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The bacterial type six secretion system (T6SS) is an injectisome responsible for the translocation of effector molecules directly into host cells or competing bacteria. The system is widely distributed among proteobacteria and is found in both clinically relevant strains as well as environmental stains and represents an important system for the study of both microbial ecology and virulence. The apparatus itself is believed to have arisen from a combination of genes from bacteria and bacteriophage due to seqeuence and structural identity between T6SS components and structural bacteriophage proteins. The current model of the T6SS apparatus consists essentially of an inverted phage body that is attached to the donor cell membrane complex. The phage-like structure can contract and force a sharp needle point complex along with effector proteins into the target cell. The phage derived components have received a considerable amount of attention and the mode of assembly is relatively well understood. However, little detailed information on the assembly and function of the membrane embedded complex is available. The first major goal of this thesis was to structurally characterize the proteins of the membrane embedded complex of the type six secretion system. The structures of IglE and TssL from Francisella sp. were solved and represent a platform for further characterization of the T6SS assembly and function. The periplasmic domain of a TssL homologue from P. aeruginosa was also solved and this structure represents a subset of evolved TssL proteins that bind peptidoglycan through an unknown mechanism. Biochemical and structural analysis probed this system but came short of a definitive model for peptidoglycan binding. However, the data collected from this study will further the field of peptidoglycan binding modules and help to characterise differences among T6SSs. The translocated proteins of the T6SS are often bactericidal and attack the peptidoglycan, lipid bilayer or DNA of the target cell. However, one secreted substrate, Tse2 from Pseudomonas aeruginosa is targeted to other neighbouring cells of the same species. This toxin shares no sequence identity with any known protein but has been shown to be toxic to not only bacteria but also yeast and mammalian cells. The structure of the complex between Tse2 and its immunity protein was solved and led to two interrelated discoveries. The first was that the molecular details behind the immunity protein inhibiting Tse2 where it binds directly to the active site. The second was that based on structural identity with ADP-ribosylating toxins, the active site of Tse2 was identified. These results carry the study of this protein forward significantly although the precise function of Tse2 remains unknown. This structure is the first co-structure of a cytotoxic T6SS substrate and has significant implications for the cell in terms of handling the toxin for delivery rather than self intoxication. / Graduate

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Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The moderately thermophilic (45 to 50DC), highly acidophilic (pH 1.5 to 2.5), chemolithoautotrophic Acidithiobaci/lus caldus strain "f' was isolated from a biooxidation process used to treat nickel ore concentrates. Trans-Alternating Field Electrophoresis (TAFE) analysis of total DNA from the At. caldus cells revealed two plasmids of approximately 14 and 45-kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404 which suggested that pTC-F14 was a broad host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open-reading frames, and a replicon organization like that of the broad host-range IncQ plasmids. Three of the open-reading frames encoded replication proteins with amino acid sequence identities similar to that of the IncQ-like plasmid pTF-FC2 (RepA, 81%; RepB, 78%; RepC, 74%). This high level of relatedness suggested that the two replicons had evolved from a common ancestor. Since closely related replicons are usually incompatible, the compatible replicons of pTC-F14 and pTFFC2 raised the question of how the replicons of the two sister plasmids had evolved such that they can now co-exist in the same host cell line. Further incompatibility testing with the IncQ-like plasmid pIEll08 and the IncQ prototype plasmid RSF10101R11621R300B determined that pTC-F14 was compatible with pIEI108, but incompatible with the IncQ prototype plasmid. It was found that the RepB and RepA replication proteins ofpTF-FC2 and pIEll08 were able to complement the pTC-FI4 orthologs only ifpTC-F14 RepC was present in trans. The RepC protein ofpTC-F14 was thus plasmid-template specific, while the RepA and RepB proteins were less plasmid-template specific. A five nucleotide possible iteron-discriminating region in the direct repeats of IncQ-like plasmid oriV regions has been identified (Tietze, E. (1998) Plasmid 39: 165-181). The iteron sequence ofpTC-F14 differs from pTF-FC2 and pIE 1108 by three nucleotides in this iteron-discriminating region. It was therefore proposed that co-evolution of the iterons and the RepC protein to a point where the RepC protein no longer recognizes the iteron sequence of a closely related sister plasmid is the mechanism by which replicons evolve to become compatible in the same host cell. The incompatibility determinant of the IncQ prototype plasmid RSFlOlOIR11621R300B was also sought, and subsequently localized to the region encoding the IncQ prototype plasmid's repAC genes. Interference with the initiation of pTC-F14 replication by the IncQ prototype plasmid was demonstrated by growth inhibition of a replication-deficient M13 bacteriophage into which oriVpTC-F14 had been cloned. Secondly, the IncQ prototype derivative pKE462 displaced a ColEloriVpTC- F14 construct in complementation assays, and a construct containing only the pTC-F14 repBAC genes similarly displaced the pKE462 plasmid. As the oriVRSFIOIO region was not incompatible with a pTC-F14 replicon, this suggested that it was not the oriV region which was expressing incompatibility, but the products of the IncQ prototype plasmid repAC genes. It is proposed that incompatibility between pTC-F14 and the IncQ prototype plasmid was the consequence of the repAC gene products binding to the iterons of the related rep licon, and that these products are unable to initiate replication. The compatible phenotypes expressed by members of the IncQ plasmid family indicates the inadequacy of using plasmid incompatibility as a classification system. Alignment of the amino acid sequences of the three replication protein orthologs clearly showed that the IncQ plasmid family was divided into two groups. To account for replication protein relatedness and the incompatibility phenotype expressed, it is now proposed that that members of the IncQ family be classified into subdivisions that reflect the different IncQ-like replicons identified in this study. Investigation of pTC-F14 replicon regulation identified a putative promoter sequence which is believed to regulate the initiation of a 5.l-5.7-kb polycistronic transcript that encodes all the replication proteins of the pTC-F14 replicon and the MobB and MobA proteins of the IncP-type mobilization module. The large polycistronic transcript appears to regulated by the RepB protein of the pTC-F14 replicon, and is not subject to cross-regulation by related IncQ plasmids. This suggested that the RepB primase function was not plasmid specific, but that its regulatory function was replicon specific. A second putative promoter sequence identified upstream of the pTC-F14 pasAB operon was, however, cross-regulated by the closely related pTF-FC2 plasmid. The pTC-F14 pas operon encodes two proteins with high amino acid sequence identity (PasA, 81 %; PasB, 72 %) to the plasmid addiction system ofpTF-FC2. This is the second time a plasmid addiction system of this type has been found on an IncQ-like plasmid. / AFRIKAANSE OPSOMMING: Die matig termofiliese (45 to 50°C), hoogs asidofiliese (pH 1.5 to 2.5), chemolitooutotrofiese Acidithiobaci/lus caldus ras "f' is geïsoleer vanaf 'n biooksiderende proses wat gebruik word om gekonsentreerde nikkel-erts te behandel. Trans- Afwisselende Veld Elektroforese (TAVE) analise van totale DNA vanaf die At. caldus selle, het twee plasmiede van ongeveer 14 en 45-kb. onthul. Die 14-kb plasmied, genaamd pTC-F14, is gekloneer en deur vervanging van die kloneringsvektor met 'n kanamisien weerstandsgeen is daar gewys dat hierdie plasmied in staat is tot outonome replikasie in Escherichia coli. Outonome replikasie is ook gedemonstreer in Pseudomonas putida en Agrobacterium tumefaciens LBA 4404 wat suggereer dat pTC-F14 'n wye gasheer-reeks plasmied is. Volgorde analise van die pTC-F14 replikon area het vyf oop leesrame onthul, en 'n replikon organisasie soortgelyk aan dié van die wye gasheer-reeks IncQ plasmiede. Drie van die oop leesrame kodeer vir replikasie proteïene met aminosuur volgordes ooreenstemmend met dié van die IncQ-tipe plasmied pTF-FC2 (RepA, 81%; RepB, 78%; RepC, 74%). Hierdie hoë vlak van verwantskap stel voor dat die twee replikons vanaf 'n gemeenskaplike voorouer ontwikkel het. Aangesien naby-verwante replikons gewoonlik onverenigbaar is, het die verenigbaarheid van die replikons van pTC-F14 en pTF-FC2 die vraag laat onstaan van hoe die replikons van twee susterplasmiede ontwikkel het, sodat hulle nou gelyktydig in dieselfde gasheer sellyn kan voortbestaan. Verdere onverenigbaarheid toetsing van die IncQ-tipe plasmied pIE1108 en die IncQ prototipe plasmied RSF10101R11621R300B, het bepaal dat pTCF14 verenigbaar is met pIE1108, maar onverenigbaar met die IncQ prototipe plasmied. Daar is gevind dat die RepB en RepA replikasie proteïene van pTF-FC2 en pIE1108 in staat was om die pTC-F14 ortoloë te komplementeer, slegs as pTC-F14 RepC in trans teenwoordig was. Die RepC proteïen van pTC-F14 is dus plasmiedtemplaat spesifiek, terwyl die RepA en RepB proteïene minder plasmied-templaat spesifiek is. 'n Moontlike iteron-onderskeidende vyf-nukleotied area in die direkte herhalings van die IncQ-tipe plasmied oril/ areas, is geïdentifiseer (Tietze, E. (1998) Plasmid 39: 165-181). Die iteron volgorde van pTC-F14 verskil van pTF-FC2 en pIEll08 met drie nukleotiedes in hierdie iteron-onderskeidende area. Om hierdie rede is daar voorgestel dat ko-evolusie van iterons en die RepC proteïen, tot by 'n punt waar die RepC proteïen nie meer die iteron volgorde van 'n naby-verwante susterplasmied herken nie, die meganisme is waardeur replikons ontwikkel om verenigbaar te word in dieselfde gasheersel. Die onverenigbaarheidsbepaler van die IneQ prototipe plasmied RSFIOIOIR11621R300B is ook ondersoek en gelokaliseer tot die area wat kodeer vir die IneQ prototipe plasmied se repAC gene. Inmenging met die inisiasie van pTC-F14 replikasie deur die IneQ prototipe plasmied is gedemonstreer deur groei vertraging van 'n replikasie-gebrekkige M13 bakteriofaag waarin die oriVpTC-F14 gekloneer is. Tweedens is die ColEl-oriVpTc-FI4 konstruk vervang deur die IneQ prototipe-afgeleide pKE462 in komplementasie proewe, en is die pKE462 plasmied op soortgelyke wyse vervang deur 'n konstruk wat slegs die pTC-F14 repBAC gene bevat. Aangesien die oriVRSF1010 area nie verenigbaar was met 'n pTC-F14 replikon nie, stel dit voor dat dit nie die oriV area is wat onverenigbaarheid uitdruk nie, maar die produkte van die IneQ prototipe plasmied se repAC gene. Dit is voorgestel dat onverenigbaarheid tussen pTC-F14 en die IneQ prototipe plasmied die gevolg is van die repAC geenprodukte wat bind aan die iterons van die verwante replikon en dat hierdie produkte nie in staat is om replikasie te inisieer nie. Die verenigbare fenotipes wat deur die lede van die IneQ plasmied familie uitgedruk word, dui aan op die ontoereikendheid van die gebruik van plasmied onverenigbaarheid as 'n klassifikasie sisteem. Vergelyking van die aminosuur volgordes van die drie replikasie proteïen ortoloë wys duidelik daarop dat die IneQ plasmied familie in twee groepe verdeel is. Om verantwoording te doen vir die replikasie proteïen verwantskap en die onverenigbare fenotipe wat uitgedruk is, word daar nou voorgestel dat die lede van die IneQ familie geklassifiseer word in subafdelings wat die verskillende IneQ-tipe replikons geïdentifiseer in hierdie studie, reflekteer. Ondersoek na die pTC-F14 replikon regulering het 'n moontlike promotor volgorde geïdentifiseer. Daar word gemeen dat hierdie promotor die inisiasie van 'n 5.l-5.7-kb polisistroniese transkrip reguleer, wat kodeer vir al die replikasie proteïene van die pTC-F14 replikon en die MobB en Mob A proteïene van die IneP-tipe mobilisasie module. Die groot polisistroniese transkrip blyk om gereguleer te word deur die RepB proteïen van die pTC-F14 replikon, en word nie gekruis-reguleer deur die IneQ plasmiede nie. Dit stel voor dat die RepB primase se funksie nie plasmiedspesifiek is nie, maar dat die reguleerbare funksie replikon-spesifiek is. 'n Tweede moontlike promotor volgorde wat stroom-op van die pTC-F14 pasAB operon geïdentifiseer is, is egter gekruis-reguleer deur die pTF-FC2 plasmied. Die pTC-F14 pas operon kodeer vir twee proteïene met hoë aminosuur volgorde verwantskappe (PasA, 81 %; PasB, 72 %) aan die plasmied-verslaafde sisteem van pTF-FC2. Dit is die tweede keer dat hierdie tipe plasmied-verslaafde sisteem in 'n IncQ-tipe plasmied gevind is.

published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Botany / Master / Master of Philosophy

published_or_final_version / Microbiology / Master / Master of Medical Sciences

No description available.