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Pseudomonas putida is a promising strain for lignin valorisation. However, there is a dearth of stable and efficient systems for secreting enzymes to enhance the process. Therefore, a novel secretion system for recombinant lignin-depolymerising peroxidase was developed. By adopting a flagellar type III secretion system, P. putida KT-M2, a secretory host strain, was constructed and an optimal secretion signal fusion partner was identified. Application of the dye-decolourising peroxidase of P. putida to this system resulted in efficient oxidation activity of the cell-free supernatant against various chemicals, including lignin model compounds. This peroxidase-secreting strain was examined to confirm its lignin utilisation capability, resulting in the efficient assimilation of various lignin substrates with 2.6-fold higher growth than that of the wild-type strain after 72 h of cultivation. Finally, this novel system will lead efficient bacterial lignin breakdown and utilization through enzyme secretion, paving the way for sustainable lignin-consolidated bioprocessing.

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Export of type 3 secretion (T3S) substrates is traditionally evaluated using trichloroacetic acid (TCA) precipitation of cultured cell supernatants followed by western blot analysis of the secreted substrates. In our lab, we have developed ß-lactamase (Bla), lacking its Sec secretion signal, as a reporter for the export of flagellar proteins into the periplasm via the flagellar T3S system. Bla is normally exported into the periplasm through the SecYEG translocon. Bla must be secreted into the periplasm in order to fold into an active conformation, where it acts to cleave ß-lactams (such as ampicillin) to confer ampicillin resistance (ApR) to the cell. The use of Bla as a reporter for flagellar T3S allows the relative comparison of translocation efficiency of a particular fusion protein in different genetic backgrounds. In addition, it can also be used as a positive selection for secretion. Graphical overview Utilization of ß-lactamase (Bla) lacking its Sec secretion signal and fused to flagellar proteins to assay the secretion of exported flagellar substrates, into the periplasm, through the flagellar T3S system. A. Bla is normally transported into the periplasm space through the Sec secretion pathway, where it folds into an active conformation and allows resistance to ampicillin (ApR). B. Bla, lacking its Sec secretion signal, is fused to flagellar proteins to assay the secretion of exported flagellar proteins into the periplasm through the flagellar T3S system.

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Pectobacterium carotovorum subsp. carotovorum (Pcc) is a Gram-negative phytopathogenic bacterium that produces carocin, a low-molecular-weight bacteriocin that can kill related strains in response to factors in the environment such as UV exposure or nutritional deficiency. The function of the catabolite activator protein (CAP), also known as the cyclic AMP receptor protein (CRP), as a regulator of carocin synthesis was examined. The crp gene was knocked out as part of the investigation, and the outcomes were assessed both in vivo and in vitro. Analysis of the DNA sequence upstream of the translation initiation site of carocin S3 revealed two putative binding sites for CRP that were confirmed using a biotinylated probe pull-down experiment. This study revealed that the deletion of crp inhibited genes involved in extracellular bacteriocin export via the flagellar type III secretion system and impacted the production of many low-molecular-weight bacteriocins. The biotinylated probe pull-down test demonstrated that when UV induction was missing, CRP preferentially attached to one of the two CAP sites while binding to both when UV induction was present. In conclusion, our research aimed to simulate the signal transduction system that controls the expression of the carocin gene in response to UV induction.

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The flagellum is a sophisticated nanomachine responsible for motility in Gram-negative bacteria. Flagellar assembly is a strictly choreographed process, in which the motor and export gate are formed first, followed by the extracellular propeller structure. Extracellular flagellar components are escorted to the export gate by dedicated molecular chaperones for secretion and self-assembly at the apex of the emerging structure. The detailed mechanisms of chaperone-substrate trafficking at the export gate remain poorly understood. Here, we structurally characterized the interaction of Salmonella enterica late-stage flagellar chaperones FliT and FlgN with the export controller protein FliJ. Previous studies showed that FliJ is absolutely required for flagellar assembly since its interaction with chaperone-client complexes controls substrate delivery to the export gate. Our biophysical and cell-based data show that FliT and FlgN bind FliJ cooperatively, with high affinity and on specific sites. Chaperone binding completely disrupts the FliJ coiled-coil structure and alters its interactions with the export gate. We propose that FliJ aids the release of substrates from the chaperone and forms the basis of chaperone recycling during late-stage flagellar assembly.

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The plant pathogen Pectobacterium carotovorum subsp. carotovorum (previously Erwinia carotovora subsp. carotovora) causes soft rot and stem rot diseases in a variety of crops, including Chinese cabbage, potato, and tomato. The flagellar-type III secretion systems were used by Pcc's virulence mechanism to export proteins or bacteriocins to the outside of the cell. DGC, a virulence factor that cyclizes c-di-GMP, a common secondary signal in physiological processes and toxin control systems of many bacteria, was discovered in Pcc's genomic DNA. The dgc gene in Pcc was blocked using the method of homologous recombination in our study. In the in vivo setting, the results demonstrated that the dgc knockout strain does not release low molecular weight bacteriocins. The bacteriocin gene (carocin S2, carocin S3, carocin S4) and the flagellar-type III secretion system genes were also unable to be transcribed by the dgc knockout strain in the transcription experiment. We also observed that the amount of bacteriocin expressed changed when the amount of L-glutamine in the environment exceeded a particular level. These data suggested that L-glutamine influenced physiological processes in Pcc strains in some way. We hypothesized a relationship between dgc and the genes involved in Pcc LMWB external export via the flagellar-type secretion system based on these findings. In this study, the current findings led us to propose a mechanism in which DGC's cyclic di-GMP might bind to receptor proteins and positively regulate bacteriocin transcription as well as the synthesis, mobility, and transport of toxins.

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The lack of functional flagella and the ability to prey upon other microorganisms are well-known traits of Lysobacter enzymogenes, a plant beneficial bacterial species. Here, we report a possible link between these two traits in the model strain L. enzymogenes OH11 (OH11). The genome of OH11 encompasses several homologous genes involved in the flagellum formation but it lacks a functional fliC, encoding the flagellin. Despite the lack of the main component of the flagellum, OH11 genome includes genes involved in the flagellar type III secretion system (FT3SS), which is commonly deployed by flagellated bacteria to transport flagellar subunit proteins. To understand the role played by FT3SS in OH11, we showed that the remaining FT3SS genes were expressed under laboratory conditions. Subsequently, we showed that the identified FT3SS genes involved in the secretion of the hook-capping protein FlgD, suggesting OH11 likely possessed a functional FT3SS system. Blocking FT3SS in OH11 via inactivation of the ATPase FliI impaired the secretion of the proteins Le3970 (protease), Le4493 (ß-1,3-glucanase A) and Le1659 (halo acid dehalogenase family), that showed a toxic activity against the yeast Saccharomyces cerevisiae. The possible link between FT3SS and OH11 antagonism towards S. cerevisiae was also confirmed by loss of toxicity in both mutants of ΔfliI and ΔflhB that lacks the FT3SS structural gene flhB when co-cultured with the yeast strain. The design of synthetic proteins toxic against the Gram-negative bacterium Ralstonia solanacearum further supported the involvement of FT3SS in the ability of OH11 to parasitize other microorganisms. Overall, these results revealed a possible cooption of components of FT3SS system in the competition with other microorganisms in the plant beneficial bacterium OH11 and highlighted a functional divergence of FT3SS between flagellated and non-flagellated bacteria.

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During assembly of the bacterial flagellum, structural subunits synthesized inside the cell must be exported across the cytoplasmic membrane before they can crystallize into the nascent flagellar structure. This export process is facilitated by a specialized Flagellar Type III Secretion System (fT3SS) located at the base of each flagellum. Here, we describe three methods-isothermal titration calorimetry, photo-crosslinking using unnatural amino acids, and a subunit capture assay-used to investigate the interactions of flagellar structural subunits with the membrane export machinery component FlhB.

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