he 56-kb ocfC gene cluster is required for occidiofungin production by Burkholderia contaminans strain MS14. However, the function of the ocfC gene remains unknown. Sequence analysis showed the putative protein encoded by ocfC shares significant identities to glycosyltransferase. Plate bioassays showed that occidiofungin production by the ocfC mutant (ocfC::nptII) was significantly reduced as compared with strain MS14. The occidiofungin produced by the ocfC mutant is xyloseree, which suggests the ocfC gene encodes a xylosyltransferase to add a xylose to the peptide backbone. Single variant optimization of culture condition and medium compositions was also performed in this study. The results indicated that occidiofungin production was promoted with higher cell density inoculum, additional casamino acid, xylose, urea, zinc ions, and at pH 5. The findings have provided insights into development of pharmaceutical drug and agricultural biofungicide.

The Burkholderia cepacia complex (Bcc) are a group of closely related species known for their intrinsic multidrug resistance, large multipart genomes and ability to infect people with cystic fibrosis. The clinical relevance of the Bcc and their large multipart genomes make the study of their essential genes of broad interest. Essential genes are those required for survival in standard laboratory conditions this makes them potential targets for novel antibiotics against a group of species where few existing antibiotics are effective. Furthermore, while essential gene studies have been carried out in a number of bacterial species, only one of these species had multiple chromosomes and none had a genome as large as the Bcc. In my research I identified essential genes in B. cenocepacia K56-2, a member of the Bcc, by using transposon mutagenesis to deliver a rhamnose inducible promoter randomly into the genome and screening for a conditional growth (CG) phenotype. The utility of the CG mutant library was confirmed by showing that, when grown in suboptimal concentrations of rhamnose, only mutants that under-expressed the target of the antibiotic were hypersensitive. The CG mutant library included transposon insertions upstream from widely conserved, well-characterized essential genes suggesting that the system is capable of recovering essential gene mutants. A number of genes with either no or mixed records of essentiality in other microorganisms were also recovered. Among these was one of the three electron transfer flavoproteins (ETFs) in B. cenocepacia. The ETFs are a family of proteins found in a large number of eukaryotic, archaeal and bacterial species, which are required for the metabolism of specific substrates or for symbiotic nitrogen fixation in some bacteria. Despite these non-essential functions, high throughput screens have identified ETFs as putatively essential in several species. I showed that ETF expression is required for both viability and growth both on complex media and on media containing a variety of single carbon sources. Furthermore, cells depleted of ETF were determined to be nonviable and the morphologic shape of the cells changed from short rods to small spheres. In depth studies of essential genes are only possible for organisms with sequenced genomes. Of the 18 named species that currently comprise the Bcc, only 7 have been sequenced limiting the possibility of cross species comparative genomics. Therefore, I have assembled the first draft genomes of B. contaminans isolates, a species that has emerged as the dominant Bcc species recovered from the CF populations of Argentina and Spain. Identifying and characterizing essential genes in the Bcc, and sequencing additional Bcc species for comparative genomics are important first steps in understanding these clinically important bacteria. / February 2016

No description available.

Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, and phenylpyruvate could be used as sole carbon sources by wild type B. cenocepacia K56-2 but not by a PA catabolism defective mutant. These intermediates also induced a PA-inducible reporter system. Furthermore, proteomic analysis utilizing iTRAQ were used to study the protein expression of B. cenocepacia K56-2 grown in the amino acid-rich SCFM. Our results showed the over-expression of several proteins involved in amino acid and carbohydrate transport and metabolism. Interestingly, our results also showed the over-expression of flagellin and membrane efflux protein which are involved in the virulence of B. cenocepacia.

Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, and phenylpyruvate could be used as sole carbon sources by wild type B. cenocepacia K56-2 but not by a PA catabolism defective mutant. These intermediates also induced a PA-inducible reporter system. Furthermore, proteomic analysis utilizing iTRAQ were used to study the protein expression of B. cenocepacia K56-2 grown in the amino acid-rich SCFM. Our results showed the over-expression of several proteins involved in amino acid and carbohydrate transport and metabolism. Interestingly, our results also showed the over-expression of flagellin and membrane efflux protein which are involved in the virulence of B. cenocepacia.

Plaques formed by DC1 on B. cepacia LMG 18821 and B. cenocepacia PC184 are surrounded by large and expanding halos when production of the exopolysaccharide (EPS) cepacian is induced. This plaque morphology indicates that DC1 putatively carries an EPS depolymerase enzyme. Plaque halos were absent when DC1 infected a PC184 cepacian knockout mutant and a non-mucoid LMG 18821 mutant, constructed using plasposon mutagenesis. The virulence of these mutants compared to wildtype PC184 and LMG 18821 was determined using the Galleria mellonella infection model. No major changes to virulence were observed for the LMG 18821 mutant. But, the PC184 cepacian knockout mutant was attenuated for virulence suggesting that this carbohydrate pathway may play a role in pathogenesis. The gene(s) involved in halo formation remain unknown although attempts were made to determine the gene(s) involved by cloning and expressing DC1 fragments in E. coli and assaying for EPS degradation. / Microbiology and Biotechnology

Members of the Burkholderia cepacia complex (Bcc) are plant and human opportunistic pathogens. Essentially all Bcc isolates demonstrate in vitro broad-spectrum antibiotic resistance. In fact, many clinical isolates are resistant to all currently available antibiotics, rendering therapy ineffective. There is a substantial need to develop new antimicrobial therapies. The potential use of phage-tail-like high molecular weight bacteriocins, or "tailocins", as alternative anti-bacterial agents against Bcc was investigated. A tailocin, designated Bcep0425, produced by B.cenocepacia strain BC0425 was determined to have broad host range activity against members of the Bcc. Targeted mutagenesis of genes involved in the biosynthesis of the bacterial lipopolysaccharide (LPS) was conducted to determine the receptor site and it was determined that L-rhamnose and alpha-glucose associated with the LPS core were the receptors. Genetic analysis and targeted mutagenesis of the tailocin encoding genes was conducted in the host strain, B. cenocepacia BC0425, to determine the genetic organization of the tailocin Bcep0425 gene cluster and to confirm gene functions. We report for the first time genes involved in replication and integration that are associated with a pyocin/tailocin gene cluster. Additionally, a new class (IV) of holin was identified as part of the lysis cassette. Genetic analysis of the tailocin encoding genes revealed a high degree of similarity to defective phages identified in sequenced Burkholderia genomes. Two novel transcriptional regulators, bctN and bctR, along with recA were found to be involved in the induction of Bcep0425. Numerous studies have focused on the characterization of pyocins from Pseudomonas, but there have been no molecular investigations of tailocins from Burkholderia. This constitutes the first molecular characterization of a phage tail-like bacteriocin from Burkholderia.

Thesis (Ph.D.) - James Cook University, 2006. / Typescript (photocopy) Bibliography: leaves 187-206.

Stuttgart, Univ., Diss., 2000.

Species in the genus Burkholderia are found in a wide variety of environments ranging from plant rhizospheres to the human respiratory tract. Even though they have great biotechnological and bioremediative potential in preventing some plant diseases, promoting crop production, and degrading toxic compounds, some species of Burkholderia can be serious pathogens to those who have compromised immune systems, particularly those with Cystic Fibrosis (CF). Many Burkholderia species have two distinct copies of the hfq gene, a rare phenomenon in bacteria studied to date. As a global regulatory protein, Hfq has been shown to act as an RNA chaperone involved in stress responses, survival, and virulence in a variety of other bacteria via riboregulation. To address the significance of possessing two distinct Hfq proteins, sequence and expression analyses of the two corresponding B. cenocepacia genes were performed. RT-PCR revealed that both hfq genes were expressed constitutively and that neither hfq1 nor hfq2 appear to be transcribed as part of an operon. Phylogenetic analysis indicated that Bc-Hfq2 clustered with Hfq proteins from other Beta-Proteobacteria, while Bc-Hfq1 shared a common ancestor with the Hfq from Delta-Proteobacteria. This incongruence with 16S rRNA gene phylogeny suggests that the B. cenocepacia hfq1 gene may have been acquired through horizontal transfer. Further analysis of the two B.cenocepacia proteins indicated that Bc-Hfq2 was able to partially complement an E. coli hfq mutant, while the effect of heterologously expressing hfq1 could not be determined. A corresponding B. cenocepacia hfq2 deletion strain was constructed utilizing homologous recombination. This mutant showed extended lag phases when grown at 37°C and 41°C and a slower growth rate at a pH of 5 compared to the wild type. These data suggest that Hfq plays an important and yet still not fully understood role in the stress response of Burkholderia species.