BACKGROUND:RNA polymerase (RNAP) is highly conserved and essential for prokaryotic housekeeping activities, making it an important target for the development of new antibiotics. The rpoB gene, encoding a β-subunit of bacterial RNAP, has a well-known association with rifampicin resistance. However, the roles of other RNAP component genes such as rpoA, encoding an α-subunit of RNAP, in antibiotic resistance remain unexplored.OBJECTIVES:To characterize the antibiotic resistance-related role of RpoA.METHODS:We measured the expression of the MexEF-OprN efflux pump in an RpoA mutant using a transcriptional reporter. The MICs of various antibiotics for this RpoA mutant were determined.RESULTS:We uncover a novel role of antibiotic susceptibility for an RpoA mutant in Pseudomonas aeruginosa. We found that a single amino acid substitution in RpoA resulted in reduced activity of the MexEF-OprN efflux pump, which is responsible for the exportation of various antibiotics, including ciprofloxacin, chloramphenicol, ofloxacin and norfloxacin. This attenuated efflux pump activity, caused by the RpoA mutation, conferred the bacteria further susceptibility to antibiotics regulated by MexEF-OprN. Our work further revealed that certain clinical P. aeruginosa isolates also contained the same RpoA mutation, providing clinical relevance to our findings. Our results elucidate why this new antibiotic-susceptible function of RpoA mutants would have remained undetected in conventional screens for mutants involving antibiotic resistance.CONCLUSIONS:The discovery of antibiotic susceptibility in an RpoA mutant implicates a new therapeutic approach for treating clinical isolates of P. aeruginosa with RpoA mutations, using specific antibiotics regulated by MexEF-OprN. More generally, our work suggests that RpoA could serve as a promising candidate target for anti-pathogen therapeutic purposes.

Quorum-sensing (QS) coordinates the expression of virulence factors in Pseudomonas aeruginosa, an opportunistic pathogen known for causing severe infections in immunocompromised patients. QS has a master regulator, the lasR gene, but in clinical settings, P. aeruginosa isolates have been found that are QS-active but LasR-null. In this study, we developed an experimental evolutionary approach to identify additional QS-reprogramming determinants. We began the study with a LasR-null mutant with an extra copy of mexT, a transcriptional regulator gene that is known to be able to reprogram QS in laboratory LasR-null strains. In this strain, spontaneous single mexT mutations are expected to have no or little phenotypic consequences. Using this novel method, which we have named "targeted gene duplication followed by mutant screening", we identified QS-active revertants with mutations in genes other than mexT. One QS-active revertant had a point mutation in rpoA, a gene encoding the α-subunit of RNA polymerase. QS activation in this mutant was found to be associated with the downregulated expression of mexEF-oprN efflux pump genes. Our study therefore uncovers a new functional role for RpoA in regulating QS activity. Our results indicate that a RpoA-dependent regulatory circuit controlling the expression of the mexEF-oprN operon is critical for QS-reprogramming. In conclusion, our study reports on the identification of non-MexT proteins associated with QS-reprogramming in a laboratory strain, shedding light on possible QS activation mechanisms in clinical P. aeruginosa isolates.

LasR is a master regulator of quorum-sensing (QS) in Pseudomonas aeruginosa. LasR-null mutants commonly appear in lung isolates from chronically infected cystic fibrosis (CF) patients. However, numerous such CF isolates have a QS-active phenotype, but factors underlying QS-reprogramming in LasR-null mutants remain largely unknown. Mutations in the transcriptional regulator gene mexT are well known to be responsible for QS-reprogramming in a laboratory LasR-null mutant strain, however, simultaneous occurrence of lasR and mexT mutations is rare in CF isolates. To identify QS-reprogramming determinants, we developed an experimental evolution approach, for which a QS-inactive LasR null mutant with an extra copy of mexT was engineered. In such a strain, spontaneous single mexT mutations are expected to have no or little phenotypic consequences. This novel method, named “targeted gene duplication followed by mutant screening” (TGD-MS), resulted in the identification of QS-active revertants with mutations in genes other than mexT . We characterized a QS-active revertant with a point mutation in rpoA, a gene encoding the α-subunit of RNA polymerase. QS activation in this mutant was found to be associated with the down-regulated expression of mexEF-oprN efflux pump genes. Our study therefore uncovers a new functional role for RpoA in regulating QS activity. Furthermore, our results suggest that a regulatory circuit controlling the expression of the mexEF-oprN operon is critical for QS-reprogramming. In conclusion, our study reports on the identification of non-MexT proteins associated with QS-reprogramming in a laboratory strain, shedding light on possible QS activation mechanisms in clinical P. aeruginosa isolates.

We develop a biotin-based tandem labeling approach to improve detection sensitivity of DNA probe. Through DNA polymerase-mediated overhand filling, the 3'end of DNA probe was tandemly labeled with biotin molecules. The intensity of biotin signals could be flexibly manipulated by controlling the introduced length of poly(A) in the 5' overhang.

Exopolysaccharides (EPS) play critical roles in rhizobium-plant interactions. However, the EPS biosynthesis pathway in Bradyrhizobium diazoefficiens USDA110 remains elusive. Here we used transposon (Tn) mutagenesis with the aim to identify genetic elements required for EPS biosynthesis in B. diazoefficiens USDA110. Phenotypic screening of Tn5 insertion mutants grown on agar plates led to the identification of a mutant with a transposon insertion site in the blr2358 gene. This gene is predicted to encode a phosphor-glycosyltransferase that transfers a phosphosugar onto a polyprenol phosphate substrate. The disruption of the blr2358 gene resulted in defective EPS synthesis. Accordingly, the blr2358 mutant showed a reduced capacity to induce nodules and stimulate the growth of soybean plants. Glycosyltransferase genes related to blr2358 were found to be well conserved and widely distributed among strains of the Bradyrhizobium genus. In conclusion, our study resulted in identification of a gene involved in EPS biosynthesis and highlights the importance of EPS in the symbiotic interaction between USDA110 and soybeans.

Many Gram-negative bacteria communicate by using homoserine lactones (HSLs) as quorum sensing (QS) signals in a cell density-dependent manner. In addition to fatty acyl-HSL (acyl-HSL) signals, certain strains, most of them associated with plants, produce non-canonical aryl-HSLs such as cinnamoyl-HSL. However, the role of aryl-HSL in endophytic associations remained elusive. Bradyrhizobium sp. strain ORS278 possesses a LuxI-LuxR type QS system and produces cinnamoyl-HSL as a QS signal. Here, we report that strain ORS278 promotes growth of domesticated rice (Oryza sativa). QS mutants unable to produce cinnamoyl-HSL exhibited reduced plant-growth promoting activity in comparison to the parent strain ORS278. Likewise, the QS mutants were impaired in their ability to colonize rice roots. These findings suggest that genes controlled by cinnamoyl-HSL play an important role in the association between rice and ORS278. However, biofilm production was not visibly altered in these mutants. In conclusion, our study highlights the importance of aryl-HSLs in endophytic plant-bacteria interactions.