Truncated type IV pilin PilA108 activates the intramembrane protease AlgW to cleave MucA and PilA108 itself in vitro

Li, Ronghui; Withers, Ryan; Dai, Jingcheng; Ruan, Jing; Li, Wei; Dai, Yunfeng; An, Weixing; Yu, Dianzhen; Wei, Hongbin; Xia, Ming; Chun-yuan, Tian; Yu, Hongwei D.; Qiu, Dongru

doi:10.1007/s00203-016-1248-y

Cited by 4 publications

(4 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, inactivation of rpoN1 resulted in the downregulation of type IV prepilin gene tapA in the RN12T4 mutant compared to the wild-type strain (Fig. 6A, B, and C), which may partly account for the decrease of swarming motility in this mutant (24)(25)(26)(27). Furthermore, the transcriptional start site mapping of tapA was determined by primer extension analysis and revealed that the conserved Ϫ24/Ϫ12 motifs (GG and GC) characteristic of 54 -dependent promoters were present upstream of the open reading frame (Fig.…”

Section: Resultsmentioning

confidence: 94%

RpoN (σ ⁵⁴ ) Is Required for Floc Formation but Not for Extracellular Polysaccharide Biosynthesis in a Floc-Forming Aquincola tertiaricarbonis Strain

Xia

Zhang

et al. 2017

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Some bacteria are capable of forming flocs, in which bacterial cells become self-flocculated by secreted extracellular polysaccharides and other biopolymers. The floc-forming bacteria play a central role in activated sludge, which has been widely utilized for the treatment of municipal sewage and industrial wastewater. Here, we use a floc-forming bacterium, Aquincola tertiaricarbonis RN12, as a model to explore the biosynthesis of extracellular polysaccharides and the regulation of floc formation. A large gene cluster for exopolysaccharide biosynthesis and a gene encoding the alternative sigma factor RpoN1, one of the four paralogues, have been identified in floc formation-deficient mutants generated by transposon mutagenesis, and the gene functions have been further confirmed by genetic complementation analyses. Interestingly, the biosynthesis of exopolysaccharides remained in the rpoN1-disrupted flocculation-defective mutants, but most of the exopolysaccharides were secreted and released rather than bound to the cells. Furthermore, the expression of exopolysaccharide biosynthesis genes seemed not to be regulated by RpoN1. Taken together, our results indicate that RpoN1 may play a role in regulating the expression of a certain gene(s) involved in the self-flocculation of bacterial cells but not in the biosynthesis and secretion of exopolysaccharides required for floc formation.IMPORTANCE Floc formation confers bacterial resistance to predation of protozoa and plays a central role in the widely used activated sludge process. In this study, we not only identified a large gene cluster for biosynthesis of extracellular polysaccharides but also identified four rpoN paralogues, one of which (rpoN1) is required for floc formation in A. tertiaricarbonis RN12. In addition, this RpoN sigma factor regulates the transcription of genes involved in biofilm formation and swarming motility, as previously shown in other bacteria. However, this RpoN paralogue is not required for the biosynthesis of exopolysaccharides, which are released and dissolved into culture broth by the rpoN1 mutant rather than remaining tightly bound to cells, as observed during the flocculation of the wild-type strain. These results indicate that floc formation is a regulated complex process, and other yet-to-be identified RpoN1-dependent factors are involved in self-flocculation of bacterial cells via exopolysaccharides and/or other biopolymers.

show abstract

Section: Resultsmentioning

confidence: 94%

RpoN (σ ⁵⁴ ) Is Required for Floc Formation but Not for Extracellular Polysaccharide Biosynthesis in a Floc-Forming Aquincola tertiaricarbonis Strain

Xia

Zhang

et al. 2017

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Boucher et al found that AlgU and RpoN have overlapping binding sequences at P algD , and that overexpression of rpoN inhibited mucoidy in PAO578 (20). However, the muc23 mutation was later characterized to be pilA 108 , which produces a truncated type IV pilin with an FTF tail at the C terminus of PilA108 to activate AlgW, which initiates the proteolytic cascade to degrade the full length of MucA (39). Similarly, Yin et al reported that overexpression of rpoN using pHERD20T resulted in the loss of mucoidy in PAO581 (37).…”

Section: Discussionmentioning

confidence: 99%

Pyrimidine Biosynthesis Regulates the Small-Colony Variant and Mucoidy in Pseudomonas aeruginosa through Sigma Factor Competition

Ahmar

Kirby

2019

J Bacteriol

Self Cite

View full text Add to dashboard Cite

Mucoidy due to alginate overproduction by the Gram-negative bacteriumPseudomonas aeruginosafacilitates chronic lung infections in patients with cystic fibrosis (CF). We previously reported that disruption inde novosynthesis of pyrimidines resulted in conversion to a nonmucoid small-colony variant (SCV) in the mucoidP. aeruginosastrain (PAO581), which has a truncated anti-sigma factor, MucA25, that cannot sequester sigma factor AlgU (AlgT). Here, we showed that supplementation with the nitrogenous bases uracil or cytosine in growth medium complemented the SCV to normal growth, and nonmucoidy to mucoidy, in thesemucA25mutants. This conversion was associated with an increase in intracellular levels of UMP and UTP suggesting that nucleotide restoration occurred via a salvage pathway. In addition, supplemented pyrimidines caused an increase in activity of the alginate biosynthesis promoter (PalgD), but had no effect on PalgU, which controls transcription ofalgU. Cytosolic levels of AlgU were not influenced by uracil supplementation, yet levels of RpoN, a sigma factor that regulates nitrogen metabolism, increased with disruption of pyrimidine synthesis and decreased after supplementation of uracil. This suggested that an elevated level of RpoN in SCV may block alginate biosynthesis. To support this, we observed that overexpressingrpoNresulted in a phenotypic switch to nonmucoidy in PAO581 and in mucoid clinical isolates. Furthermore, transcription of an RpoN-regulated promoter increased in the mutants and decreased after uracil supplementation. These results suggest that the balance of RpoN and AlgU levels may regulate growth from SCV to mucoidy through sigma factor competition for PalgD.IMPORTANCEChronic lung infections withP. aeruginosaare the main cause of morbidity and mortality in patients with cystic fibrosis. This bacterium overproduces a capsular polysaccharide called alginate (also known as mucoidy), which aids in bacterial persistence in the lungs and in resistance to therapeutic regimens and host immune responses. The current study explores a previously unknown link between pyrimidine biosynthesis and mucoidy at the level of transcriptional regulation. Identifying/characterizing this link could provide novel targets for the control of bacterial growth and mucoidy. Inhibiting mucoidy may improve antimicrobial efficacy and facilitate host defenses to clear the noncapsulatedP. aeruginosabacteria, leading to improved prognosis for patients with cystic fibrosis.

show abstract

“…Large scale transposon mutagenesis was conducted on the wild type A. tertiaricarbonis RN12 strain by using the mariner transposon delivery vector pmini Himar RB1 as previously described [ 2 , 12 , 13 ]. A series of floc-formation-deficient mutants, whose liquid cultures were turbid and planktonic rather than flocculated as observed in the cultures of the wild type RN12 strain, had been isolated and the transposon insertion site was mapped in most of the floc-deficient mutants.…”

Section: Resultsmentioning

confidence: 99%

An RpoN-dependent PEP-CTERM gene is involved in floc formation of an Aquincola tertiaricarbonis strain

Xia

Chen

et al. 2023

BMC Microbiol

Self Cite

View full text Add to dashboard Cite

Background The floc is a characteristic of microbial aggregate growth, displaying cloudy suspensions in water. Floc formation has been demonstrated in a series of bacteria and the floc-forming bacteria play a crucial role in activated sludge (AS) process widely used for municipal sewage and industrial wastewater treatment over a century. It has been demonstrated that some exopolysaccharide biosynthesis genes and the sigma factor (sigma54 or rpoN) were required for floc forming in some bacteria. However, the mechanism underlying the floc formation stills need to be elucidated. Results In this study, we demonstrate that a TPR (Tetratricopeptide repeats) protein-encoding gene prsT is required for floc formation of Aquincola tertiaricarbonis RN12 and an upstream PEP-CTERM gene (designated pepA), regulated by RpoN1, is involved in its floc formation but not swarming motility and biofilm formation. Overexpression of PepA could rescue the floc-forming phenotype of the rpoN1 mutant by decreasing the released soluble exopolysaccharides and increasing the bound polymers. Conclusion Our results indicate that the wide-spread PEP-CTERM proteins play an important role in the self-flocculation of bacterial cells and may be a component of extracellular polymeric substances required for floc-formation.

show abstract

Truncated type IV pilin PilA108 activates the intramembrane protease AlgW to cleave MucA and PilA108 itself in vitro

Abstract: polypeptide with the C-terminal motif WVF, could induce mucoid conversion in the PAO1 strain. In all, our results provided a model of activation of AlgW by another protein ending with proper motifs. Our data suggest that in addition to MucA cleavage, AlgW may cleave other substrates.

Cited by 4 publications

References 33 publications

RpoN (σ ⁵⁴ ) Is Required for Floc Formation but Not for Extracellular Polysaccharide Biosynthesis in a Floc-Forming Aquincola tertiaricarbonis Strain

RpoN (σ ⁵⁴ ) Is Required for Floc Formation but Not for Extracellular Polysaccharide Biosynthesis in a Floc-Forming Aquincola tertiaricarbonis Strain

Pyrimidine Biosynthesis Regulates the Small-Colony Variant and Mucoidy in Pseudomonas aeruginosa through Sigma Factor Competition

An RpoN-dependent PEP-CTERM gene is involved in floc formation of an Aquincola tertiaricarbonis strain

Contact Info

Product

Resources

About

Truncated type IV pilin PilA108 activates the intramembrane protease AlgW to cleave MucA and PilA108 itself in vitro

Abstract: polypeptide with the C-terminal motif WVF, could induce mucoid conversion in the PAO1 strain. In all, our results provided a model of activation of AlgW by another protein ending with proper motifs. Our data suggest that in addition to MucA cleavage, AlgW may cleave other substrates.

Cited by 4 publications

References 33 publications

RpoN (σ 54 ) Is Required for Floc Formation but Not for Extracellular Polysaccharide Biosynthesis in a Floc-Forming Aquincola tertiaricarbonis Strain

RpoN (σ 54 ) Is Required for Floc Formation but Not for Extracellular Polysaccharide Biosynthesis in a Floc-Forming Aquincola tertiaricarbonis Strain

Pyrimidine Biosynthesis Regulates the Small-Colony Variant and Mucoidy in Pseudomonas aeruginosa through Sigma Factor Competition

An RpoN-dependent PEP-CTERM gene is involved in floc formation of an Aquincola tertiaricarbonis strain

Contact Info

Product

Resources

About

RpoN (σ ⁵⁴ ) Is Required for Floc Formation but Not for Extracellular Polysaccharide Biosynthesis in a Floc-Forming Aquincola tertiaricarbonis Strain

RpoN (σ ⁵⁴ ) Is Required for Floc Formation but Not for Extracellular Polysaccharide Biosynthesis in a Floc-Forming Aquincola tertiaricarbonis Strain