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The understanding of protein-protein interaction mechanisms is key to the atomistic description of cell signaling pathways and for the development of new drugs. In this context, the mechanism of intrinsically disordered proteins folding upon binding has attracted attention. The VirB9 C-terminal domain (VirB9Ct) and the VirB7 N-terminal motif (VirB7Nt) associate with VirB10 to form the outer membrane core complex of the Type IV Secretion System injectisome. Despite forming a stable and rigid complex, VirB7Nt behaves as a random coil, while VirB9Ct is intrinsically dynamic in the free state. Here we combined NMR, stopped-flow fluorescence, and computer simulations using structure-based models to characterize the VirB9Ct-VirB7Nt coupled folding and binding mechanism. Qualitative data analysis suggested that VirB9Ct preferentially binds to VirB7Nt by way of a conformational selection mechanism at lower temperatures. However, at higher temperatures, energy barriers between different VirB9Ct conformations are more easily surpassed. Under these conditions the formation of non-native initial encounter complexes may provide alternative pathways toward the native complex conformation. These observations highlight the intimate relationship between folding and binding, calling attention to the fact that the two molecular partners must search for the most favored intramolecular and intermolecular interactions on a rugged and funnelled conformational energy landscape, along which multiple intermediates may lead to the final native state.

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Many bacteria kill rival species by translocating toxic effectors into target cells. Effectors are often encoded along with cognate immunity proteins that could (i) protect against "friendly-fire" (trans-intoxication) from neighboring sister cells and/or (ii) protect against internal cis-intoxication (suicide). Here, we distinguish between these two mechanisms in the case of the bactericidal Xanthomonas citri Type IV Secretion System (X-T4SS). We use a set of X. citri mutants lacking multiple effector/immunity protein (X-Tfe/X-Tfi) pairs to show that X-Tfis are not absolutely required to protect against trans-intoxication by wild-type cells. Our investigation then focused on the in vivo function of the lysozyme-like effector X-TfeXAC2609 and its cognate immunity protein X-TfiXAC2610. In the absence of X-TfiXAC2610, we observe X-TfeXAC2609-dependent and X-T4SS-independent accumulation of damage in the X. citri cell envelope, cell death, and inhibition of biofilm formation. While immunity proteins in other systems have been shown to protect against attacks by sister cells (trans-intoxication), this is an example of an antibacterial secretion system in which the immunity proteins are dedicated to protecting cells against cis-intoxication.

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The genus Xanthomonas includes more than 30 phytopathogenic species that infect a wide range of plants and cause severe diseases that greatly impact crop productivity. These bacteria are highly adapted to the soil and plant environment, being found in decaying material, as epiphytes, and colonizing the plant mesophyll. Signal transduction mechanisms involved in the responses of Xanthomonas to environmental changes are still poorly characterized. Xanthomonad genomes typically encode several representatives of the extracytoplasmic function σ (σECF) factors, whose physiological roles remain elusive. In this work, we functionally characterized the Xanthomonas citri pv. citri EcfL, a σECF factor homologous to members of the iron-responsive FecI-like group. We show that EcfL is not required or induced during iron starvation, despite presenting the common features of other FecI-like σECF factors. EcfL positively regulates one operon composed of three genes that encode a TonB-dependent receptor involved in cell surface signaling, an acid phosphatase, and a lectin-domain containing protein. Furthermore, we demonstrate that EcfL is required for full virulence in citrus, and its regulon is induced inside the plant mesophyll and in response to acid stress. Together, our study suggests a role for EcfL in the adaptation of X. citri to the plant environment, in this way contributing to its ability to cause citrus canker disease. IMPORTANCE The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide variety of economically important plants worldwide. Bacterial adaptation to the plant and soil environment relies on their repertoire of signal transduction pathways, including alternative sigma factors of the extracytoplasmic function family (σECF). Here, we describe a new σECF factor found in several Xanthomonas species, demonstrating its role in Xanthomonas citri virulence to citrus plants. We show that EcfL regulates a single operon containing three genes, which are also conserved in other Xanthomonas species. This study further expands our knowledge on the functions of the widespread family of σECF factors in phytopathogenic bacteria.

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Many soil-, water-, and plant-associated bacterial species from the orders Xanthomonadales, Burkholderales, and Neisseriales carry a type IV secretion system (T4SS) specialized in translocating effector proteins into other gram-negative species, leading to target cell death. These effectors, known as X-Tfes, carry a carboxyl-terminal domain of ∼120 residues, termed XVIPCD, characterized by several conserved motifs and a glutamine-rich tail. Previous studies showed that the XVIPCD is required for interaction with the T4SS coupling protein VirD4 and for T4SS-dependent translocation. However, the structural basis of the XVIPCD-VirD4 interaction is unknown. Here, we show that the XVIPCD interacts with the central all-alpha domain of VirD4 (VirD4AAD). We used solution NMR spectroscopy to solve the structure of the XVIPCD of X-TfeXAC2609 from Xanthomonas citri and to map its interaction surface with VirD4AAD Isothermal titration calorimetry and in vivo Xanthomonas citri versus Escherichia coli competition assays using wild-type and mutant X-TfeXAC2609 and X-TfeXAC3634 indicate that XVIPCDs can be divided into two regions with distinct functions: the well-folded N-terminal region contains specific conserved motifs that are responsible for interactions with VirD4AAD, while both N- and carboxyl-terminal regions are required for effective X-Tfe translocation into the target cell. The conformational stability of the N-terminal region is reduced at and below pH 7.0, a property that may facilitate X-Tfe unfolding and translocation through the more acidic environment of the periplasm.

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Type IV pili (T4P) are thin and flexible filaments found on the surface of a wide range of Gram-negative bacteria that undergo cycles of extension and retraction and participate in a variety of important functions related to lifestyle, defense and pathogenesis. During pilus extensions, the PilB ATPase energizes the polymerization of pilin monomers from the inner membrane. In Xanthomonas citri, two cytosolic proteins, PilZ and the c-di-GMP receptor FimX, are involved in the regulation of T4P biogenesis through interactions with PilB. In vivo fluorescence microscopy studies show that PilB, PilZ and FimX all colocalize to the leading poles of X. citri cells during twitching motility and that this colocalization is dependent on the presence of all three proteins. We demonstrate that full-length PilB, PilZ and FimX can interact to form a stable complex as can PilB N-terminal, PilZ and FimX C-terminal fragments. We present the crystal structures of two binary complexes: i) that of the PilB N-terminal domain, encompassing sub-domains ND0 and ND1, bound to PilZ and ii) PilZ bound to the FimX EAL domain within a larger fragment containing both GGDEF and EAL domains. Evaluation of PilZ interactions with PilB and the FimX EAL domain in these and previously published structures, in conjunction with mutagenesis studies and functional assays, allow us to propose an internally consistent model for the PilB-PilZ-FimX complex and its interactions with the PilM-PilN complex in the context of the inner membrane platform of the X. citri Type IV pilus.

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Bacteria of the Xanthomonas genus are mainly phytopathogens of a large variety of crops of economic importance worldwide. Xanthomonas spp. rely on an arsenal of protein effectors, toxins and adhesins to adapt to the environment, compete with other microorganisms and colonize plant hosts, often causing disease. These protein effectors are mainly delivered to their targets by the action of bacterial secretion systems, dedicated multiprotein complexes that translocate proteins to the extracellular environment or directly into eukaryotic and prokaryotic cells. Type I to type VI secretion systems have been identified in Xanthomonas genomes. Recent studies have unravelled the diverse roles played by the distinct types of secretion systems in adaptation and virulence in xanthomonads, unveiling new aspects of their biology. In addition, genome sequence information from a wide range of Xanthomonas species and pathovars have become available recently, uncovering a heterogeneous distribution of the distinct families of secretion systems within the genus. In this review, we describe the architecture and mode of action of bacterial type I to type VI secretion systems and the distribution and functions associated with these important nanoweapons within the Xanthomonas genus.

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Several Xanthomonas species have a type IV secretion system (T4SS) that injects a cocktail of antibacterial proteins into neighbouring Gram-negative bacteria, often leading to rapid lysis upon cell contact. This capability represents an obvious fitness benefit since it can eliminate competition while the liberated contents of the lysed bacteria could provide an increase in the local availability of nutrients. However, the production of this Mega Dalton-sized molecular machine, with over a hundred subunits, also imposes a significant metabolic cost. Here we show that the chromosomal virB operon, which encodes the structural genes of this T4SS in X. citri, is regulated by the conserved global regulator CsrA. Relieving CsrA repression from the virB operon produced a greater number of T4SSs in the cell envelope and an increased efficiency in contact-dependent lysis of target cells. However, this was also accompanied by a physiological cost leading to reduced fitness when in co-culture with wild-type X. citri. We show that T4SS production is constitutive despite being downregulated by CsrA. Cells subjected to a wide range of rich and poor growth conditions maintain a constant density of T4SSs in the cell envelope and concomitant interbacterial competitiveness. These results show that CsrA provides a constant though partial repression on the virB operon, independent of the tested growth conditions, in this way controlling T4SS-related costs while at the same time maintaining X. citri's aggressive posture when confronted by competitors.

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The class D ß-lactamase OXA-143 has been described as an efficient penicillinase, oxacillinase, and carbapenemase. The D224A variant, known as OXA-231, was described in 2012 as exhibiting less activity toward imipenem and increased oxacillinase activity. Additionally, the P227S mutation was reported as a case of convergent evolution for homologous enzymes. To investigate the impact of both mutations (D224A and P227S), we describe in this paper a deep investigation of the enzymatic activities of these three homologues. OXA-143(P227S) presented enhanced catalytic activity against ampicillin, oxacillins, aztreonam, and carbapenems. In addition, OXA-143(P227S) was the only member capable of hydrolyzing ceftazidime. These enhanced activities were due to a combination of a higher affinity (lower Km) and a higher turnover number (higher kcat). We also determined the crystal structure of apo OXA-231. As expected, the structure of this variant is very similar to the published OXA-143 structure, except for the two M223 conformations and the absence of electron density for three solvent-exposed loop segments. Molecular dynamics calculations showed that both mutants experience higher flexibility compared to that of the wild-type form. Therefore, our results illustrate that D224A and P227S act as deleterious and positive mutations, respectively, within the evolutionary path of the OXA-143 subfamily toward a more efficient carbapenemase.

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Xanthomonas citri pv. citri (X. citri pv. citri) is the causal agent of Asiatic citrus canker and infects economically important citrus crops. X. citri pv. citri contains one type VI secretion system (T6SS) required for resistance to predation by the soil amoeba Dictyostelium discoideum and induced by the ECF sigma factor EcfK in the presence of amoeba. In this work, we describe the analysis of T6SS gene expression during interaction with host plants. We show that T6SS genes and the cognate positive regulator ecfK are upregulated during growth in the plant surface (epiphytic) and maintain low expression levels during growth inside plant mesophyll. In addition, expression of the virulence-associated T3SS is also induced during epiphytic growth and shows a temporal induction pattern during growth inside plant leaves. The T6SS is not required for adhesion to leaf surface and biofilm formation during the first stages of plant colonization nor for killing of yeasts cells. Since the phyllosphere is colonized by eukaryotic predators of bacteria, induction of the X. citri pv. citri anti-amoeba T6SS during epiphytic growth suggests the presence of an environmental signal that triggers the resistance phenotype.

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Bacteria have been constantly competing for nutrients and space for billions of years. During this time, they have evolved many different molecular mechanisms by which to secrete proteinaceous effectors in order to manipulate and often kill rival bacterial and eukaryotic cells. These processes often employ large multimeric transmembrane nanomachines that have been classified as types I-IX secretion systems. One of the most evolutionarily versatile are the Type IV secretion systems (T4SSs), which have been shown to be able to secrete macromolecules directly into both eukaryotic and prokaryotic cells. Until recently, examples of T4SS-mediated macromolecule transfer from one bacterium to another was restricted to protein-DNA complexes during bacterial conjugation. This view changed when it was shown by our group that many Xanthomonas species carry a T4SS that is specialized to transfer toxic bacterial effectors into rival bacterial cells, resulting in cell death. This review will focus on this special subtype of T4SS by describing its distinguishing features, similar systems in other proteobacterial genomes, and the nature of the effectors secreted by these systems and their cognate inhibitors.