Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System

Singer, Hanna M.; Erhardt, Marc; Steiner, Andrew M.; Zhang, Min Min; Yoshikami, Doju; Bułaj, Grzegorz; Olivera, Baldomero M.; Hughes, Kelly T.

doi:10.1128/mbio.00115-12

Cited by 35 publications

(47 citation statements)

References 39 publications

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“…To ensure that we did not underestimate their numbers, we also counted hooks engineered to encode an HA epitope tag (40); hooks are short and shear resistant (41). Immunostaining with HA antibodies coupled to Alexa-Fluor 488 was used to visualize the hooks in cells propagated under the same conditions as in Fig.…”

Section: Resultsmentioning

confidence: 99%

More than Motility: Salmonella Flagella Contribute to Overriding Friction and Facilitating Colony Hydration during Swarming

Partridge

Harshey

2012

Journal of Bacteriology

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We show in this study that Salmonella cells, which do not upregulate flagellar gene expression during swarming, also do not increase flagellar numbers per m of cell length as determined by systematic counting of both flagellar filaments and hooks. Instead, doubling of the average length of a swarmer cell by suppression of cell division effectively doubles the number of flagella per cell. The highest agar concentration at which Salmonella cells swarmed increased from the normal 0.5% to 1%, either when flagella were overproduced or when expression of the FliL protein was enhanced in conjunction with stator proteins MotAB. We surmise that bacteria use the resulting increase in motor power to overcome the higher friction associated with harder agar. Higher flagellar numbers also suppress the swarming defect of mutants with changes in the chemotaxis pathway that were previously shown to be defective in hydrating their colonies. Here we show that the swarming defect of these mutants can also be suppressed by application of osmolytes to the surface of swarm agar. The "dry" colony morphology displayed by che mutants was also observed with other mutants that do not actively rotate their flagella. The flagellum/motor thus participates in two functions critical for swarming, enabling hydration and overriding surface friction. We consider some ideas for how the flagellum might help attract water to the agar surface, where there is no free water. Swarming bacteria may be divided into two categories: robust swarmers, which can navigate across a hard agar surface (1.5% agar and above), and temperate swarmers, which can swarm only on a softer agar surface (0.5 to 0.8% agar) (1-3). Robust swarmers include polarly flagellated bacteria, which induce peritrichous flagellation upon surface contact, such as Azospirillum, Rhodospirillum, and Vibrio species, as well as the peritrichously flagellated Proteus species (4-6). These bacteria display a hyperflagellated and hyperelongated swarm cell morphology, which is dramatically different from their broth-grown (swimming) counterparts. Transcriptome studies have shown a swarming-specific gene expression program in the robust swarmers (7-10). In polarly flagellated bacteria, frictional forces on the surface are surmised to slow flagellar rotation and signal altered gene expression (11,12), an observation solidified by experiments demonstrating a direct relationship between motor speed and lateral flagellar (laf) gene transcription (13). How motor speed might be sensed and transduced to activate laf expression is unknown. This phenomenon is apparently unique to polarly flagellated bacteria.Temperate swarmers include Escherichia coli and Bacillus, Pseudomonas, Rhizobium, Salmonella, Serratia, and Yersinia species. Among these, Bacillus subtilis displays increased flagellar numbers and cell length (14), but this morphology is not as dramatic as that seen in the robust swarmers. In a Bacillus cereus transcriptome, several genes, including flagellar genes, were seen to be differentially regulat...

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Section: Resultsmentioning

confidence: 99%

More than Motility: Salmonella Flagella Contribute to Overriding Friction and Facilitating Colony Hydration during Swarming

Partridge

Harshey

2012

Journal of Bacteriology

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“…Both the identified minimal export signal of the flagellin and fulllength anti-s 28 factor FlgM have been successfully used as export signals for substrates which could subsequently cleaved by TEV protease [190,191]. As in the injectisome, both the peptide sequence and an mRNA signal seem to play a role in substrate recognition [189].…”

Section: Export Signalsmentioning

confidence: 99%

“…Both types of T3SS have been used for this purpose: the injectisome was used to export various proteins, including spider silk proteins [261], and has recently been shown to be a useful tool for the production of biopolymer-forming proteins, with increased export levels upon overexpression of the master transcriptional regulator [262]. Similarly, the flagellar T3SS was shown to be able to export a variety of different protein types [191] and subsequently optimized for protein secretion [263]. The injectisome, a system evolutionarily optimized for protein translocation, has also been used to directly transport proteins into host cells, especially to deliver vaccines [264][265][266][267], but also for immunomodulatory cytokines [268].…”

Section: Applications Of the T3ss In Medicine And Biotechnologymentioning

confidence: 99%

Type III secretion systems: the bacterial flagellum and the injectisome

Diepold

Armitage

2015

Phil. Trans. R. Soc. B

189

175

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One contribution of 12 to a theme issue 'The bacterial cell envelope'. The flagellum and the injectisome are two of the most complex and fascinating bacterial nanomachines. At their core, they share a type III secretion system (T3SS), a transmembrane export complex that forms the extracellular appendages, the flagellar filament and the injectisome needle. Recent advances, combining structural biology, cryo-electron tomography, molecular genetics, in vivo imaging, bioinformatics and biophysics, have greatly increased our understanding of the T3SS, especially the structure of its transmembrane and cytosolic components, the transcriptional, post-transcriptional and functional regulation and the remarkable adaptivity of the system. This review aims to integrate these new findings into our current knowledge of the evolution, function, regulation and dynamics of the T3SS, and to highlight commonalities and differences between the two systems, as well as their potential applications.

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“…The FlgM type III secretion system can be used to express and purify recombinant proteins (16). Here we have identified factors that improve yields of secreted FlgM.…”

mentioning

confidence: 99%

Analysis of Factors That Affect FlgM-Dependent Type III Secretion for Protein Purification with Salmonella enterica Serovar Typhimurium

et al. 2014

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Conditions known to affect flagellar gene expression, FlgM stability, and flagellar T3S were tested either alone or in combination to determine their effects on levels of secreted FlgM. These conditions included mutations that affect activity of the flagellar FlhD 4 C 2 master regulatory protein complex or the FlgM T3S chaperone 28 , the removal of Salmonella pathogenicity island 1 (Spi1), the removal of flagellar late secretion substrates that could compete with FlgM for secretion, and changes in the ionic strength of the growth medium. Conditions that enhanced FlgM secretion were combined in order to maximize levels of secreted FlgM. An optimized FlgM secretion strain was used to secrete and isolate otherwise difficult-to-produce proteins and peptides fused to the C terminus of FlgM. These include cysteine-rich, hydrophobic peptides (conotoxins ␦-SVIE and MrVIA), nodulespecific, cysteine-rich antimicrobial peptides (NCR), and a malaria surface antigen domain of apical membrane antigen AMA-1.

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Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System

Cited by 35 publications

References 39 publications

More than Motility: Salmonella Flagella Contribute to Overriding Friction and Facilitating Colony Hydration during Swarming

More than Motility: Salmonella Flagella Contribute to Overriding Friction and Facilitating Colony Hydration during Swarming

Type III secretion systems: the bacterial flagellum and the injectisome

Analysis of Factors That Affect FlgM-Dependent Type III Secretion for Protein Purification with Salmonella enterica Serovar Typhimurium

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