Identification of the Genes Required for the Culture of Liberibacter crescens, the Closest Cultured Relative of the Liberibacter Plant Pathogens
Here Tn5 random transposon mutagenesis was used to identify the essential elements for culturing Liberibacter crescens BT-1 that can serve as antimicrobial targets for the closely related pathogens of citrus, Candidatus Liberibacter asiaticus (Las) and tomato and potato, Candidatus Liberibacter solanacearum (Lso). In order to gain insight on the virulence, metabolism, and culturability of the pathogens within the genus Liberibacter, a mini-Tn5 transposon derivative system consisting of a gene specifying resistance to kanamycin, flanked by a 19-base-pair terminal repeat sequence of Tn5, was used for the genome-wide mutagenesis of L. crescens BT-1 and created an insertion mutant library. By analyzing the location of insertions using Sanger and Illumina Mi-Seq sequencing, 314 genes are proposed as essential for the culture of L. crescens BT-1 on BM-7 medium. Of those genes, 76 are not present in the uncultured Liberibacter pathogens and, as a result, suggest molecules necessary for the culturing these pathogens. Those molecules include the aromatic amino acids, several vitamins, histidine, cysteine, lipopolysaccharides, and fatty acids. In addition, the 238 essential genes of L. crescens in common with L. asiaticus are potential targets for the development of therapeutics against the disease.
Department of Microbiology and Cell Science University of Florida
B.S. (1976) Rutgers University, Cook College, New Brunswick, NJ
M.S. (1978) University of Maryland, College Park
Ph.D. (1981) University of Missouri, Columbia
Postdoctoral (1981-1982) University of Wisconsin-Madison
Description of Research
Colonization of plants by endophytic bacteria.
Endophytic bacteria are defined as those bacteria that enter plants without causing disease or any organized symbiotic structures. There are three important reasons to study bacterial endophytes. First, these bacteria often enhance plant growth under field and greenhouse conditions. Second, these strain can fix nitrogen and we hope to find ways to improve the nitrogen nutrition with such strains. And third, some human pathogenic bacteria can colonize the interior of plants.
We have learned that strains within the same bacterial species can differ radically in the their ability to enter plant hosts. We are seeking to understand the basis of this strain-specificity. We also know that an endophyte can have a broad-host-range and colonize monocots as well as dicots. For most of this work, we use a model endophytic bacterium called Klebsiella pneumoniae 342, also referred to as Kp342. A single cell of Kp342 in the inoculum is sufficient to fully colonize several plant hosts. This strain fixes nitrogen and can enhance the growth of a number of plant hosts.
Microbial diversity and ecology.
I work with several colleagues at the University of Wisconsin-Madison on an NSF-supported Microbial Observatory of the North Temperate Lakes Long Term Ecological Research site (http://microbes.limnology.wisc.edu). We have observed tremendous variation in microbial diversity in lakes across time and space. Different lakes vary greatly in microbial diversity but within lake variability across time is also very significant. In one of our lakes, changes in microbial diversity appears to be correlated with biological changes that occur in the lake over time rather than changes in water chemistry. We have developed two new tools for microbial diversity research. These are Automated Ribosomal Intergenic Spacer Analysis (ARISA) and a web-based tool for T-RFLP analysis.
Currently we are characterizing the taxa found in our study lakes and plan to culture those bacteria that are either unusual or are important in productivity.