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Bacterial type VII secretion systems secrete a wide range of extracellular proteins that play important roles in bacterial viability and in interactions of pathogenic mycobacteria with their hosts. Mycobacterial type VII secretion systems consist of five subtypes, ESX-1-5, and have four substrate classes, namely, Esx, PE, PPE, and Esp proteins. At least some of these substrates are secreted as heterodimers. Each ESX system mediates the secretion of a specific set of Esx, PE, and PPE proteins, raising the question of how these substrates are recognized in a system-specific fashion. For the PE/PPE heterodimers, it has been shown that they interact with their cognate EspG chaperone and that this chaperone determines the designated secretion pathway. However, both structural and pulldown analyses have suggested that EspG cannot interact with the Esx proteins. Therefore, the determining factor for system specificity of the Esx proteins remains unknown. Here, we investigated the secretion specificity of the ESX-1 substrate pair EsxB_1/EsxA_1 in Mycobacterium marinum Although this substrate pair was hardly secreted when homologously expressed, it was secreted when co-expressed together with the PE35/PPE68_1 pair, indicating that this pair could stimulate secretion of the EsxB_1/EsxA_1 pair. Surprisingly, co-expression of EsxB_1/EsxA_1 with a modified PE35/PPE68_1 version that carried the EspG5 chaperone-binding domain, previously shown to redirect this substrate pair to the ESX-5 system, also resulted in redirection and co-secretion of the Esx pair via ESX-5. Our results suggest a secretion model in which PE35/PPE68_1 determines the system-specific secretion of EsxB_1/EsxA_1.

The pathogen Mycobacterium tuberculosis employs a range of ESX-1 substrates to manipulate the host and build a successful infection. Although the importance of ESX-1 secretion in virulence is well established, the characterization of its individual components and the role of individual substrates is far from complete. Here, we describe the functional characterization of the Mycobacterium marinum accessory ESX-1 proteins EccA1, EspG1 and EspH, i.e. proteins that are neither substrates nor structural components. Proteomic analysis revealed that EspG1 is crucial for ESX-1 secretion, since all detectable ESX-1 substrates were absent from the cell surface and culture supernatant in an espG1 mutant. Deletion of eccA1 resulted in minor secretion defects, but interestingly, the severity of these secretion defects was dependent on the culture conditions. Finally, espH deletion showed a partial secretion defect; whereas several ESX-1 substrates were secreted in normal amounts, secretion of EsxA and EsxB was diminished and secretion of EspE and EspF was fully blocked. Interaction studies showed that EspH binds EspE and therefore could function as a specific chaperone for this substrate. Despite the observed differences in secretion, hemolytic activity was lost in all M. marinum mutants, implying that hemolytic activity is not strictly correlated with EsxA secretion. Surprisingly, while EspH is essential for successful infection of phagocytic host cells, deletion of espH resulted in a significantly increased virulence phenotype in zebrafish larvae, linked to poor granuloma formation and extracellular outgrowth. Together, these data show that different sets of ESX-1 substrates play different roles at various steps of the infection cycle of M. marinum., Author summary M. tuberculosis is a facultative intracellular pathogen that has an intimate relationship with host macrophages. Proteins secreted by the ESX-1 secretion system play an important role in this interaction, for instance by orchestrating the escape from the phagosome into the cytosol of the macrophage. However, the exact role of the ESX-1 substrates is unknown, due to their complicated interdependency for secretion. Here, we study the function of ESX-1 accessory proteins EccA1, EspG1 and EspH in ESX-1 secretion in Mycobacterium marium, the causative agent of fish tuberculosis. We found that these proteins affect the secretion of different substrate classes, which offers an approach to study the roles of these substrate groups. An espG1 deletion broadly aborts ESX-1 secretion and thus resulted in severe attenuation in a zebrafish model for tuberculosis, whereas EccA1 is only crucial under specific growth conditions. The most surprising results were obtained for EspH. This protein seems to function as a molecular chaperone for EspE and is as such involved in the secretion of a small subset of ESX-1 substrates. Disruption of espH showed a dual character: whereas this gene is essential for the successful infection of macrophages, deletion of espH resulted in significantly increased virulence in zebrafish larvae. These data convincingly show that different subsets of ESX-1 substrates play different roles at various steps in the mycobacterial infection cycle.

Eruca sativa , commonly known as rocket salad, is one of the leading leafy vegetables, belonging to the Brassicaceae family that include other species of agricultural importance, such as broccoli, cabbage, cauliflower, and mustard, amongst others. It is widely cultivated and consumed, being its popularity due to its peculiar flavour and nutritional value. On the other hand, to maintain agricultural production, the indiscriminate use of chemical fertilizers, pesticides, and herbicides results in damage to soil and biodiversity reduction and may result in adverse health effects. An effective alternative is the use of biofertilizers based on plant growth-promoting rhizobacteria (PGPR). In this study, we evaluated the use of a Rhizobium sp. strain as biofertilizer for E. sativa , performing different assays to demonstrate its potential. According to the obtained results, the partial sequencing of 16S rRNA gene classified this strain into the genus Rhizobium. This strain is not able to solubilize phosphate under in vitro conditions. However, this strain was an excellent producer of indole-3-acetic acid (IAA) and its precursors. Moreover, this strain produced siderophores in a low concentration. In vitro inoculation of E. sativa plants with this strain resulted in a significative increase of the number of secondary roots at 6 and 8 days post-inoculation compared to the uninoculated treatment. Therefore, our results support the possible inclusion of this strain of Rhizobium sp. in formulations as potential biofertilizer for E. sativa crops.