Disruption of MiaA provides insights into the regulation of phenazine biosynthesis under suboptimal growth conditions in Pseudomonas chlororaphis 30-84

Abstract

Many products of secondary metabolism are activated by quorum sensing (QS), yet even at cell densities sufficient for QS, their production may be repressed under suboptimal growth conditions via mechanisms that still require elucidation. For many beneficial plant-associated bacteria, secondary metabolites such as phenazines are important for their competitive survival and plant-protective activities. Previous work established that phenazine biosynthesis in Pseudomonas chlororaphis 30-84 is regulated by the PhzR/PhzI QS system, which in turn is regulated by transcriptional regulator Pip, two-component system RpeA/RpeB and stationary phase/stress sigma factor RpoS. Disruption of MiaA, a tRNA modification enzyme, altered primary metabolism and growth leading to widespread effects on secondary metabolism, including reduced phenazine production and oxidative stress tolerance. Thus, the miaA mutant provided the opportunity to examine the regulation of phenazine production in response to altered metabolism and growth or stress tolerance. Despite the importance of MiaA for translation efficiency, the most significant effect of miaA disruption on phenazine production was the reduction in the transcription of phzR, phzI and pip, whereas neither the transcription nor translation of RpeB, a transcriptional regulator of pip, was affected. Constitutive expression of rpeB or pip in the miaA mutant completely restored phenazine production, but it resulted in further growth impairment. Constitutive expression of RpoS alleviated sensitivity to oxidative stress resulting from RpoS translation inefficiency in the miaA mutant, but it did not restore phenazine production. Our results support the model that cells curtail phenazine biosynthesis under suboptimal growth conditions via RpeB/Pip-mediated regulation of QS.

An upstream sequence modulates phenazine production at the level of transcription and translation in the biological control strain Pseudomonas chlororaphis 30-84

“…Since RsmE is a post-transcriptional regulator, we constructed the translational fusion reporter plasmids pJMYX6 (control) and pJMYX7 (90-bp deletion) ( Table 1 and Fig 4B ). Sequence spanning -124 to +175 relative to the TSS of phzX (includes first 20 codons of PhzX) with and without with the 90-bp sequence in the 5’-UTR of phenazine biosynthetic operon were fused in frame with the 8 th codon of lacZ to create the translational fusions pJMYX6 (control) and pJMYX7 (90-bp deletion), respectively, in pME6015 [ 34 , 45 ]. The β-galactosidase activity (translational expression) of pJMYX7 (1743 ± 46 MU) was 3.2-fold higher than pJMYX6 (540 ± 63 MU) in the wild-type background ( Fig 4C ), which is similar to the relative fold change in transcriptional expression between pJMYX1 and pJMYX2 in the wild-type ( Fig 4A ).…”

“…One hypothesis is that the cluster of phenazine regulatory ( phzI/phzR ) and biosynthetic genes (including phzO ) share a common inheritance among 2OHPCA producers, with selection pressure for maintaining the conserved sequence. It was noted previously that despite the benefits of phenazine production to the ecological fitness of phenazine producers [ 1 , 13 , 16 , 17 , 39 ], phenazine production is stressful for the producing strain [ 45 , 58 ]. It is interesting to speculate that some phenazine derivatives are more stressful to the producer than others.…”

Effect of Producing Different Phenazines on Bacterial Fitness and Biological Control in Pseudomonas chlororaphis 30-84

“…Standard methods were used for plasmid DNA isolation, transformation, cloning, restriction enzyme digestions, agarose gel electrophoresis, and T4 DNA ligation ( Sambrook and Russell, 2001 ). Plasmids were introduced into P. chlororaphis 30-84 and its derivatives using triparental mating as previously described ( Pierson and Thomashow, 1992 ; Yu et al, 2017 ) Polymerase chain reaction (PCR) was carried out using Taq DNA polymerase (New England BioLabs, Ipswich, MA, USA). DNA sequencing was performed at the Laboratory for Genome Technology within the Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA.…”

Phenazines in plant‐beneficial Pseudomonas spp.: biosynthesis, regulation, function and genomics

“…piscium PCL1391 to various stress factors, including subinhibitory concentration of antibiotics (Girard and Rigali, 2011). In P. chlororaphis 30-84, the two-component system RpeA/RpeB positively regulates Pip and modulates pip expression under suboptimal growth conditions (Yu et al, 2017). Interestingly, the expression of rpeA and rpeB were not altered in gacA or rpoS mutants and the expression of gacA, gacS and rpoS were also unaltered in a rpeB mutant .…”