QuiC2 represents a functionally distinct class of dehydroshikimate dehydratases identified in Listeria species including Listeria monocytogenes

Many Listeria species including L. monocytogenes contain the pathway for the biosynthesis of protocatechuate from shikimate and quinate. The qui1 and qui2 operons within these Listeria spp. encode enzymes for this pathway. The diversion of shikimate pathway intermediates in some Listeria species to produce protocatechuate suggests an important biological role for this compound to these organisms. A total of seven ORFs, including quiC2, were identified within qui1 and qui2, however only three proteins encoded by the operons have been functionally annotated. The final step in Listeria's protocatechuate biosynthesis involves the conversion of dehydroshikimate by a dehydroshikimate dehydratase (DSD). In this study, we demonstrate that QuiC2 functions as a DSD in Listeria spp. through biochemical and structural analyses. Moreover, we show that QuiC2 forms a phylogenetic cluster distinct from other functionally annotated DSDs. The individual phylogenetic clusters of DSD are represented by enzymes that produce protocatechuate for distinct biological processes. Similarly, QuiC2 is expected to produce protocatechuate for a novel biological process. We postulate that protocatechuate produced by DSDs found within the QuiC2 phylogenetic cluster provides an ecological niche for representative organisms.     

Hydroaromatic metabolism in Rhodococcus rhodochrous: purification and characterisation of its NAD-dependent quinate dehydrogenase

Quinate grown cells of Rhodococcus rhodochrous N75 metabolized both quinate and shikimate via protocatechuate to succinate and acetyl CoA. The initial enzyme of the hydroaromatic pathway, quinate dehydrogenase was purified 188-fold to electrophoretic homogeneity. The enzyme is a monomer with a native relative molecular mass of 44,000 and is NAD-dependent. The enzyme is highly stereospecific with regard to hydroaromatic substrates, oxidising only the axial hydroxyl group at C-3 of (-)-isomers of quinate, shikimate, dihydroshikimate and t-3,t-4-dihydroxycyclohexane-c-1-carboxylate, but shows activity with several NAD analogues.     

Chorismate‐dependent transcriptional regulation of quinate/shikimate utilization genes by LysR‐type transcriptional regulator QsuR in Corynebacterium glutamicum: carbon flow control at metabolic branch point

The qsu operon of Corynebacterium glutamicum comprises four genes (qsuABCD) that underpin the microorganism's quinate/shikimate utilization pathways. The genes encode enzymes that catalyse reactions at the metabolic branch point between the biosynthesis route for synthesis of aromatic compounds and the catabolic route for degradation of quinate and shikimate for energy production. A qsuR gene located immediately upstream of qsuA encodes a protein (QsuR) which activates the operon in the presence of quinate or shikimate. Three observations support chorismate, an intermediate of the biosynthesis route, as a direct effector of QsuR: First, induction of qsuA mRNA in the presence of either quinate or shikimate disappears upon deletion of the gene encoding chorismate synthase. Second, chorismate accumulates when the operon is induced. Third, a DNase I‐protected segment by QsuR is shortened in the presence of chorismate. The QsuR tetramer senses the accumulation of chorismate and activates qsu genes that promote the quinate/shikimate catabolic instead of the aromatic compounds biosynthetic route. Such chorismate‐dependent control of carbon flow has not been previously described.     

Purification and Characterization of Membrane-Bound 3-Dehydroshikimate Dehydratase from Gluconobacter oxydans IFO 3244, A New Enzyme Catalyzing Extracellular Protocatechuate Formation

3-Dehydroshikimate dehydratase (DSD) is the first known enzyme catalyzing aromatization from 3-dehydroshikimate (DSA) to protocatechuate (PCA). Differently from cytosolic DSD (sDSD), a membrane-bound 3-dehydroshikimate dehydratase (mDSD) was found for the first time in the membrane fraction of Gluconobacter oxydans IFO 3244, and DSA was confirmed to be the direct precursor of PCA. In contrast to weak and instable sDSD, the abundance of mDSD in the membrane fraction suggested the metabolic significance of mDSD as the initial step in aromatization. mDSD was solubilized only by a detergent and was readily purified to high homogeneity. Its molecular weight was estimated to be 76,000. Purified mDSD showed a sole peak at 280 nm in the absorption spectrum and no critical cofactor requirements. The Km of DSA was measured at 0.5 mM, and the optimum pH was observed at pH 6–8. mDSD appeared to react only with DSA, and was inert to other compounds, such as 3-dehydroquinate, quinate, and shikimate.     

Discovery of a novel periplasmic protein that forms a complex with a trimeric autotransporter adhesin and peptidoglycan

Trimeric autotransporter adhesins (TAAs), fibrous proteins on the cell surface of Gram‐negative bacteria, have attracted attention as virulence factors. However, little is known about the mechanism of their biogenesis. AtaA, a TAA of Acinetobacter sp. Tol 5, confers nonspecific, high adhesiveness to bacterial cells. We identified a new gene, tpgA, which forms a single operon with ataA and encodes a protein comprising two conserved protein domains identified by Pfam: an N‐terminal SmpA/OmlA domain and a C‐terminal OmpA_C‐like domain with a peptidoglycan (PGN)‐binding motif. Cell fractionation and a pull‐down assay showed that TpgA forms a complex with AtaA, anchoring it to the outer membrane (OM). Isolation of total PGN‐associated proteins showed TpgA binding to PGN. Disruption of tpgA significantly decreased the adhesiveness of Tol 5 because of a decrease in surface‐displayed AtaA, suggesting TpgA involvement in AtaA secretion. This is reminiscent of SadB, which functions as a specific chaperone for SadA, a TAA in Salmonella species; however, SadB anchors to the inner membrane, whereas TpgA anchors to the OM through AtaA. The genetic organization encoding the TAA–TpgA‐like protein cassette can be found in diverse Gram‐negative bacteria, suggesting a common contribution of TpgA homologues to TAA biogenesis.     

Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase.

The Escherichia coli aroE gene encoding shikimate dehydrogenase was sequenced. The deduced amino acid sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced protein. The complete polypeptide chain has 272 amino acid residues and has a calculated Mr of 29,380. E. coli shikimate dehydrogenase is homologous to the shikimate dehydrogenase domain of the fungal arom multifunctional enzymes and to the catabolic quinate dehydrogenase of Neurospora crassa.     

Defensin‐like peptide3 from black solder fly: Identification,characterization, and key amino acids for anti‐Gram‐negative bacteria

An antibacterial peptide was isolated from a black soldier fly, Hermetia illucens. The molecular mass of this peptide was established as 4247.37 by matrix‐assisted laser desorption/ionization‐time of flight mass (MALD‐TOF MS) spectrometry. The amino acid sequence of the mature peptide was determined by N‐terminal sequencing using Edman degradation, combined with cDNA sequencing of the previously reported defensin‐like peptide (DLP) 3. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP3 had potent activity against Gram‐positive and negative bacteria, but DLP4 had only anti‐Gram‐positive activity as previously reported. Recombinant DLP3 and DLP4 were overexpressed in Escherichia coli, and antibacterial activities were identical to DLPs purified from H. illucens hemolyph. In silico analysis revealed that only six amino acid sequences were different between DLP3 and DLP4, but antibacterial activity against Gram‐negative bacteria differed. Therefore these amino acid variants may be key amino acids (Gly‐10, Val‐18, Met‐23, Arg‐25, Asp‐32, Arg‐40) related to killing Gram‐negative bacteria.     

Chemotaxis to aromatic and hydroaromatic acids: comparison of Bradyrhizobium japonicum and Rhizobium trifolii

Rhizobia are bacteria well known for their ability to fix nitrogen in symbiosis with leguminous plants. Members of diverse rhizobial species grow at the expense of hydroaromatic and aromatic compounds commonly found in plant cells and plant litter. Using a quantitative capillary assay to measure chemotaxis, we tested the ability of hydroaromatic acids, selected aromatic acids, and their metabolites to serve as chemoattractants for two distantly related rhizobial species, Bradyrhizobium japonicum and Rhizobium trifolii. Slow-growing B. japonicum I-110 demonstrated positive chemotaxis to shikimate, quinate, protocatechuate, and vanillate; threshold concentrations for the compounds were as low as 10(-6) M. The dicarboxylic acids succinate and beta-ketoadipate, metabolites in the catabolism of many aromatic compounds, were positive chemoattractants with low threshold concentrations as well. Taxis to beta-ketoadipate occurred constitutively and, of the tested compounds, beta-ketoadipate gave the strongest peak response. Taxis to shikimate or quinate was induced by growth on either substrate but not by growth on protocatechuate or succinate. In contrast, fast-growing R. trifolii 2066 was only weakly attracted to quinate and other aromatic and dicarboxylic acids that were strong attractants for B. japonicum. The R. trifolii strain exhibited positive chemotaxis to shikimate, but the threshold concentration of shikimate required to elicit a response (10(-4) M) was 2 orders of magnitude higher than that for the B. japonicum strain.     

The pca-pob supraoperonic cluster of Acinetobacter calcoaceticus contains quiA, the structural gene for quinate-shikimate dehydrogenase.

An 18-kbp Acinetobacter calcoaceticus chromosomal segment contains the pcaIJFBDKCHG operon, which is required for catabolism of protocatechuate, and pobSRA, genes associated with conversion of p-hydroxybenzoate to protocatechuate. The genetic function of the 6.5 kbp of DNA between pcaG and pobS was unknown. Deletions in this DNA were designed by removal of fragments between restriction sites, and the deletion mutations were introduced into A. calcoaceticus by natural transformation. The mutations prevented growth with either quinate or shikimate, growth substrates that depend upon qui gene function for their catabolism to protocatechuate. The location of quiA, a gene encoding quinate-shikimate dehydrogenase, was indicated by its expression in one of the deletion mutants, and the position of the gene was confirmed by determination of its 2,427-bp nucleotide sequence. The deduced amino acid sequence of QuiA confirmed that it is a member of a family of membrane-associated, pyrrolo-quinoline quinone-dependent dehydrogenases, as had been suggested by earlier biochemical investigations. Catabolism of quinate and skikimate is initiated by NAD(+)-dependent dehydrogenases in other microorganisms, so it is evident that different gene pools were called upon to provide the ancestral enzyme for this metabolic step.     

Mutations in the N‐terminal kinase‐like domain of the repressor of photomorphogenesis SPA1 severely impair SPA1 function but not light responsiveness in Arabidopsis

The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark‐grown plants. While both COP1 and the four SPA proteins contain coiled‐coil and WD‐repeat domains, SPA proteins differ from COP1 in carrying an N‐terminal kinase‐like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N‐terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N‐terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This ΔN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N‐terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase‐like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase‐like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase‐like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N‐terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N‐terminal domain. Together, these results uncover an important, but complex role of the SPA1 N‐terminus in the suppression of photomorphogenesis.     

Conversion of Quinate to 3-Dehydroshikimate by Ca-Alginate-Immobilized Membrane of Gluconobacter oxydans IFO 3244 and Subsequent Asymmetric Reduction of 3-Dehydroshikimate to Shikimate by Immobilized Cytoplasmic NADP-Shikimate Dehydrogenase

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.     

Molecular characterization and heterologous expression of quinate dehydrogenase gene from <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> IFO3244

The quinate dehydrogenase (QDH) from Gluconobacter oxydans IFO3244 exhibits high affinity for quinate, suggesting its application in shikimate production. Nucleotide sequence analysis of the qdh gene revealed a full-length of 2475-bp encoding an 824-amino acid protein. The qdh gene has the unusual TTG translation initiation codon. Conserved regions and a signature sequence for the quinoprotein family were observed. Phylogenetic analysis demonstrated relatedness of QDH from G. oxydans to other quinate/shikimate dehydrogenases with the highest similarity (56%) with that of Acinetobacter calcoaceticus ADP1 and lower similarity (36%) with a membrane-bound glucose dehydrogenase of Escherichia coli. The function of the gene coding for QDH was confirmed by heterologous gene expression in pyrroloquinoline quinone-synthesizing Pseudomonas putida HK5.     

Evolution of a secondary metabolic pathway from primary metabolism: shikimate and quinate biosynthesis in plants

The shikimate pathway synthesizes aromatic amino acids essential for protein biosynthesis. Shikimate dehydrogenase (SDH) is a central enzyme of this primary metabolic pathway, producing shikimate. The structurally similar quinate is a secondary metabolite synthesized by quinate dehydrogenase (QDH). SDH and QDH belong to the same gene family, which diverged into two phylogenetic clades after a defining gene duplication just prior to the angiosperm/gymnosperm split. Non‐seed plants that diverged before this duplication harbour only a single gene of this family. Extant representatives from the chlorophytes (Chlamydomonas reinhardtii), bryophytes (Physcomitrella patens) and lycophytes (Selaginella moellendorfii) encoded almost exclusively SDH activity in vitro. A reconstructed ancestral sequence representing the node just prior to the gene duplication also encoded SDH activity. Quinate dehydrogenase activity was gained only in seed plants following gene duplication. Quinate dehydrogenases of gymnosperms, represented here by Pinus taeda, may be reminiscent of an evolutionary intermediate since they encode equal SDH and QDH activities. The second copy in P. taeda maintained specificity for shikimate similar to the activity found in the angiosperm SDH sister clade. The codon for a tyrosine residue within the active site displayed a signature of positive selection at the node defining the QDH clade, where it changed to a glycine. Replacing the tyrosine with a glycine in a highly shikimate‐specific angiosperm SDH was sufficient to gain some QDH function. Thus, very few mutations were necessary to facilitate the evolution of QDH genes.     

Structure of bacteriophage SPN1S endolysin reveals an unusual two‐module fold for the peptidoglycan lytic and binding activity

Bacteriophage SPN1S infects the pathogenic Gram‐negative bacterium Salmonella typhimurium and expresses endolysin for the release of phage progeny by degrading peptidoglycan of the host cell walls. Bacteriophage SPN1S endolysin exhibits high glycosidase activity against peptidoglycans, resulting in antimicrobial activity against a broad range of outer membrane‐permeabilized Gram‐negative bacteria. Here, we report a crystal structure of SPN1S endolysin, indicating that unlike most endolysins from Gram‐negative bacteria background, the α‐helical protein consists of two modular domains, a large and a small domain, with a concave groove between them. Comparison with other structurally homologous glycoside hydrolases indicated a possible peptidoglycan binding site in the groove, and the presence of a catalytic dyad in the vicinity of the groove, one residue in a large domain and the other in a junction between the two domains. The catalytic dyad was further validated by antimicrobial activity assay against outer membrane‐permeabilized Escherichia coli. The three‐helix bundle in the small domain containing a novel class of sequence motif exhibited binding affinity against outer membrane‐permeabilized E. coli and was therefore proposed as the peptidoglycan‐binding domain. These structural and functional features suggest that endolysin from a Gram‐negative bacterial background has peptidoglycan‐binding activity and performs glycoside hydrolase activity through the catalytic dyad.     

Sequence‐independent processing site of the C‐terminal domain (CTD) influences maturation of the RgpB protease from Porphyromonas gingivalis

The Gram‐negative periodontal pathogen Porphyromonas gingivalis produces a family of outer membrane‐anchored proteases, the gingipains, shown to play an essential role in virulence of the organism. The C‐terminal domain (CTD) of gingipains and other secreted proteins is known to be the targeting signal for maturation and translocation of the protein through the outer membrane. The CTD is subsequently cleaved during the secretion process. Multiple alignment of various CTDs failed to define a consensus sequence at the putative CTD processing site. Using mutagenesis, we were able to show that cleavage at the site is not dependent on a specific residue and that recognition of the site is independent of local sequence. Interestingly, length of the junction between the CTD and adjacent Ig‐like subdomain has a critical influence on post‐translational glycan modification of the protein, whereby insertion of additional residues immediately N‐terminal to the cleavage site results in failure of glycan modification and release of soluble protease into the culture medium. Various hypotheses are presented to explain these phenomena. Knowledge of the role CTDs play in maturation of gingipains has broader application for understanding maturation of CTD homologues expressed by bacteria of the Bacteriodetes phylum.     

Two types of information processing in cercal systems of insects: directional sensitivity of giant interneurons

Summary Cercal systems of seven insect species (cricketMelanogryllus desertus, mole cricketGryllotalpa gryllotalpa, katydidsPholidoptera pustulipes andTettigonia viridissima, cockroachesPeriplaneta americana andBlatta orientalis, and locustLocusta migratoria) were examined for direction-sensitive giant interneurons (GIs) that are excited by cercal receptors but have directional preferences independent of cercus position. Such GIs are known for the cricketsAcheta domesticus andGryllus bimaculatus. Directional sensitivity diagrams (DSDs) of GIs were obtained by recording and analysing the electrical responses of abdominal connectives to sound stimuli from various directions. For each animal DSDs were plotted in the form of polar graphs for two or three positions of the stimulated cercus so that the effect of cercus position on the orientation of the DSD could be evaluated.All insects studied had GIs whose DSDs for fixed cercus positions were similar in appearance to the DSDs described for GIs of the cricketsAcheta domesticus andGryllus bimaculatus. Most of these DSDs were shaped like a figure 8 (when airflow is used as the stimulus instead of sound, each DSD has only one lobe). However, not all GIs demonstrated a constant directional preference. GIs with constant directionality were found only inMelanogryllus desertus, Pholidoptera pustulipes, Tettigonia viridissima andLocusta migratoria. In these insects DSDs from one GI plotted for different cercus positions had approximately the same orientation (Figs. 4–7). In contrast, GIs inGryllotalpa gryllotalpa, Periplaneta americana andBlatta orientalis had DSDs whose orientation changed in accordance with a change in position of the stimulated cercus (Figs. 8–10).Thus, direction-sensitive GIs investigated here can be divided into two types: (1) GIs with constant directionality (whose DSDs are fixed to the body, and (2) GIs with variable directionality (whose DSDs are fixed to the cerci). To date, in each species only GIs of the same type have been encountered. This may be an indication that cercal systems can be divided into two categories according to how they process information. However, since we have not tested all GIs in each species, we cannot rule out the possibility that a species might have both types of GIs.Abbreviations DSD directional sensitivity diagram - GI giant interneuron - TAG terminal abdominal ganglion     

The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu

Sufu (Suppressor of Fused), a two‐domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu‐like proteins have previously been identified based on sequence similarity to the N‐terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu‐like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu‐like protein. The structure revealed a striking similarity to the N‐terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ～15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu‐like proteins that are present in ～200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam.