Cyclic Di-GMP and VpsR Induce the Expression of Type II Secretion in Vibrio cholerae.

Vibrio cholerae is a human pathogen that alternates between growth in environmental reservoirs and infection of human hosts, causing severe diarrhea. The second messenger cyclic di-GMP (c-di-GMP) mediates this transition by controlling a wide range of functions, such as biofilms, virulence, and motility. Here, we report that c-di-GMP induces expression of the extracellular protein secretion (eps) gene cluster, which encodes the type II secretion system (T2SS) in V. cholerae Analysis of the eps genes confirmed the presence of two promoters located upstream of epsC, the first gene in the operon, one of which is induced by c-di-GMP. This induction is directly mediated by the c-di-GMP-binding transcriptional activator VpsR. Increased expression of the eps operon did not impact secretion of extracellular toxin or biofilm formation but did increase expression of the pseudopilin protein EpsG on the cell surface.IMPORTANCE Type II secretion systems (T2SSs) are the primary molecular machines by which Gram-negative bacteria secrete proteins and protein complexes that are folded and assembled in the periplasm. The substrates of T2SSs include extracellular factors, such as proteases and toxins. Here, we show that the widely conserved second messenger cyclic di-GMP (c-di-GMP) upregulates expression of the eps genes encoding the T2SS in the pathogen V. cholerae via the c-di-GMP-dependent transcription factor VpsR.

Elizabethkingia anophelis: molecular manipulation and interactions with mosquito hosts.

Flavobacteria (members of the family Flavobacteriaceae) dominate the bacterial community in the Anopheles mosquito midgut. One such commensal, Elizabethkingia anophelis, is closely associated with Anopheles mosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements of E. anophelis have not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation of E. anophelis, including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function in E. anophelis. A flavobacterial expression system based on the promoter PompA was integrated into the E. anophelis chromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-tagged E. anophelis associated with mosquitoes at successive developmental stages and propagated in Anopheles gambiae and Anopheles stephensi but not in Aedes triseriatus mosquitoes. Feeding NanoLuc-tagged cells to A. gambiae and A. stephensi in the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected in Aedes triseriatus mosquitoes under the same conditions. Of the initial E. anophelis cells provided to larvae, 23%, 71%, and 85% were digested in A. stephensi, A. gambiae, and Aedes triseriatus, respectively, demonstrating that E. anophelis adapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells in A. stephensi significantly increased when arginine was added to a sugar diet, showing it to be an important amino acid for E. anophelis. Animal erythrocytes promoted E. anophelis growth in vivo and in vitro, indicating that this bacterium could obtain nutrients by participating in erythrocyte lysis in the mosquito midgut.

The 1.59Å resolution structure of the minor pseudopilin EpsH of Vibrio cholerae reveals a long flexible loop.

The type II secretion complex exports folded proteins from the periplasm to the extracellular milieu. It is used by the pathogenic bacterium Vibrio cholerae to export several proteins, including its major virulence factor, cholera toxin. The pseudopilus is an essential component of the type II secretion system and likely acts as a piston to push the folded proteins across the outer membrane through the secretin pore. The pseudopilus is composed of the major pseudopilin, EpsG, and four minor pseudopilins, EpsH, EpsI, EpsJ and EpsK. We determined the x-ray crystal structure of the head domain of EpsH at 1.59Å resolution using molecular replacement with the previously reported EpsH structure, 2qv8, as the template. Three additional N-terminal amino acids present in our construct prevent an artifactual conformation of residues 160-166, present in one of the two monomers of the 2qv8 structure. Additional crystal contacts stabilize a long flexible loop comprised of residues 104-135 that is more disordered in the 2qv8 structure but is partially observed in our structure in very different positions for the two EpsH monomers in the asymmetric unit. In one of the conformations the loop is highly extended. Modeling suggests the highly charged loop is capable of contacting EpsG and possibly secreted protein substrates, suggesting a role in specificity of pseudopilus assembly or secretion function.

Molecular characterization of a cold-active recombinant xylanase from Flavobacterium johnsoniae and its applicability in xylan hydrolysis.

A novel xylanase gene, xyn10A, was cloned from Flavobacterium johsoniae, overexpressed in a flavobacterial expression system, the recombinant enzyme purified by Ni-affinity chromatography, and enzyme structure and activity analyzed. Xyn10A was found to be a modular xylanase with an Fn3 accessory domain on its N-terminal and a catalytic region on the C-terminal. The optimum pH and temperature for Xyn10A was 8.0 and 30 °C, but Xyn10A retained 50% activity at 4 °C, indicating that Xyn10A is a cold-active xylanase. A Fn3-deletion xylanase had relative activity ca. 3.6-fold lower than the wild-type, indicating that Fn3 promotes xylanase activity. The Fn3 region also contributed to stability of the enzyme at elevated temperatures. However, Fn3 did not bind this xylanase to insoluble substrates. The enzyme hydrolyzed xylo-oligosaccharides into xylobiose, and xylose with xylobiose as the main product, confirming that Xyn10A is a strict endo-ß-1,4-xylanase. Xyn10A also hydrolyzed birchwood and beechwood xylan to yield mainly xylose, xylobiose and xylotriose.

Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species.

The type II secretion system (T2SS) functions as a transport mechanism to translocate proteins from the periplasm to the extracellular environment. The ExeA homologue in Aeromonas hydrophila, GspA(Ah), is an ATPase that interacts with peptidoglycan and forms an inner membrane complex with the ExeB homologue (GspB(Ah)). The complex may be required to generate space in the peptidoglycan mesh that is necessary for the transport and assembly of the megadalton-sized ExeD homologue (GspD(Ah)) secretin multimer in the outer membrane. In this study, the requirement for GspAB in the assembly of the T2SS secretin in Aeromonas and Vibrio species was investigated. We have demonstrated a requirement for GspAB in T2SS assembly in Aeromonas salmonicida, similar to that previously observed in A. hydrophila. In the Vibrionaceae species Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus, gspA mutations significantly decreased assembly of the secretin multimer but had minimal effects on the secretion of T2SS substrates. The lack of effect on secretion of the mutant of gspA of V. cholerae (gspA(Vc)) was explained by the finding that native secretin expression greatly exceeds the level needed for efficient secretion in V. cholerae. In cross-complementation experiments, secretin assembly and secretion in an A. hydrophila gspA mutant were partially restored by the expression of GspAB from V. cholerae in trans, further suggesting that GspAB(Vc) performs the same role in Vibrio species as GspAB(Ah) does in the aeromonads. These results indicate that the GspAB complex is functional in the assembly of the secretin in Vibrio species but that a redundancy of GspAB function may exist in this genus.

In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae.

The type II secretion system is a multi-protein complex that spans the cell envelope of Gram-negative bacteria and promotes the secretion of proteins, including several virulence factors. This system is homologous to the type IV pilus biogenesis machinery and contains five proteins, EpsG-K, termed the pseudopilins that are structurally homologous to the type IV pilins. The major pseudopilin EpsG has been proposed to form a pilus-like structure in an energy-dependent process that requires the ATPase, EpsE. A key remaining question is how the membrane-bound EpsG interacts with the cytoplasmic ATPase, and if this is a direct or indirect interaction. Previous studies have established an interaction between the bitopic inner membrane protein EpsL and EpsE; therefore, in this study we used in vivo cross-linking to test the hypothesis that EpsG interacts with EpsL. Our findings suggest that EpsL may function as a scaffold to link EpsG and EpsE and thereby transduce the energy generated by ATP hydrolysis to support secretion. The recent discovery of structural homology between EpsL and a protein in the type IV pilus system implies that this interaction may be conserved and represent an important functional interaction for both the type II secretion and type IV pilus systems.

Development of an efficient expression system for Flavobacterium strains.

Strong promoters were isolated from Flavobacterium johnsoniae in a promoter-trap vector incorporating a gfp reporter system, and were used to express fluorescent protein markers (including GFP, YFP, mOrange and mStrawberry) and insecticidal protein genes in Flavobacterium strains. Sequence analysis of trapped DNA fragments showed conserved Bacteroidetes promoter motifs (TTG-N(19)-TAnnTTTG) located upstream of putative open reading frames. Plasmids harboring these genomic DNA fragments from F. johnsoniae promoted strong production of fluorescent proteins in Flavobacterium hibernum but not in Escherichiacoli. The most potent promoter (PompA) identified in this work was cloned upstream of genes encoding fluorescent proteins, and these were co-expressed in Flavobacterium strains. The p42 and p51 genes (binary toxins from Bacillus sphaericus) when translationally fused to the 3'-end of gfp showed strong expression. Flavobacteria expressing these genes exhibited toxicity against larvae of the mosquitoes Culex quinquefasciatus, Anopheles gambiae, and Ochlerotatus triseriatus. However, transformants with the transcriptional fusion construct between cry11A with p20 from Bacillus thuringiensis did not express Cry11A protein indicating that constitutive expression of cry11A may be problematic in Flavobacterium.

Cell envelope perturbation induces oxidative stress and changes in iron homeostasis in Vibrio cholerae.

The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189:8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.

Mutational analysis of the ompA promoter from Flavobacterium johnsoniae.

Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other members of the phylum Bacteroidetes, but not of proteobacteria, and they function poorly in Escherichia coli. In order to analyze the Flavobacterium promoter structure systematically, we investigated the -33 consensus element, -7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal -33/-7 motifs (TTTG/TANNTTTG) were identical to Bacteroides fragilis sigma(ABfr) consensus -33/-7 promoter elements but lacked similarity to the E. coli sigma(70) promoter elements. The length of the spacer separating the -33 and -7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.

Organization of a partial S10 operon and its transcriptional analysis in Flavobacterium hibernum strain W22.

A cluster of six genes from Flavobacterium hibernum strain W22, fus-rpsJ-rplC-rplD-rplW-rplB, was cloned and sequenced. A short fragment upstream of rplC, but not of rpsJ, showed strong promoter activity in flavobacteria. TATCTTTG and TTG motifs that are conserved in Flavobacterium promoters were found immediately upstream of the transcription start point of rplC, at the -7 and -33 positions, respectively. RT-PCR analysis of the transcripts revealed that rpsJ and rplC are expressed as separate transcriptional units, whereas rplC and rplD-rplW-rplB are cotranscribed as a single mRNA, in contrast to the situation in the Gammaproteobacteria, which have a single transcriptional unit of 11 ribosomal S10 genes.

Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae.

The type 2 secretion system (T2SS) occurring in Gram-negative bacteria is composed of 12-15 different proteins which form large assemblies spanning two membranes and secreting several virulence factors in folded state across the outer membrane. The T2SS component EpsC of Vibrio cholerae plays an important role in this machinery. While anchored in the inner membrane, by far the largest part of EpsC is periplasmic, containing a so-called homology region (HR) domain and a PDZ domain. Here we report studies on the structure and function of both periplasmic domains of EpsC. The crystal structures of two variants of the PDZ domain of EpsC from V. cholerae were determined at better than 2 A resolution. Compared to the short variant, the longer variant contains an additional N-terminal helix, and reveals a significant difference in the position of helix alphaB with respect to the beta-sheet. Both our structures show that the PDZ domain of EpsC adopts a more open form than in previously reported structures of other PDZ domains. Most interestingly, in the crystals of the short EpsC-PDZ domain the peptide binding groove interacts with an alpha-helix from a neighboring subunit burying approximately 921 A2 solvent accessible surface. This makes it possible that the PDZ domain of this bacterial protein binds proteins in a manner which is altogether different from that seen in any other PDZ domain so far. We also determined that the HR domain of EpsC is primarily responsible for the interaction with the secretin EpsD, while the PDZ is not, or much less, so. This new finding, together with studies of others, leads to the suggestion that the PDZ domain of EpsC may interact with exoproteins to be secreted while the HR domain plays a key role in linking the inner-membrane sub-complex of the T2SS in V. cholerae to the outer membrane secretin.

The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae.

Gram-negative bacteria use type II secretion systems for the transport of virulence factors and hydrolytic enzymes through the outer membrane. These sophisticated multi-protein complexes reach from the pore in the outer membrane via the pseudopilins in the periplasm and a multi-protein inner-membrane sub-complex, to an ATPase in the cytoplasm. The human pathogen Vibrio cholerae uses such a secretion machinery, called the Eps-system, for the export of its major virulence factor cholera toxin into the intestinal tract of the human host. Here, we describe the 2.4 A structure of the hetero-tetrameric complex of the N-terminal domain of the ATPase EpsE and the cytoplasmic domain of the inner membrane protein EpsL, which constitute the major cytoplasmic components of the Eps-system. A stable fragment of EpsE in complex with the cytoplasmic domain of EpsL was identified via limited proteolysis and facilitated the crystallization of the complex. This first structure of a complex between two different proteins of the type II secretion system reveals that the N-terminal domain of EpsE and the cytoplasmic domain of EpsL form a hetero-tetramer, in which EpsL is the central dimer and EpsE binds on the periphery. The dimer of EpsL in this complex is very similar to the dimer seen in the crystal structure of the native cytoplasmic domain of EpsL, suggesting a possible physiological relevance despite a relatively small 675 A2 buried solvent accessible surface. The N-terminal domain of EpsE, which forms a compact domain with an alpha+beta-fold, places its helix alpha2 in a mostly hydrophobic cleft between domains II and III of EpsL burying 1700 A2 solvent accessible surface. This extensive interface involves several residues whose hydrophobic or charged nature is well conserved and is therefore likely to be of general importance in type II secretion systems.

The structure of the cytoplasmic domain of EpsL, an inner membrane component of the type II secretion system of Vibrio cholerae: an unusual member of the actin-like ATPase superfamily.

The type II secretion system (T2SS) is used by several Gram-negative bacteria for the secretion of hydrolytic enzymes and virulence factors across the outer membrane. In these secretion systems, a complex of 12-15 so-called "Gsp proteins" spans from a regulatory ATPase in the cytoplasm, via several signal or energy transducing proteins in the inner membrane and the pseudopilins in the periplasm, to the actual pore in the outer membrane. The human pathogen Vibrio cholerae employs such an assembly, called the Eps system, for the export of its major virulence factor, cholera toxin, from its periplasm into the lumen of the gastro-intestinal tract of the host. Here, we report the atomic structure of the major cytoplasmic domain of the inner membrane-spanning EpsL protein from V. cholerae. EpsL is the binding partner of the regulatory ATPase EpsE as well as of EpsM and pseudopilins, and is therefore a critical link between the cytoplasmic and the periplasmic part of the Eps-system. The 2.7A resolution structure was determined by a combination of Se-Met multiple anomalous dispersion (MAD) and multiple isomorphous replacement with anomalous scattering (MIRAS) phasing methods. The 28kDa cytoplasmic domain of EpsL (cyto-EpsL) consists of three beta-sheet-rich domains. With domains I and III similar to the RNaseH-fold, cyto-EpsL unexpectedly shows structural homology with the superfamily of actin-like ATPases. cyto-EpsL, however, is an unusual member of this superfamily as it misses the canonical actin domains 1B and 2B, which are common yet variable in this superfamily. Moreover, cyto-EpsL has an additional domain II, which has the topology of an SHS2-fold module. Within the superfamily this fold module has been observed only for domain 1C of the cell division protein FtsA, in which it mediates protein-protein interactions. This domain II displays great flexibility and contributes to a pronounced negatively charged canyon on the surface of cyto-EpsL. Functional data as well as structural homology and sequence conservation suggest that domain II interacts with EpsE, the major cytoplasmic binding partner of EpsL.

The crystal structure of the periplasmic domain of the type II secretion system protein EpsM from Vibrio cholerae: the simplest version of the ferredoxin fold.

The terminal branch of the general secretion pathway (Gsp or type II secretion system) is used by several pathogenic bacteria for the secretion of their virulence factors across the outer membrane. In these secretion systems, a complex of 12-15 Gsp proteins spans from the pore in the outer membrane via several associated signal or energy-transducing proteins in the inner membrane to a regulating ATPase in the cytosol. The human pathogen Vibrio cholerae uses such a system, called the Eps system, for the export of the cholera toxin and other virulence factors from its periplasm into the lumen of the gastrointestinal tract of the host. Here, we report the atomic structure of the periplasmic domain of the EpsM protein from V.cholerae, which is a part of the interface between the regulating part and the rest of the Eps system. The crystal structure was determined by Se-Met MAD phasing and the model was refined to 1.7A resolution. The monomer consists of two alphabetabeta-subdomains forming a sandwich of two alpha-helices and a four-stranded antiparallel beta-sheet. In the dimer, a deep cleft with a polar rim and a hydrophobic bottom made by conserved residues is located between the monomers. This cleft contains an extra electron density suggesting that this region might serve as a binding site of an unknown ligand or part of a protein partner. Unexpectedly, the fold of the periplasmic domain of EpsM is an undescribed circular permutation of the ferredoxin fold.