An Integrated Approach to Functional Genomics: Construction of a Novel Reporter Gene Fusion Library forSinorhizobium meliloti

Abstract

As a means of investigating gene function, we developed a robust transcription fusion reporter vector to measure gene expression in bacteria. The vector, pTH1522, was used to construct a random insert library for the Sinorhizobium meliloti genome. pTH1522 replicates in Escherichia coli and can be transferred to, but cannot replicate in, S. meliloti. Homologous recombination of the DNA fragments cloned in pTH1522 into the S. meliloti genome generates transcriptional fusions to either the reporter genes gfp ؉ and lacZ or gusA and rfp, depending on the orientation of the cloned fragment. Over 12,000 fusion junctions in 6,298 clones were identified by DNA sequence analysis, and the plasmid clones were recombined into S. meliloti. Reporter enzyme activities following growth of these recombinants in complex medium (LBmc) and in minimal medium with glucose or succinate as the sole carbon source allowed the identification of genes highly expressed under one or more growth condition and those expressed at very low to background levels. In addition to generating reporter gene fusions, the vector allows Flp recombinase-directed deletion formation and gene disruption, depending on the nature of the cloned fragment. We report the identification of genes essential for growth on complex medium as deduced from an inability to recover recombinants from pTH1522 clones that carried fragments internal to gene or operon transcripts. A database containing all the gene expression activities together with a web interface showing the precise locations of reporter fusion junctions has been constructed (www.sinorhizobium.org).

The LysR-type PcaQ protein regulates expression of a protocatechuate-inducible ABC-type transport system in Sinorhizobium meliloti

“…The intergenic region between pcaI and pcaR was amplified using primers (59-ATCAGATCTGAGTTCGTCGACGATCTCC-39 and 59-TAGGTACCCAGGTTGATCGACATCACC-39). The amplified product was cloned into the reporter plasmid pTH1705 (Cowie et al, 2006) via BglII and KpnI to create pcaI : : gfp+/lacZ. The resulting plasmid (pTH1960) is unable to replicate in S. meliloti; however, it carries an~1 kb region that is homologous to the S. meliloti pcaI-R intergenic region.…”

“…After sequencing (to confirm the absence of mutations within the coding sequence), the plasmid was transferred via conjugation into S. meliloti strain RmP1811 (RmP110 pcaQ : : V smb20568 : : gfp+/lacZ), in which expression of pcaQ has been disrupted through the integration of an Sp/Sm antibiotic-resistance cassette. RmP1811 also contains a transcriptional fusion (smb20568 : : gfp+/lacZ) created through the integration of plasmid pFL1131 (Cowie et al, 2006), which allows expression of smb20568 to be monitored via b-galactosidase assays.…”

Cyclopropane fatty acyl synthase in Sinorhizobium meliloti

“…The expression of the gusA gene, encoding b-glucuronidase, was measured using S. meliloti cultures grown in three different media: LBmc, MOPS-buffered minimal medium with 2 and 0 mM P i , and M9 at pH 7.0 and pH 5.5. To 100 ml of cell culture, 400 ml of GusA assay buffer [50 mM Na 3 PO 4 pH 7.4, 1 mM EDTA, 10 mM DTT, 0.01 % SDS and 0.44 mg ml 21 of substrate p-nitrophenyl-a-D-glucopyranoside (pNPG) (Cowie et al, 2006)] was added, mixed well and incubated at room temperature until the development of yellow colour. The reaction was stopped by the addition of 500 ml of a solution of Na 2 CO 3 (1 M); mixtures were spun down for 2 min at 10 000 g to remove cell debris and the supernatant was used to measure A 405 .…”

“…All assays were conducted using cultures grown to the early stationary phase; assays performed on mid-exponential-phase cultures showed approximately half the activities for both cfa1 and cfa2 (data not shown), suggesting a twofold increase in promoter activities during stationary phase. To eliminate the possibility that the cfa1 or cfa2 mutations may influence their transcription, we also measured cfa1 and cfa2 transcripion using cfa1-and cfa2-gusA transcriptional fusions (6448 and 869; Cowie et al, 2006) which do not disrupt the respective cfa genes. Data from these strains were similar to those in Fig.…”

“…In the last two decades, extensive research has been conducted to understand the biochemistry and genetics of nitrogenfixing bacteria and their symbiosis with leguminous plants, given the importance of this process for sustainable agriculture (Cowie et al, 2006;Galibert et al, 2001;MacLean et al, 2007;Weidner et al, 2003). Soil bacteria encounter numerous adverse conditions in the environment, including increased soil salinity, acidic pH conditions and limited inorganic phosphate (P i ) (Cheng et al, 2005;Garau et al, 2005;Ibragimova et al, 2006).…”