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International audience; Ferredoxin-NAD(P) + oxidoreductases are important enzymes for redox balancing in n-butanol production by Clostridium acetobutylicum, but the encoding genes remain unknown. Here, the authors identify the long sought-after genes and increase n-butanol production by optimizing the levels of the two enzymes.Clostridium acetobutylicum is a promising biocatalyst for the renewable production of n-butanol. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it has previously demonstrated that the activities of both ferredoxin-NADP(+) reductase and ferredoxin-NAD(+) reductase, whose encoding genes remain unknown, are necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purify, identify and partially characterize the proteins responsible for both activities and demonstrate the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrate the yield of butanol formation is limited by the level of expression of CA_C0764, the ferredoxin-NADP(+) reductase encoding gene and the bcd operon, encoding a ferredoxin-NAD(+) reductase. The integration of these enzymes into metabolic engineering strategies introduces opportunities for developing a homobutanologenic C. acetobutylicum strain.

International audience; Pseudomonas aeruginosa is an opportunistic bacterium of which the main virulence factor is the Type III Secretion System. The ATPase of this machinery, PscN (SctN), is thought to be localized at the base of the secretion apparatus and to participate in the recognition, chaperone dissociation and unfolding of exported T3SS proteins. In this work, a protein-protein interaction ELISA revealed the interaction of PscN with a wide range of exported T3SS proteins including the needle, translocator, gate-keeper and effector. These interactions were further confirmed by Microscale Thermophoresis that also indicated a preferential interaction of PscN with secreted proteins or protein-chaperone complex rather than with chaperones alone, in line with the release of the chaperones in the bacterial cytoplasm after the dissociation from their exported proteins. Moreover, we suggest a new role of the gate-keeper complex and the ATPase in the regulation of early substrates recognition by the T3SS. This finding sheds a new light on the mechanism of secretion switching from early to middle substrates in P. aeruginosa .

Pseudomonas aeruginosais an opportunistic bacterium of which the main virulence factor is the Type III Secretion System. The ATPase of this machinery, PscN (SctN), is thought to be localized at the base of the secretion apparatus and to participate in the recognition, chaperone dissociation and unfolding of exported T3SS proteins. In this work, a protein-protein interaction ELISA revealed the interaction of PscN with a wide range of exported T3SS proteins including the needle, translocator, gate-keeper and effector. These interactions were further confirmed by Microscale Thermophoresis that also indicated a preferential interaction of PscN with secreted proteins or protein-chaperone complex rather than with chaperones alone, in line with the release of the chaperones in the bacterial cytoplasm after the dissociation from their exported proteins. Moreover, we suggest a new role of the gate-keeper complex and the ATPase in the regulation of early substrates recognition by the T3SS. This finding sheds a new light on the mechanism of secretion switching from early to middle substrates inP. aeruginosa.HighlightsT3SS substrates are secreted sequentially but information on the switches are missingInteraction of the T3SS ATPase with secreted proteins were investigated by different approachesMicroscale Thermophoresis revealed a lower affinity for chaperones alone compared to complexesThe Gate-keeper complex binds to the ATPase and increases its affinity for the needle complexA new role of the Gate-keeper complex is proposed, directly acting on the T3SS ATPase

Pseudomonas aeruginosa is responsible for high-morbidity infections of cystic fibrosis patients and is a major agent of nosocomial infections. One of its most potent virulence factors is a type III secretion system (T3SS) that injects toxins directly into the host cell cytoplasm. ExsB, a lipoprotein localized in the bacterial outer membrane, is one of the components of this machinery, of which the function remained elusive until now. The localization of the exsB gene within the exsCEBA regulatory gene operon suggested an implication in the T3SS regulation, while its similarity with yscW from Yersinia spp. argued in favor of a role in machinery assembly. The present work shows that ExsB is necessary for full in vivo virulence of P. aeruginosa . Furthermore, the requirement of ExsB for optimal T3SS assembly and activity is demonstrated using eukaryotic cell infection and in vitro assays. In particular, ExsB promotes the assembly of the T3SS secretin in the bacterial outer membrane, highlighting the molecular role of ExsB as a pilotin. This involvement in the regulation of the T3S apparatus assembly may explain the localization of the ExsB-encoding gene within the regulatory gene operon.

International audience; Gram-negative bacteria utilize a dedicated membrane-embedded apparatus, the type III secretion system (T3SS), to inject proteins into host cells. The passage of the proteins across the target membrane is accomplished by a proteinaceous pore-the translocon-formed within the host-cell cytoplasmic membrane. Translocators bound to their chaperones can be expressed in Escherichia coli and subsequently dissociated from the chaperone by guanidine treatment. The pore formation properties of the translocators can then be studied by an in-vitro liposome leakage assay. Sulforhodamine-B is encapsulated within lipid vesicles during liposome preparation. At high concentration, this fluorochrome exhibits self-quenching limiting fluorescence emission. Upon pore formation, liposome leakage leads to the dilution of Sulforhodamine-B in the medium and fluorescence emission increases. Alternatively, fluorochromes coupled to large dextran molecules can be encapsulated in order to estimate pore dimensions. Here we describe protein expression and purification, dye-liposome preparation, and leakage assay conditions.

Gram-negative bacteria utilize a dedicated membrane-embedded apparatus, the type III secretion system (T3SS), to inject proteins into host cells. The passage of the proteins across the target membrane is accomplished by a proteinaceous pore-the translocon-formed within the host-cell cytoplasmic membrane. Translocators bound to their chaperones can be expressed in Escherichia coli and subsequently dissociated from the chaperone by guanidine treatment. The pore formation properties of the translocators can then be studied by an in-vitro liposome leakage assay. Sulforhodamine-B is encapsulated within lipid vesicles during liposome preparation. At high concentration, this fluorochrome exhibits self-quenching limiting fluorescence emission. Upon pore formation, liposome leakage leads to the dilution of Sulforhodamine-B in the medium and fluorescence emission increases. Alternatively, fluorochromes coupled to large dextran molecules can be encapsulated in order to estimate pore dimensions. Here we describe protein expression and purification, dye-liposome preparation, and leakage assay conditions.

International audience; Pseudomonas aeruginosa is an opportunistic human pathogen that employs a finely tuned type III secretion system (T3SS) to inject toxins directly into the cytoplasm of target cells. ExsB is a 15.6-kDa protein encoded in a T3SS transcription regulation operon that displays high sequence similarity to YscW, a lipoprotein from Yersinia spp. whose genetic neighborhood also involves a transcriptional regulator, and has been shown to play a role in the stabilization of the outer membrane ring of the T3SS. Here, we show that ExsB is expressed in P. aeruginosa upon induction of the T3SS, and subcellular fractionation studies reveal that it is associated with the outer membrane. The high-resolution crystal structure of ExsB shows that it displays a compact β-sandwich fold with interdependent β-sheets. ExsB possesses a large patch of basic residues that could play a role in membrane recognition, and its structure is distinct from that of MxiM, a lipoprotein involved in secretin stabilization in Shigella, as well as from those of Pil lipoproteins involved in pilus biogenesis. These results reveal that small lipoproteins involved in formation of the outer membrane secretin ring display clear structural differences that may be related to the different functions they play in these systems.