid. The various species of the genus Haemophilus are characterised by their requirements for two growth factors, X factor or haemin and V factor or nicotinamide adenine dinucleotide (NAD).IT201

Etude de la transformation plasmidique naturelle d'Escherichia coli et de ses relations éventuelles avec la compétence programmée pour la transformation génétique et la compétence dite nutritionnelle

Bien que la bactérie Escherichia coli ne soit pas connue pour être naturellement transformable, mon travail de thèse montre que l'on peut obtenir des transformants plasmidiques spontanément sur boîte. Cette transformation n'est pas induite par les cations divalents au contraire de la transformation ‘artificielle chimique’ (Sun et al., FEMS Microbiol. Lett. 2006. 265: 249–255). Les bactéries transformables utilisent une machinerie protéique transmembranaire, évolutivement conservée, pour internaliser l'ADN exogène sous forme simple brin. E.coli possède l'ensemble des gènes codant pour cette machinerie. J'ai inactivé les gènes clés de cette machinerie, dont hofQ (canal transmembranaire externe) et ycaI (canal transmembranaire interne), et observé qu'aucun de ces mutants n'est affecté pour la transformation sur boîte. L'ADN plasmidique ne pénètre donc pas via la machinerie de transformation, mais plutôt sous forme double brin ce que suggèrent les courbes de réponse à la concentration d'ADN (Sun et al., J. Bacteriol. 2009. 191: 713-719). Le troisième volet de ma thèse a consisté à tenter de mieux caractériser un phénomène appelé 'compétence nutritionnelle', appellation qui désigne la capacité d'utiliser l'ADN comme source de carbone. Pour établir si différents gènes de la machinerie de transformation étaient impliqués, j'ai cherché à reproduire les expériences publiées de croissance de la souche ZK126 sur milieu minimum M63 contenant de l'ADN. Malgré de nombreuses tentatives et contrôles, je n'ai pas pu reproduire ces expériences, ce qui m'a amené à clore mon mémoire de thèse par une discussion critique des données publiées relatives à la compétence nutritionnelle de E. coli.While Escherichia coli is not considered to belong to naturally transformable species, I established a transformation system allowing spontaneous plasmid transformation on plate (Sun et al., FEMS Microbiol. Lett. 2006. 265: 249–255). Transformation is not induced by divalent cations in contrast to chemically-induced 'artificial transformation' (Sun et al., J. Bacteriol. 2009. 191: 713-719). As DNA uptake in naturally transformable bacteria relies on a conserved multiprotein machinery and the E. coli genome contains all genes encoding this machinery, I investigated whether key genes are required for plasmid transformation. None of the mutants I constructed, including hofQ and ycaI which encode putative outer and inner membrane channel proteins were affected, indicating that plasmid DNA is not taken up via the transformation machinery. We proposed that plasmid DNA instead enters the cytoplasm as double stranded material as suggested by response curves to DNA concentration (Sun et al., J. Bacteriol. 2009. Ibid.). In the last part of my thesis, I reinvestigated so-called ‘nutritional competence’ of E. coli. Previously work reported that E. coli cells are able to use DNA as the sole carbon source. I wished to establish whether this phenomenon relies on the above-mentioned DNA uptake machinery. I therefore tried to reproduce the published growth experiments of E. coli ZK126 on M63 minimal medium with DNA. Despite numerous attempts and controls, I could not observe any growth. This failure to reproduce published observations led me to conclude my thesis by an in-depth discussion of the three articles dedicated to so-called nutritional competence of E. coli

Molecular cloning, nucleotide sequencing, and construction of a Pasteurella haemolytica biotype A serotype 2 aroA mutant

http://www.worldcat.org/oclc/3846996

Analysis of plasmid DNA from Pasteurella haemolytica and construction of cloning vectors

Pasteurella haemolytica, a Gram-negative bacterium, is the principal causative agent of pneumonic pasteurellosis in cattle and sheep. The presence of plasmids in various isolates of this species and their relationship to antibiotic resistance and to the health status of the source animal were investigated. Plasmids conferring resistance to ampicillin were analyzed and the plasmid-encoded p-lactamase enzymes characterized. A suitable plasmid was identified for the construction of shuttle cloning vectors for P. haemolytica. Thirty five typable and untypable isolates of P. haemolytica from cattle or sheep were screened for the presence of plasmids and for resistance to a range of antibiotics. Eight strains (four of serotype Al, three of serotype A2 and one untypable) contained plasmid DNA. Isolates of the same serotype had similar plasmid profiles, which were different from those of the other serotypes. All but one of the plasmid-bearing strains were isolated from pneumonic, or from animals in contact with pneumonic, cattle or sheep. In A2 and untypable strains, there was no obvious correlation between antibiotic resistance and the presence of a specific plasmid. In contrast, all plasmid-bearing Al strains exhibited ampicillin resistance, which was shown by transfer studies (transformation and conjugation) to be plasmid-mediated. Plasmid DNA prepared from ampicillin-resistant E. coli transformants was not routinely detected on ethidium-bromide-stained agarose gels, but could be amplified to detectable levels by treatment of cultures with chloramphenicol or by modifying the growth conditions. Terrific broth or broth supplemented with 1% yeast nitrogen base was found to be the best growth medium for plasmid amplification. All Apft plasmids had a similar size (~4. 3 kb), and one of them (pPH843) was highly amplifiable and more stable in E. coll compared to the other plasmids (pPH2, pPH33 and pPH821). An improved method was developed for the large-scale purification of covalently closed circular plasmid DNA molecules over a wide size range. The protocol used an alkaline-lysis procedure followed by acid-phenol extraction and included several modifications to previously reported methods. Two principal modifications were the replacement of NaCl by MgCl in the extraction buffer and vortexing instead of shaking the crude DNA suspensions to more efficiently remove chromosomal and other non-CCC plasmid DNA and to improve yield of purified CCC DNA forms. The Apft plasmids from P, haemolytica were identical by restriction enzyme analysis. Restriction analysis and hybridization data indicated that these plasmids were closely related to the prototype ROB-1 p-lactamase-encoding plasmid, originally isolated from Haemophilus influenzae, but a part of the DNA sequence <~0. 7 kb) from the Haemophilus plasmid was not present in the Pasteurella plasmid. This suggested that one plasmid had been derived from the other during the course of evolution. From substrate profiles and isoelectric focusing data, the p-lactamases encoded by the P. haemolytica plasmids were found to be indistinguishable from the ROB-1 0- lactamase. A restriction map of plasmid pPH843 was constructed and the putative Apft, oriV, and mob regions of the plasmid were located by deletion and fusion experiments. The plasmid was converted into a series of possible cloning vectors (pAKAlb, pAKA15-l, pAKA16, pAKA16-l and pAKA17) by insertion at different points in the plasmid of the lacZ alpha-peptide-coding gene and a multiple cloning site (for insertional inactivation of beta-galactosidase activity) from the E. coli vector pIC20H and an IncP mobilization function from plasmid RK2. Transconjugants obtained by transfer of the constructs from E, coli to P. haemolytlca were found to be unstable on subculture, although they were stable upon transfer to another E, coli strain. This indicated that P. haemolytlca might have a restriction/modification system which affected plasmid DNA previously propagated in E. coli