The Use of Signature Tagged Mutagenesis in a Burkholderia cepacia Pulmonary Infection Animal Model

HUNT, TRACEY A. * , CORA KOOI, PAMELA A. SOKOL, and MIGUEL A. VALVANO
University of Western Ontario, London, Ontario, Canada. and University of Calgary, Calgary, Alberta, Canada.
[email protected]   Visit the Valvano lab

Burkholderia cepacia infections cause elevated morbidity and mortality among cystic fibrosis (CF) patients.  The seriousness of these lung infections is complicated by the ability of B. cepacia to spread by patient-to-patient transmission, as well as the multiple antimicrobial agents to which B. cepacia is resistant.  As a result, B. cepacia is rarely eradicated once a CF patient has been colonized.  Although many virulence factors have been postulated to play a role in infections caused by B. cepacia, the mechanisms by which this pathogen causes disease remain largely unknown.  We hypothesize that B. cepacia possesses unique virulence factors which allow it to persist and survive within the host.  Some bacterial genes may be preferentially expressed following interactions with the host and, consequently, potential virulence factors that are studied using in vitro approaches may not be relevant in vivo.  We have adapted the negative selection strategy of signature tagged mutagenesis (STM) to identify bacterial genes that are expressed in vivo using the rat agar bead model for B. cepacia pulmonary infections.  A PCR-based STM system developed by Lehoux et al. (1999) improves upon the traditional hybridization procedure and allows for a more rapid and method of screening.  STM uses a pool of genetically engineered transposon mutants, each containing a unique oligonucleotide tag incorporated onto the chromosome.  We have adapted the PCR-based technique to construct a pool of unique plasposons for use in B. cepacia containing 41 unique tags.  Using real-time PCR, strains harbouring mutations in genes essential in vivo will not be detected following infection in the rat model and can, therefore, be putatively identified as genes required for survival.  This modified method of STM has allowed us to identify genes important for virulence of B. cepacia in a rat model of pulmonary infection.  The PCR-based STM approach can reveal putative virulence determinants of B. cepacia which are important for in vivo infections and may potentially serve as targets for drug therapy. (Abstract received Sep 30)

This poster was presented previously at the American Society for Microbiology 102nd General Meeting, Salt Lake City, Utah, May 2002.

Identification and Mutagenesis of a Catalase/Peroxidase Gene in Burkholderia cepacia

MATT D. LEFEBRE* and MIGUEL A. VALVANO
University Of Western Ontario, London, Ontario, Canada.  Department Of Microbiology And Immunology.
[email protected]   Visit the Valvano lab

B. cepacia is an aerobic, gram-negative microorganism that was originally described as the cause of soft rot in onions. Over the past 20 years the bacterium has emerged as an important opportunistic pathogen, primarily in cystic fibrosis patients.  Our laboratory has shown that B. cepacia can survive intracellularly in macrophages and amoebae. Catalase and catalase/peroxidase enzymes are involved in detoxifying toxic oxygen intermediates and may permit B. cepacia to survive exposure to hydrogen peroxide generated by host cells in response to infection.  This study focused on the cloning, characterization and disruption of a catalase/peroxidase gene from a clinical isolate of  B. cepacia.   We have cloned a 2.3 kb gene encoding a catalase/peroxidase protein roughly 85 kDa in size that shows homology to similar proteins from other high G+C bacteria.  DNA sequence analysis revealed 2 potential start codons separated by roughly 150 base pairs that maintained the open reading frame of the gene.  A potential iron regulated binding element exists in the sequence between the 2 proposed start codons.   Complementation studies using both forms of the gene controlled by the regulatable BAD promoter were able to restore catalase activity to the E. coli double kat knockout mutant UM2.  Activity gels from extracts obtained from the wild type C5424 strain transformed with constructs expressing both forms of the gene suggest that the first GTG start codon is the actual start site for translation.    A catalase/peroxidase mutant was generated using an internal fragment of the gene to facilitate a single cross-over inactivation event.  The enzymatic profiles of the mutant strain as assessed by native PAGE did not appear to be significantly different from the wild type strain (WT).  Survival following exposure to hydrogen peroxide in vitro showed that the mutant and WT strains behaved in a similar fashion when grown in iron rich media. However, the mutant showed a 2 log decrease in survival compared to the WT strain when grown in iron-limiting conditions.  In accordance to these results, the mutant strain showed a decreased ability to grow under iron limiting conditions as compared to the WT strain.
(Abstract received Sep 30)

This poster was previously presented at the UA/UC 10th Biennial Conference on Infectious Diseases, Banff, Alberta, April 21-24, 2002

K. A. VIGEANT*, L. D. TATAR*, and M. A. VALVANO
Department of Microbiology and Immunology, University of Western Ontario, London, CANADA, N6A 5C1
[email protected]  and  [email protected]         Visit the Valvano lab

The O-specific polysaccharide, or O-antigen, is the most external component of the lipopolysaccharide in Gram-negative bacteria and plays a role in bacterial virulence. WecA mediates the transfer of N-acetyl glucosamine (GlcNAc) to undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc, and may also interact with components important in the subsequent steps of O-antigen synthesis and translocation to the cell surface. In this work, we constructed a functional WecA protein derivative carrying a C-terminal FLAG epitope tag and a 5x His tag, and used it to perform amino acid substitutions of aspartic acid residues D156 and D159, which are highly conserved in all the WecA homologues. We originally hypothesized that these amino acids were involved in coordination with divalent metal cations. Our strategy involved introducing replacements that modified the amino acid, including the replacement of the aspartic acids with E (glutamic acid), N (asparagine), C (cysteine), A (alanine), H (histidine), and G (glycine) residues. These amino acid substitutions were analyzed by examining their ability to complement 07 LPS synthesis in vivo in a wecA-deficient strain and by an in vitro transfer assay using membrane extracts under various concentrations of magnesium and manganese. Results from Mg++ and Mn++ KM and VMAX determination suggest that these sites are not individually responsible for metal binding, and that they may participate in recognition of the nucleotide sugar substrate. We also constructed a WecA-GFP tagged at its C-terminus to further investigate its membrane topology and localization. The presence of green fluorescent membranes in bacteria expressing the WecA-GFP recombinant protein confirmed that the C-terminus of WecA is located in the cytosol. Sucrose density gradient experiments suggest that WecA may be localized at the attachment sites between IM and OM.  (Abstract received Oct 3)

SILVIA T. CARDONA* AND MIGUEL A. VALVANO
University Of Western Ontario, London, Ontario, Canada.  Department Of Microbiology and Immunology.
[email protected]       Visit the Valvano lab

The extraordinary ability of Burkholderia cepacia to resist the majority of clinically useful antibiotics requires the identification and characterization of new genes required for survival (essential genes) that could serve as novel targets for antimicrobial treatment in cystic fibrosis (CF) patients. We recently identified by homology-based searches 95 B. cepacia genes of unknown function that are similar to putative essential genes from Haemophilus influenzae. Many of these genes show a high degree of conservation across microbial genomes. We are developing a methodology to validate these putative essential genes of B. cepacia based on their functional conservation in E. coli. Our strategy consists of deleting an essential gene in E. coli in the presence of the homologous gene of B. cepacia which is cloned under the control of an inducible promoter. As a model system we used the gmhA gene which its product is required for growth in the presence of Noboviocin (Nb). Taking advantage of the Red system-based inactivation of chromosomal genes in E. coli we were able to delete the gmhA gene of E. coli in the presence of the cloned complementing gene. We are currently extending this methodology to other putative essential genes of unknown function. (Abstract received Oct 8)

ERICA L. FRANCIS*, AND ELIZABETH W. ALM
Central Michigan University, Mt. Pleasant, MI 48859
[email protected]

Due to the increasing incidence of antibiotic resistance among bacteria, it is important to look at reservoirs of
antibiotic resistance in the environment.  Recreational beaches may provide a reservoir of antibiotic resistant
strains of bacteria, as well as a contact point between the bacteria and the public.  The minimum inhibitory
concentrations (MIC) of four antibiotics used in human and veterinary medicine (cephalothin, ampicillin, trimethoprim, and amoxicillin � clavulanic acid (2/1)) were determined for 24 Escherichia coli isolates from six bathing beaches (18 isolated from 20-cm sand cores, and six from water), in St. Clair County, Michigan.  The MICs were determined using antibiotic-impregnated Etest® strips.  The MIC of the antibiotics did not differ among E. coli isolated from beaches in different watershed types (industrial, urban/agricultural, or forested ANOVA, P>0.34).  The multiple antibiotic resistant isolates were found in 5-10 cm and 15-20 cm strata.  The 1-5 cm sand stratum, which normally had the highest abundance of E. coli, showed the lowest incidence of antibiotic resistant E. coli.  These results, though not statistically significant, were obtained from a limited sample size.  Ongoing work with more isolates may better elucidate patterns of resistance in the environment.  The presence of antibiotic resistant E. coli at our study sites suggests that they originated in human or domestic livestock fecal material.  If bacteria are persisting in the environment and if horizontal gene transfer is occurring in the sand, this has serious implications for human health.  (Abstract received Oct 9)

Anthony July, Brandy Buckbee*, Jacob Meganck, Jerry Sanders.
University Michigan-Flint, Biology Department, 303 East Kearsley St, Flint MI 48502-1950.
[email protected]

The degradation of industrial waste has become a significant topic in light of the manufacturing processes and/or accidental spills.  The bioremediation of organic compounds usually occurs through a large consortium of microorganisms.  Therefore we isolated a variety of microorganisms that were tolerant of these chemicals and could also utilize them as a carbon source as an initial step to develop a more effective means for bioremediation of petrochemicals.  We have isolated four distinct microorganisms from complex soil environments.  These bacteria can utilize gasoline as their sole carbon source.  These bacterium have been screened for the presence of accessory genetic elements, and have been characterized based upon structural composition and have had standard biochemical analysis.  (Abstract received Oct 10)

Findings presented in this poster were previously presented as a talk at Michigan Academy of Science, Arts & Letters, March,2002.

J. R. COLE, T. G. LILBURN(1), R. J. FARRIS, P. R. SAXMAN, S. CHANDRA,
B. CHAI*, Q. WANG, S. KULAM, D. M. MCGARRELL, G. M. GARRITY, T. M. SCHMIDT,
J. M. TIEDJE
Michigan State University, East Lansing, MI 48824
(1)American Type Culture Collection, Manassas, VA
[email protected]        http://rdp.cme.msu.edu

The Ribosomal Database Project - II (RDP-II) provides data, tools and services related to ribosomal RNA sequences to the research community. Through its website (http://rdp.cme.msu.edu), RDP-II offers aligned and
annotated rRNA sequence data, analysis services, and phylogenetic inferences (trees) derived from these data. RDP-II release 8.1 (May 21, 2001) contains 16,277 prokaryotic, 5201 eukaryotic, and 1503 mitochondrial small subunit rRNA sequences in aligned and annotated format. The current public beta release of 9.0 debuts a new regularly updated alignment of over 50,000 annotated (eu)bacteria small subunit rRNA sequences. This alignment was created using a modified version of RNACAD, a stochastic context-free grammar based alignment program by M.P.S. Brown(1), and marks a major departure from prior RDP releases by incorporating secondary-structure information directly in the internal model. We compared the two trees using the Kishino-Hasegawa-Templeton
statistical test(2). Neither tree was a significantly better fit of either the RDP's hand-tuned alignment or of the automated alignment, indicating the two alignments are equally valid at this level of resolution. In addition, this automated method should reduce the potential for human error or unintended bias in the alignment process.
Also included in the beta release are a new Hierarchy Browser, which place sequences into a hierarchy consistent with the taxonomy used in the second edition of Bergey�s Manual of Systematic Bacteriology (3), and the addition of Weighted-neighbor joining option to the phylogenetic tree building and visualization tool (Phylip Interface). The RDP-II email address for questions or comments is [email protected]. (Abstract received Oct 11)

DR. BENLI CHAI, RIBOSOMAL DATABASE PROJECT
MICHIGAN STATE UNIVERSITY�S CENTER FOR MICROBIAL ECOLOGY

(1) M. P. S. Brown, Small subunit ribosomal RNA modeling using stochastic context-free grammars, Proceedings of the Eighth
International Conference on Intelligent System for Molecular Biology,  pp. 57-66 (2000).

(2) H. Kishino and M. Hasegawa, Evaluation of the Maximum-Likelihood estimate of the evolutionary tree topologies from DNA-sequence data, and the branching order in Hominoidea, Journal of Molecular Evolution, 29, 170-179 (1989).

(3) G.M. Garrity, M.Winters, A.W.Kuo, and D.B.Searles, Taxonomic Outline of the Prokaryotes. Bergey�s Manual of Systematic
Bacteriology, Second Edition. Release 2.0, January 2002. Springer-Verlag, New York.

ADRIANE J. MATTHEWS AND ELIZABETH W. ALM
Central Michigan University  Mt. Pleasant, MI  [email protected]

Fecal contamination of public recreational waters is a growing problem around the world, as many harmful human pathogens are associated with fecal waste.  Currently in Michigan, monitoring of public recreational waters for the fecal indicator bacterium Escherichia coli is carried out on a voluntary basis.  The sand environment, however, is not monitored.  The purpose of this study was to observe the spatial arrangement of fecal bacteria in a natural beach sand environment to determine if viable cells persist in the sand and whether fecal bacteria participate in biofilms.  Glass slides were buried in sand at a Lake Huron beach in St. Clair County Michigan, collected throughout the summer of 2002, and analyzed by microscopy.  Scanning laser confocal microscopy was used to observe the Gram reaction and viability of bacteria in the sand.  Overall, the bacteria in the sand biofilms were viable and predominantly Gram negative.  Antibody staining was used to observe E.coli 0157:H7 and Salmonella spp. in the sand using epifluorescence microscopy.  Both E. coli 0157:H7 and Salmonella spp. were observed in the sand biofilms.    The presence of viable pathogens in the sand indicates that bacteria may be persisting in the sand environment, where they could pose a human health threat.  (Abstract received Oct 11)