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Development of Chemically Defined Media Reveals Citrate as Preferred Carbon Source for Liberibacter Growth.

ABSTRACT: Liberibacter crescens is the closest cultured relative of four important uncultured crop pathogens. Candidatus L. asiaticus, L. americanus, and L. africanus are causal agents of citrus greening disease, otherwise known as huanglongling (HLB). Candidatus L. solanacearum is responsible for potato Zebra chip disease. Cultures of L. crescens grow slowly on BM-7 complex medium, while attempts to culture the Ca. Liberibacter pathogens in BM-7 have failed. Developing a defined medium for the growth of L. crescens will be useful in the study of Liberibacter metabolism and will improve the prospects for culturing the Ca. Liberibacter pathogens. Here, M15 medium is presented and described as the first chemically defined medium for the growth of L. crescens cultures that approaches the growth rates obtained with BM-7. The development of M15 was a four step process including: (1) the identification of Hi-Graces Insect medium (Hi-GI) as an essential, yet undefined component in BM-7, for the growth of L. crescens, (2) metabolomic reconstruction of Hi-GI to create a defined medium for the growth of L. crescens cultures, and (3) the discovery of citrate as the preferred carbon and energy source for L. crescens growth. The composition of M15 medium includes inorganic salts as in the Hi-GI formula, amino acids derived from the metabolomic analyses of Hi-GI, and a 10-fold increase in vitamins compared to the Hi-GI formula, with exception choline chloride, which was increased 5000-fold in M15. Since genome comparisons of L. crescens and the Ca. Liberibacter pathogens show that they are very similar metabolically. Thus, these results imply citrate and other TCA cycle intermediates are main energy sources for these pathogens in their insect and plant hosts. Thus, strategies to reduce citrate levels in the habitats of these pathogens may be effective in reducing Ca. Liberibacter pathogen populations thereby reducing symptoms in the plant host.

SUBMITTER: Cruz-Munoz M

PROVIDER: S-EPMC5895721 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Development of Chemically Defined Media Reveals Citrate as Preferred Carbon Source for Liberibacter Growth.

Cruz-Munoz Maritsa M Petrone Joseph R JR Cohn Alexa R AR Munoz-Beristain Alam A Killiny Nabil N Drew Jennifer C JC Triplett Eric W EW

Frontiers in microbiology 20180405

Liberibacter crescens is the closest cultured relative of four important uncultured crop pathogens. Candidatus L. asiaticus, L. americanus, and L. africanus are causal agents of citrus greening disease, otherwise known as huanglongling (HLB). Candidatus L. solanacearum is responsible for potato Zebra chip disease. Cultures of L. crescens grow slowly on BM-7 complex medium, while attempts to culture the Ca. Liberibacter pathogens in BM-7 have failed. Developing ...[more]

PMID: 29675013

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Project description:Lactic acid bacteria are increasingly becoming important dietary supplements due to their health benefits when consumed in adequate quantity. The increasing attention on these important microbes has necessitated an in-depth understanding of their physiological processes, such as nutritional requirements and growth patterns, to better harness their probiotic potentials. This study was carried out to determine the nutritional requirements for the growth of L. salivarius ZJ614 and L. reuteri ZJ625 from a chemically defined medium and evaluate growth kinetics by fitting different sigmoidal growth models. The complete CDM contains 49 nutritional ingredients such as glucose, Tween 80®, mineral salts, buffers, amino acids, vitamins, and nucleotides at defined concentrations. In addition, the minimal nutritional requirements of the isolates were determined in a series of single-omission experiments (SOEs) to compose the MDM. Growth curve data were generated by culturing in an automated 96-well micro-plate reader at 37°C for 36 h, and photometric readings (optical density: OD600) were taken. The data were summarized in tables and charts using Microsoft Excel, while growth evaluation was carried out using open-source software (Curveball) on Python. The results revealed that omission of the amino acids, vitamins, and nucleotides groups resulted in 2.0, 20.17, and 60.24% (for L. salivarius ZJ614) and 0.95, 42.7, and 70.5% (for L. reuteri ZJ625) relative growths, respectively. Elimination of the individual CDM components also indicates varying levels of growth by the strains. The growth curve data revealed LogisticLag2 and Baranyi-Roberts models as the best fits for L. reuteri ZJ625 and L. salivarius ZJ614, respectively. All the strains showed appreciable growth on the CDM and MDM as observed in de Man-Rogosa-Sharpe (MRS) broth. We also described the growth kinetics of L. reuteri ZJ625 and L. salivarius ZJ614 in the CDM, and the best models revealed the estimated growth parameters.

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Development of Chemically Defined Media Reveals Citrate as Preferred Carbon Source for Liberibacter Growth.

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Development of Chemically Defined Media Reveals Citrate as Preferred Carbon Source for <i>Liberibacter</i> Growth.

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