Search Results (6)

Motility and biofilm formation are two crucial traits in the process of rhizosphere colonization by pseudomonads. The regulation of both traits requires a complex signaling network that is coordinated by the AmrZ-FleQ hub. In this review, we describe the role of this hub in the adaption to the rhizosphere. The study of the direct regulon of AmrZ and the phenotypic analyses of an amrZ mutant in Pseudomonas ogarae F113 has shown that this protein plays a crucial role in the regulation of several cellular functions, including motility, biofilm formation, iron homeostasis, and bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) turnover, controlling the synthesis of extracellular matrix components. On the other hand, FleQ is the master regulator of flagellar synthesis in P. ogarae F113 and other pseudomonads, but its implication in the regulation of multiple traits related with environmental adaption has been shown. Genomic scale studies (ChIP-Seq and RNA-Seq) have shown that in P. ogarae F113, AmrZ and FleQ are general transcription factors that regulate multiple traits. It has also been shown that there is a common regulon shared by the two transcription factors. Moreover, these studies have shown that AmrZ and FleQ form a regulatory hub that inversely regulate traits such as motility, extracellular matrix component production, and iron homeostasis. The messenger molecule c-di-GMP plays an essential role in this hub since its production is regulated by AmrZ and it is sensed by FleQ and required for its regulatory role. This regulatory hub is functional both in culture and in the rhizosphere, indicating that the AmrZ-FleQ hub is a main player of P. ogarae F113 adaption to the rhizosphere environment. Full article

Phenazine-1-carboxamide (PCN) is effective to control many plant pathogens, and improving PCN production would be of great significance in promoting its development as a biopesticide. This study was conducted to improve the PCN production of Pseudomonas chlororaphis H5△fleQ△relA through fermentation optimization in both shake flask and bioreactor. The PCN production of H5△fleQ△relA was improved from 2.75 ± 0.23 g/L to 5.51 ± 0.17 g/L by medium optimization in shake flask using Plackett-Burman design, the path of steepest ascent experiment and central composite design. Then, PCN production reached 8.58 ± 0.25 g/L through optimizing pH in 1 L bioreactor. After pH optimization, the transcriptional levels of ccoO_2 and ccoQ_2 genes related to microbial aerobic respiration were significantly upregulated, and the relative abundance of 3-oxo-C14-HSL was significantly enhanced 15-fold, and these changes were vital for cell activity and metabolites production. Furthermore, the PCN production reached 9.58 ± 0.57 g/L after optimization of the fed-batch fermentation strategy in 1 L bioreactor. Finally, the fermentation scale-up of the optimal medium and optimal feeding strategy were conducted in 30 L bioreactor at the optimal pH, and their PCN production reached 9.17 g/L and 9.62 g/L respectively, which were comparable to that in 1 L bioreactor. In this study, the high PCN production was achieved from the shake-flask fermentation to 30 L bioreactor, and the optimal feeding strategy improved PCN production in bioreactor without increasing total glycerol compared with in shake flask. It provides promising pathways for the optimization of processes for the production of other phenazines. Full article

Pseudomonas fluorescens, a gram-negative bacterium, has been proven to be a capable protein manufacturing factory (PMF). Utilizing its ATP-binding cassette (ABC) transporter, a type I secretion system, P. fluorescens has successfully produced recombinant proteins. However, besides the target proteins, P. fluorescens also secretes unnecessary background proteins that complicate protein purification and other downstream processes. One of the background proteins produced in large amounts is FliC, a flagellin protein. In this study, the master regulator of flagella gene expression, fleQ, was deleted from P. fluorescens Δtp, a lipase and protease double-deletion mutant, via targeted gene knockout. FleQ directs flagella synthesis, so the new strain, P. fluorescens ΔfleQ, does not produce flagella-related proteins. This not only simplifies purification but also makes P. fluorescens ΔfleQ an eco-friendly expression host because it will not survive outside a controlled environment. Six recombinant growth factors, namely, insulin-like growth factors I and II, beta-nerve growth factor, fibroblast growth factor 1, transforming growth factor beta, and tumor necrosis factor beta, prepared using our supercharging method, were successfully secreted by P. fluorescens ΔfleQ. Our findings demonstrate the potential of P. fluorescens ΔfleQ, combined with our supercharging process, as a PMF. Full article

OmpA, which encodes outer membrane protein A (OmpA), is the most abundant transcript in Stenotrophomonas maltophilia based on transcriptome analyses. The functions of OmpA, including adhesion, biofilm formation, drug resistance, and immune response targets, have been reported in some microorganisms, but few functions are known in S. maltophilia. This study aimed to elucidate the relationship between OmpA and swimming motility in S. maltophilia. KJΔOmpA, an ompA mutant, displayed compromised swimming and failure of conjugation-mediated plasmid transportation. The hierarchical organization of flagella synthesis genes in S. maltophilia was established by referencing the Pseudomonas aeruginosa model and was confirmed using mutant construction, qRT-PCR, and functional assays. Distinct from the P. aeruginosa model, rpoN, rather than fleQ and fliA, was at the top of the flagellar regulatory cascade in S. maltophilia. To elucidate the underlying mechanism responsible for ΔompA-mediated swimming compromise, transcriptome analysis of KJ and KJΔOmpA was performed and revealed rpoN downregulation in KJΔOmpA as the key element. The involvement of rpoN in ΔompA-mediated swimming compromise was verified using rpoN complementation, qRT-PCR, and function assays. Collectively, OmpA, which contributes to bacterial conjugation and swimming, is a promising target for adjuvant design in S. maltophilia. Full article

Pseudomonas simiae PICF7 is an indigenous inhabitant of the olive (Olea europaea L.) rhizosphere/root endosphere and an effective biocontrol agent against Verticillium wilt of olive (VWO), caused by the soil-borne fungus Verticillium dahliae. This study aimed to evaluate the potential involvement of selected phenotypes of strain PICF7 in root colonization ability and VWO biocontrol. Therefore, a random transposon-insertion mutant bank of P. simiae PICF7 was screened for the loss of phenotypes likely involved in rhizosphere/soil persistence (copper resistance), root colonization (biofilm formation) and plant growth promotion (phytase activity). Transposon insertions in genes putatively coding for the transcriptional regulator CusR or the chemotaxis protein CheV were found to affect copper resistance, whereas an insertion in fleQ gene putatively encoding a flagellar regulatory protein hampered the ability to form a biofilm. However, these mutants displayed the same antagonistic effect against V. dahliae as the parental strain. Remarkably, two mutants impaired in biofilm formation were never found inside olive roots, whereas their ability to colonize the root exterior and to control VWO remained unaffected. Endophytic colonization of olive roots was unaltered in mutants impaired in copper resistance and phytase production. Results demonstrated that the phenotypes studied were irrelevant for VWO biocontrol. Full article

To acclimate to different environments, gene expression has to be controlled using diverse transcriptional activators. FleQ activates σ⁵⁴-dependent transcription initiation and regulates flagellar biosynthesis and other mechanisms in several bacteria. Xanthomonas oryzae pv. oryzae (Xoo), which is a causal agent of bacterial leaf blight on rice, lacking FleQ loses swimming motility and virulence is not altered. However, other biological mechanisms related with FleQ in Xoo are unknown. In this study, we generated the FleQ-overexpressing strain, Xoo(FleQ), and knockout mutant, XooΔfleQ. To predict the mechanisms affected by FleQ, label-free shotgun comparative proteomics was carried out. Based on proteomic results, we performed diverse phenotypic assays. Xoo(FleQ) had reduced ability to elicit disease symptoms and exopolysaccharide production. Additionally, the ability of XooΔfleQ(EV) (empty vector) and Xoo(FleQ) to form biofilm was decreased. Swarming motility of XooΔfleQ(EV) was abolished, but was only reduced for Xoo(FleQ). Additionally, abnormal twitching motility was observed in both strains. Siderophore production of Xoo(FleQ) was enhanced in iron-rich conditions. The proteomic and phenotypic analyses revealed that FleQ is involved in flagellar-dependent motility and other mechanisms, including symptom development, twitching motility, exopolysaccharide production, biofilm formation, and siderophore production. Thus, this study provides fundamental information about a σ⁵⁴-dependent transcription activator in Xoo. Full article