A pathogenicity locus of Pseudomonas syringae pv. syringae identified by Tn5 mutagenesis was investigated. The mutant strain PS9024 is attenuated for disease expression in its host, Phaseolus vulgaris, but produces the hypersensitive reaction (HR) in the nonhost, tobacco (Nicotina tabacum). A cosmid clone carrying 16 kilobases (kb) of contiguous genomic DNA partially complements this mutant. Altered growth of the mutant in planta was also partially restored. Marker exchange mutagenesis with Tn3- HoHo1 at two other sites within this locus results in mutants with attenuated and severely reduced pathogenicity. The locus is complex and contains repetitive DNA sequences. Northern analysis reveals that this locus is expressed in planta, but is not expressed in a rich growth medium, and the transcript is larger than 10 kb, suggesting that the locus is transcribed as a polycistronic mRNA. Comparison of total cellular protein profiles of R32 and PS9024 using SDS-PAGE analysis further reveals that at least nine protein bands ranging from approximately 100 kD or above in size are present in the wild type strain R32, but absent from the mutant. Additionally, a protein of approximately 45 kD is absent from the mutant. The site of Tn5 insertion has been partially sequenced. The initial search of the data banks suggested a gene or genes related to the ornithine biosynthetic pathway map to this locus. Further study strongly suggest a gene that encodes a membranceassociated protein and under the control of a promoter identical to appA gene promoter maps at this site and it is involved in the process of pathogenesis. / Graduation date: 1991

Pseudomonas aeruginosa, es uno de los más importantes patógenos humanos oportunistas, que puede colonizar no solo superficies abióticas, sino también superficies bióticas. Una vez colonizadas estas superficies, dicha bacteria produce exopolisacárido originando la formación de biopelículas, en consecuencia, estas bacterias son mil veces más resistentes, no sólo a antibióticos, sino también a biocidas y desinfectantes, constituyendo un problema de salud pública. Para este trabajo de investigación se recolectaron 62 cepas aisladas de líquidos biológicos y secreciones de pacientes procedentes del Hospital Nacional Edgardo Rebagliati Martins durante el periodo de Enero a Setiembre del 2006, identificándose el 100% de ellas como cepas de Pseudomonas aeruginosa. Sólo se escogió el 81% de las cepas formadoras de biopelículas para investigar la influencia del mucolítico NAC sobre las biopelículas de Pseudomonas aeruginosa, ya que éstas presentaron mayor producción durante la cuantificación realizada por el Método de O’Toole and Kolter. / Pseudomonas aeruginosa, is one of the most important human pathogens opportunistic, that can colonizes not only abiotics surfaces, also biotics surfaces. Once colonized these surfaces produce extracellular polysaccharides originating the biofilms formation, in consequence, these bacterias are thousand times more resistant to antibiotics, biocides and desinfectants, constituting a problem of public health. For this study, were collected 62 strains isolated from biological fluids and secretions of patients from the Edgardo Rebagliati Martins National Hospital during the period from January to September 2006, indentifying 100% of them like Pseudomonas aeruginosa strains. The determination of biofilm production we preferred to use Congo Red Agar Method (ARC), for its sensibility and reproducibility which showed that 42% were biofilm producers, whereas 48% were biofilm non developed. Pseudomonas aeruginosa ATCC 9027 (non producer) were used as negative control. Only the 81% of them was chosen to investigate the influence of mucolitic N-acetylcysteine (NAC) on Pseudomonas aeruginosa biofilms, by the biggest biofim production during the quantification by O’Toole and Kolter Method.

Del mes de enero al mes de octubre del año 2008 en el Servicio de Microbiología del Hospital Nacional Guillermo Almenara Irigoyn – ESSALUD, se colectaron 186 cepas de Pseudomonas aeruginosa resistentes al Imipenem (IMP) y al Meropenem (MEM) o con resistencia Intermedia a los dos antibióticos o a uno de ellos. Utilizando el Método Fenotípico de Doble Difusión en Disco con Monodiscos de EDTA; se llegó a detectar 13 cepas de P. aeruginosa positivas para MBLs, correspondiente al 6.99 % de las cepas testadas; de las cuales, el 53.84 % de la sinergia (efecto de la metaloenzima) se manifestó en el disco de Meropenem, el 30.75 % se manifestó en ambos discos (Imipenem y Meropenem) y solo el 15.38 % se manifestó en el disco de Imipenem. El 100 % de cepas de P. aeruginosa en las que se detectó las metaloenzimas provinieron de pacientes hospitalizados, de estos aislados el 69.76 % correspondieron al sexo masculino; el 61. 53 % de casos corresponden a pacientes cuyas edades están entre 70 a 90 años, y sólo el 14.14% de casos fueron en menores de 1 año y jóvenes. Las muestras biológicas en las que aisló a P. aeruginosa productora de MBLs fueron: orina, aspirado bronquial, BAL, herida no operatoria, secreción otica, líquido de diálisis peritoneal y sangre; fue orina en la que se aisló el mayor número de casos: 46. 15 %; en el Servicio de Medicina‐1, se detecto más casos 30.76 %, seguido de Cardiología y UCI con 23.07 %. Se detectó la enzima MBLs en cepas peruanas de Pseudomonas aeruginosa y se estableció la incidencia en 6.99 %, la cual es inferior a las reportadas por otros países, por lo cual se debe hacer estudios multicentricos para establecer la incidencia real. / In the study, 186 samples of Pseudomonas aeruginosa strains resistant to the Imipenem (IMP) and the Meropenem (MEM) or with Intermediate resistance to both antibiotic and one of them were collected in the Service of Microbiology of the Guillemo Almenara Irigoyen National Hospital ‐ ESSALUD from the month of January to the month of October of the year 2008. Using the Phenotypical Method of Double Disc Diffusion with Monodiscs of EDTA; 13 positive strains of Pseudomonas aeruginosas for MBLS were detected (6.99 % of the sampled strains): of them, the 53.85% of the synergy (effect of metal‐enzyme) was present in the disc of Meropenem, the 30.75% was present in both discs (Imipenem and Meropenem) and only the 15.38% was present in the disc of Imipenem. The 100% of the P. aeruginosa strains in which metal‐enzyme was detected belonged to hospitalized patients and the 69.76% of them belonged to masculine sex. The 61.54 % of cases correspond to patients whose ages are between 70 to 90 years, and only the 15.38% of the cases were in minors of 1 year and young people. The biological samples, in which the producing P. aeruginosa of MBLs was isolated, were: urine, inhaled bronchial, BAL, non operating wound, ear secretion, liquid of dialysis peritoneal and blood; but it was in the urine in that the greater number of cases was isolated the 46. 15 %. In the Service of Medicine‐1 were detected mayor number of cases (23.07%) followed for Cardiology and UCI (23.07%). The MBLs enzyme was detected in Pseudomonas aeruginosa peruvian strains. The incident was of 6.99% that is lower tanning the incident reported in other countries. For which Multicentral studies must be done for establish the real incident.

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 27-37).

Thesis (Ph. D.)--State University of New York at Binghamton, Department of Biological Sciences, 2009. / Includes bibliographical references.

Thesis--Ph. D., University of Hong Kong. / Typewritten.

(Uncorrected OCR) Abstract of the thesis entitled MOLECULAR BIOLOGY OF A CRYPTIC DEHALOGENASE FROM Burkholderia cepacia MBA4 Submitted by Laiju Sam For the degree of Doctor of Philosophy at The University of Hong Kong in July 2000 Burkholderia cepacia MBA4 has been previously shown to produce a single dehalogenase in batch culture condition. Other cryptic dehalogenases were detected in cells grown in continuous culture. In this study, one of the cryptic dehalogenases was cloned and characterised. This cryptic haloacid dehalogenase was designated Chd 1 and expressed constitutively in Escherichia coli. The structural gene, chdJ, was isolated from a 1.7-kb PstI fragment. This fragment. contains a functional promoter since the expression of chdl in E. coli is orientation independent. The nucleotide sequence of the fragment was determined and analysed and an open reading frame for 840 amino acids was identified. The nucleotide sequence of chdl did not show any homology with those of other dehalogenase genes. Comparison of the deduced amino acid sequence, however, showed significant homology, ranging from 42-50%, with the amino acid sequences of several other dehalogenases. Phylogenetic analysis indicated that Chd 1 is closely related to dehalogenase CI and dehalogenase IVa of Pseudomonas putida CBS3 and B. cepacia MBA4 respectively. 1 The recombinant Chdl had a native molecular weight of 58,000 daltons whereas the denatured molecular weight was found to be 27,000 daltons. Thus, the enzyme exists predominantly as a dimer. When the activity towards the two stereoisomers of 2-monochloropropionic acid were considered individually, the enzyme was found to be active towards the L-isomer only. The purified enzyme was most active towards monobromoacetic acid with specific activities of 5.44, 1.5, 4.56, and 1.95 J!mole of halide released/min/mg protein for monobromoacetic acid, 2-monobromopropionic acid, monochloroacetic acid and 2-monochloropropionic acid respectively. Maximum activity of the enzyme was observed at pH 6.5. The enzyme was found to be thermolabile. chdl was cloned in the T7 RNA polymerase driven pRSET A and Irc promoter based pPROEXHT expression vectors. However, no significant expression of chdl was obtained in these systems. Chd 1 differed from other dehalogenases in having a long leader sequence. This leader sequence contains a potential signal peptidase cleavage site. This is a property of periplasmic enzymes. In order to detect the unprocessed molecules of Chd 1, chdl was expressed in the temperature-sensitive E. coli strain IT 41 (lep9-mutant). This strain contains a mutation in the leader peptidase gene. The leader peptidase is inactive in this strain at the non-permissive temperature of o 42 C. Precursor molecules of Chd 1 was detected in cultures shifted to this non- permissive temperature. Periplasmic fractions isolated from E. coli harboring chdl were found to contain the active dehalogenase. 11 / abstract / toc / Botany / Doctoral / Doctor of Philosophy

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