Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000

Abstract

The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

Transcriptomes of Ralstonia solanacearum during Root Colonization of Solanum commersonii

“…Motility and adherence genes were also up-regulated, including type IV pili ( pilG, pilH, pilN, pilM, pilY, pilW , and fimV ), as well as the transcriptional activators of the flagellum genes flhC and flhD (Kang et al, 2002 ; Tans-Kersten et al, 2004 ). Other induced genes encoding described factors that are key for bacterial virulence included hdfA (Delaspre et al, 2007 ), efe (Valls et al, 2006 ), metE (Plener et al, 2012 ), and rpoN1 (Lundgren et al, 2015 ; Ray et al, 2015 ; Table 3 ). Peroxidases, catalases ( katE, katG ) and alkyl hydroperoxide reductases ( ahpC1, ahpF ), which have been described to combat the oxidative stress response during plant infection (Rocha and Smith, 1999 ; Flores-Cruz and Allen, 2009 ; Ailloud et al, 2016 ) were also induced.…”

“…Also sharing similar up-regulation in potato (Table 3 ) and tomato are the transcriptional activators flhC and flhD (Jacobs et al, 2012 ), which regulate flagellum-encoding genes (Tans-Kersten et al, 2004 ) and the nitrogen metabolism genes narL, ptsN , and hmpX (Dalsing and Allen, 2014 ; Dalsing et al, 2015 ), implying that they all play a key role during plant infection. Additional genes induced during potato colonization had been described as key for virulence on other plant hosts, including small molecule hdfA (Delaspre et al, 2007 ), the ethylene forming enzyme efe (Valls et al, 2006 ), the methionine metabolism gene metE (Plener et al, 2012 ) and the alternative sigma factor rpoN1 (Lundgren et al, 2015 ; Ray et al, 2015 ). These factors may be also considered essential for growth in planta , irrespective of the infected species.…”

Response regulator VemR regulates the transcription of flagellar rod gene flgG by interacting with σ54 factor RpoN2 in Xanthomonas citri ssp. citri

“…A number of soil‐borne and plant‐associated bacteria possess two genes encoding RpoN proteins (Gicharu et al ., ; Kullik et al ., ; Ray et al ., ; Tian et al ., ). Although the two genes exhibit high levels of identity, the two proteins are not interchangeable (Lundgren et al ., ; Michiels et al ., ). In contrast with the rpoN1 mutant strain, inactivation of the rpoN2 gene in Rhizobium etli did not produce any phenotypic defects during free‐living growth.…”

SfnR2 Regulates Dimethyl Sulfide-Related Utilization in Pseudomonas aeruginosa PAO1

“…The msuEDC, sfnR1, sfnR2, and sfnG genes were deleted from the genome of P. aeruginosa PAO1 using the method of Choi and Schweizer (33). Cloning of gene deletion plasmids, mutant selection, and marker removal, etc., were done using established procedures (33,34). Briefly, the chromosomal mutations msuEDC::FRT-aacC1-FRT, sfnR1::FRT-aacC1-FRT, sfnR2::FRT-aacC1-FRT, and sfnG:: FRT-aacC1-FRT were generated by electroporating P. aeruginosa PAO1 with the gene deletion plasmids…”