Expression Analysis of ABC Transporters in Sinorhizobium meliloti

Abstract

Soils contain a complex mixture of compounds many of which can be transported and metabolized by microorganisms. 𝘚𝘪𝘯𝘰𝘳𝘩𝘪𝘻𝘰𝘣𝘪𝘶𝘮 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 is a soil bacterium whose gene sequence was recently determined. The diversity of the carbon and nitrogen sources than can be utilized by this organism is reflected in the large number of annotated ATP-binding cassette transporters in its genome. Although many of these genes are not necessary for survival, it is hypothesized that they arid in the competitive fitness of 𝘚. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 in the field. Many of these transporters remain uncharacterized. In this study a high throughput screen was developed to measure β-glucuronidase activity in a 96 well microtitre plate format to quantify expression of many reporter gene fusions under a variety of conditions. This system was used to analyze the expression of putative small molecule ABC transporters in 𝘚. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪. 45 𝘨𝘶𝘴𝘈 reporter gene transcriptional fusions to these transport genes were generated and recombined into the genome. These strains were grown in 96 well plates in minimal media containing a large number of carbon sources and carious legume root and seed exudates to be tested as inducers of transporter gene expression. Two transport systems were found to be induced by glucosamine and galactosamine and others were found to be induced by various sugars including mannose, arabinose, xylose and palatinose as well as protocatechuate and hydroxybenzoate. The bacteria-plant symbiosis of 𝘚. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 and alfalfa plays an important role in agriculture. To further understand the role of ABC transporters in the competition of 𝘚. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 the 𝘨𝘶𝘴𝘈 reporter fusions strains will also e inoculated onto alfalfa roots and nodules will be assayed for GusA activity to give a more complete picture of the role of ABC transporters in the competition and symbiosis of 𝘚. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪. Two substrates, galactitol and hydroxyproline found to induce transport systems were studied in depth in order to more fully understand the transport, metabolism and regulation of these compounds.