Developing a culture medium for Liberibacter asiaticus through comparative multi-omics analysis with its closest cultured relative, L. crescens

Developing a culture medium for Liberibacter asiaticus through comparative multi-omics analysis with its closest cultured relative, L. crescens

Report Date: 01/12/2016
Project: 15-027   Year: 2015
Category: CLas Bacteria
Author: Eric Triplett
Sponsor: Citrus Research and Development Foundation

Since the last report, we have developed several media for the culturing of Ca. L. asiaticus (CLas), being AM2 the most promising one. These new media build on the components of AM2. We have acquired several new ideas on culturing by using a multi-omics approach to L. crescens cultures. These methods tell us which components may be absent or missing in AM2. As a result, we have developed media AM3 through AM10. rThus, components based on the genome analysis of CLas (Duan et al, 2009), the comparative genomics of the culturable strain L. crescens with CLas (Fagen et al, 2014), and the biochemical composition of the citrus phloem (Hijaz and Killini, 2014) were added to evaluate growth of Ca. L. asiaticus. Similar to obligate bacteria, CLas appears to be able to scanvange energy in the form of ATP since it encodes for an ATP/ADP translocase able to uptake ATP from its surroundings (Duan et al, 2009). Therefore, we have added ATP to one of these media formations. CLas cannot synthesize NAD, but it is able to recycle it, so we added NAD to certain media as well (Fagen et al, 2014). Other components found in the biochemical composition of the citrus phloem were added to the media as well such as benzoic acid, threonic acid, maleic acid and quinic acid. Inoculations with psyllids guts have been done in these new media and possible growth is being evaluated using PCR and live/dead bacterial experiments. We were also evaluating other techniques to verify CLas growth such as peak-to-trough ratio (Korem et al, 2015) using digital PCR. Growing cells are active replicating with more active replicating forks, therefore, in this state the ratio near the origin of replication would be higher than near the terminus, providing a quatitative data for population growth (Korem et al, 2015). References Duan, Y., Zhou, L., Hall, D. G., Li, W., Doddapaneni, H., Lin, H., … & Dickerman, A. (2009). Complete genome sequence of citrus huanglongbing bacterium,’Candidatus Liberibacter asiaticus’ obtained through metagenomics. Molecular Plant-Microbe Interactions, 22(8), 1011-1020. Fagen, J. R., Leonard, M. T., McCullough, C. M., Edirisinghe, J. N., Henry, C. S., Davis, M. J., & Triplett, E. W. (2014). Comparative genomics of cultured and uncultured strains suggests genes essential for free-living growth of Liberibacter. PloS one, 9(1), e84469. Hijaz, F., & Killiny, N. (2014). Collection and chemical composition of phloem sap from Citrus sinensis L. Osbeck (Sweet Orange). Korem, T., Zeevi, D., Suez, J., Weinberger, A., Avnit-Sagi, T., Pompan-Lotan, M., … & Sirota-Madi, A. (2015). Growth dynamics of gut microbiota in health and disease inferred from single metagenomic samples. Science, 349(6252), 1101-1106.


Your browser does not support pdfs, click here to download the file.