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Cyclic dinucleotides (CDNs) have central roles in bacterial homeostasis and virulence by acting as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern-recognition receptors in animal cells. Here we perform a systematic biochemical screen for bacterial signalling nucleotides and discover a large family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize a diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the enzyme active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analyses of CD-NTase signalling nucleotides demonstrate that these cyclic di- and trinucleotides activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts. A bacterial family of cGAS/DncV-like nucleotidyltransferases synthesizes a diverse range of cyclic dinucleotide and trinucleotide compounds that are likely to modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts., Author(s): Aaron T. Whiteley [sup.1] [sup.2] , James B. Eaglesham [sup.1] [sup.2] , Carina C. de Oliveira Mann [sup.1] [sup.2] , Benjamin R. Morehouse [sup.1] [sup.2] , Brianna Lowey [sup.1] [...]

SpoIID is a membrane-anchored enzyme that degrades peptidoglycan and is essential for engulfment and sporulation in Bacillus subtilis. SpoIID is targeted to the sporulation septum, where it interacts with two other proteins required for engulfment: SpoIIP and SpoIIM. We changed conserved amino acids in SpoIID to alanine to determine whether there was a correlation between the effect of each substitution on the in vivo and in vitro activities of SpoIID. We identified one amino acid substitution, E88A, that eliminated peptidoglycan degradation activity and one, D210A, that reduced it, as well as two substitutions that destabilized the protein in B. subtilis (R106A and K203A). Using these mutants, we show that the peptidoglycan degradation activity of SpoIID is required for the first step of engulfment (septal thinning), as well as throughout membrane migration, and we show that SpoIID levels are substantially above the minimum required for engulfment. The inactive mutant E88A shows increased septal localization compared to the wild type, suggesting that the degradation cycle of the SpoIID/SpoIIP complex is accompanied by the activity-dependent release of SpoIID from the complex and subsequent rebinding. This mutant is also capable of moving SpoIIP across the sporulation septum, suggesting that SpoIID binding, but not peptidoglycan degradation activity, is needed for reiocalization of SpoIIP. Finally, the mutant with reduced activity (D210A) causes uneven engulfment and time-lapse microscopy indicates that the fastest-moving membrane arm has greater concentrations of SpoIIP than the slower-moving arm, demonstrating a correlation between SpoIIP protein levels and the rate of membrane migration. doi: 10.1128/JB.00127-10

Analysis of the genome of Francisella tularensis has revealed few regulatory systems, and how the organism adapts to conditions in different niches is poorly understood. The stringent response is a global stress response mediated by (p)ppGpp. The enzyme RelA has been shown to be involved in generation of this signal molecule in a range of bacterial species. We investigated the effect of inactivation of the relA gene in Francisella by generating a mutant in Francisella novicida. Under amino acid starvation conditions, the relA mutant was defective for (p)ppGpp production. Characterization showed the mutant to grow similarly to the wild-type, except that it entered stationary phase later than wild-type cultures, resulting in higher cell yields. The relA mutant showed increased biofilm formation, which may be linked to the delay in entering stationary phase, which in turn would result in higher cell numbers present in the biofilm and reduced resistance to in vitro stress. The mutant was attenuated in the J774A macrophage cell line and was shown to be attenuated in the mouse model of tularaemia, but was able to induce a protective immune response. Therefore, (p)ppGpp appears to be an important intracellular signal, integral to the pathogenesis of F. novicida. DOI 10.1099/mic.0.031021-0

Three putative hydrogenase enzyme systems in Thermoanaerobacterium saccharolyticum were investigated at the genetic, mRNA, enzymatic, and phenotypic levels. A four-gene operon containing two [FeFe]-hydrogenase genes, provisionally termed hfs (hydrogenase-Fe-S), was found to be the main enzymatic catalyst of hydrogen production, hfsB, perhaps the most interesting gene of the operon, contains an [FeFe]-hydrogenase and a PAS sensory domain and has several conserved homologues among clostridial saccharolytic, cellulolytic, and pathogenic bacteria. A second hydrogenase gene cluster, hyd, exhibited methyl viologen-linked hydrogenase enzymatic activity, but hyd gene knockouts did not influence the hydrogen yield of cultures grown in closed-system batch fermentations. This result, combined with the observation that hydB contains NAD(P)+ and FMN binding sites, suggests that the hyd genes are specific to the transfer of electrons from NAD(P)H to hydrogen ions. A third gene cluster, a putative [NiFe]-hydrogenase with homology to the ech genes, did not exhibit hydrogenase activity under any of the conditions tested. Deletion of the hfs and hydA genes resulted in a loss of detectable methyl viologen-linked hydrogenase activity. Strains with a deletion of the hfs genes exhibited a 95% reduction in hydrogen and acetic acid production. A strain with hfs and ldh deletions exhibited an increased ethanol yield from consumed carbohydrates and represents a new strategy for engineering increased ethanol yields in T. saccharolyticum. doi: 10.1128/JB.00497-09

This work is believed to be the first report on the physiological and biochemical characterization of [alpha]-L-rhamnosidases in lactic acid bacteria. A total of 216 strains representing 37 species and eight genera of food-grade bacteria were screened for [alpha]-L-rhamnosidase activity. The majority of positive bacteria (25 out of 35) were Lactobacillus plantarum strains, and activity of the L. plantarum strain NCC245 was examined in more detail. The analysis of [alpha]-L-rhamnosidase activity under different growth conditions revealed dual regulation of the enzyme activity, involving carbon catabolite repression and induction: the enzyme activity was downregulated by glucose and upregulated by L-rhamnose. The expression of the two [alpha]-L-rhamnosidase genes rhaB1 and rhaB2 and two predicted permease genes rhaP1 and rhaP2, identified in a probable operon rhaP2B2PIB 1, was repressed by glucose and induced by L-rhamnose, showing regulation at the transcriptional level. The two [alpha]-L-rhamnosidase genes were overexpressed and purified from Escherichia coli. RhaB1 activity was maximal at 50 [degrees]C and at neutral pH and RhaB2 maximal activity was detected at 60 [degrees]C and at pH 5, with high residual activity at 70 [degrees]C. Both enzymes showed a preference for the [alpha]-1,6 linkage of L-rhamnose to [beta]-D-glucose, hesperidin and rutin being their best substrates, but, surprisingly, no activity was detected towards the [alpha]-1,2 linkage in naringin under the tested conditions. In conclusion, we identified and characterized the strain L. plantarum NCC245 and its two [alpha]-L-rhamnosidase enzymes, which might be applied for improvement of bioavailability of health-beneficial polyphenols, such as hesperidin, in humans.

Agrobacterium tumefaciens requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid N-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses S-adenosylmethionine (SAM) as the methyl donor, converting it to S-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of A. tumefaciens PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid N-methyltransferase.

Conjugative transfer of plasmid DNA via close cell-cell junctions is the main route by which antibiotic resistance genes spread between bacterial strains. Relaxases are essential for conjugative transfer and act by cleaving DNA strands and forming covalent phosphotyrosine linkages. Based on data indicating that multityrosine relaxase enzymes can accommodate two phosphotyrosine intermediates within their divalent metal-containing active sites, we hypothesized that bisphosphonates would inhibit relaxase activity and conjugative DNA transfer. We identified bisphosphonates that are nanomolar inhibitors of the F plasmid conjugative relaxase in vitro. Furthermore, we used cell-based assays to demonstrate that these compounds are highly effective at preventing DNA transfer and at selectively killing cells harboring conjugative plasmids. Two potent inhibitors, clodronate and etidronate, are already clinically approved to treat bone loss. Thus, the inhibition of conjugative relaxases is a potentially novel antimicrobial approach, one that selectively targets bacteria capable of transferring antibiotic resistance and generating multidrug resistant strains. antimicrobial | bacterial conjugation | bisphosphonates | F plasmid Tral | relaxase inhibition

The conversion of [sup.14C]benzoyl-coenzyme A (CoA) to nonaromatic products in the denitrifying [beta]-proteobacterium Azoarcus evansii grown anaerobically on benzoate was investigated. With cell extracts and 2-oxoglutarate as the electron donor, benzoyl-CoA reduction occurred at a rate of 10 to 15 nmol [min.sup.-1] [mg.sup.-1] 2-Oxoglutarate could be replaced by dithionite (200% rate) and by NADPH ([tilde]10% rate); in contrast NADH did not serve as an electron donor. Anaerobic growth on aromatic compounds induced 2-oxoglutarate:acceptor oxidoreductase (KGOR), which specifically reduced NAD[P.sup.+], and NADPH:acceptor oxidoreductase. KGOR was purified by a 76-fold enrichment. The enzyme had a molecular mass of 290 [+ or -] 20 kDa and was composed of three subunits of 63 ([gamma]), 62 ([alpha]), and 37 ([beta]) kDa in a 1:1:1 ratio, suggesting an [([alpha][beta][gamma]).sub.2] composition. The native enzyme contained Fe (24 mol/mol of enzyme), S (23 mol/mol), flavin adenine dinucleotide (FAD; 1.4 mol/mol), and thiamine diphosphate (0.95 mol/mol). KGOR from A. evansii was highly specific for 2-oxoglutarate as the electron donor and accepted both NAD[P.sup.+] and oxidized viologens as electron acceptors; in contrast NA[D.sup.+] was not reduced. These results suggest that benzoyl-CoA reduction is coupled to the complete oxidation of the intermediate acetyl-CoA in the tricarboxylic acid cycle. Electrons generated by KGOR can be transferred to both oxidized ferredoxin and NAD[P.sup.+], depending on the cellular needs. N-terminal amino acid sequence analysis revealed that the open reading frames for the three subunits of KGOR are similar to three adjacently located open reading frames in Bradyrhizobium japonicum. We suggest that these genes code for a very similar three-subunit KGOR, which may play a role in nitrogen fixation. The [alpha]-subunit is supposed to harbor one FAD molecule, two [4Fe-4S] clusters, and the NADPH binding site; the [beta]-subunit is supposed to harbor one thiamine diphosphate molecule and one further [4Fe-4S] cluster; and the [gamma]-subunit is supposed to harbor the CoA binding site. This is the first study of an NAD[P.sup.+] -specific KGOR. A similar NAD[P.sup.+] -specific pyruvate oxidoreductase, which contains all domains in one large subunit, has been reported for the mitochondrion of the protist Euglena gracilis and the apicomplexan Cryptosporidium parvum.

The enzyme 2-phosphoglycolate phosphatase from Escherichia coli, encoded by the gph gene, was purified and characterized. The enzyme was highly specific for 2-phosphoglycolate and showed good catalytic efficiency ([k.sub.cat]/[K.sub.m]), which enabled the conversion of this substrate even at low intracellular concentrations. A comparison of the structural and functional features of this enzyme with those of 2-phosphoglycolate phosphatases of different origins showed a high similarity of the sequences, implying the use of the same catalytic mechanism. Western blot analysis revealed constitutive expression of the gph gene, regardless of the carbon source used, growth stage, or oxidative stress conditions. We showed that this housekeeping enzyme is involved in the dissimilation of the intracellular 2-phosphoglycolate formed in the DNA repair of 3'-phosphoglycolate ends. DNA strand breaks of this kind are caused by agents such as the radiomimetic compound bleomycin. The differential response between a 2-phosphoglycolate phosphatase-deficient mutant and its parental strain after treatment with bleomycin allowed us to connect the intracellular formation of 2-phosphoglycolate with the production of glycolate, which is subsequently incorporated into general metabolism. We thus provide evidence for a salvage function of 2-phosphoglycolate phosphatase in the metabolism of a two-carbon compound generated by the cellular DNA repair machinery.

Cysteinyl-tRNA synthetase is an essential enzyme required for protein synthesis. Genes encoding this protein have not been identified in Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, or Methanopyrus kandleri. It has previously been proposed that the prolyl-tRNA synthetase (ProRS) enzymes in these organisms recognize either proline or cysteine and can aminoacylate their cognate tRNAs through a dual-specificity mechanism. We report five crystal structures at resolutions between 2.6 and 3.2 [Angstrom]: apo M. jannaschii ProRS, and M. thermautotrophicus ProRS in apo form and in complex with cysteinyl-sulfamoyl-, prolyl-sulfamoyl-, and alanyl-sulfamoyl-adenylates. These aminoacyl-adenylate analogues bind to a single active-site pocket and induce an identical set of conformational changes in loops around the active site when compared with the ligand-free conformation of ProRS. The cysteinyl- and prolyl-adenylate analogues have similar, nanomolar affinities for M. thermautotrophicus ProRS. Homology modeling of tRNA onto these adenylate complexes places the 3'-OH of A76 in an appropriate position for the transfer of any of the three amino acids to tRNA. Thus, these structures explain recent biochemical experiments showing that M. jannaschii ProRS misacylates [tRNA.sup.Pro] with cysteine, and argue against the proposal that these archaeal ProRS enzymes possess the dual capacity to aminoacylate both [tRNA.sup.Pro] and [tRNA.sup.Cys] with their cognate amino acids.

Results demonstrate that catalytically active P450 levels in vivo can be increased by cloning Streptomyces coelicolor ferredoxin reductase and coexpressing it alongside the P450 and ferredoxin in Escherichia coli.

Research demonstrates the secretion of pro-transglutaminase by Corynebacterium glutamicum by coupling to signal peptides of the cell surface proteins of the bacterium. The pro-domain is processed by a subtilisin-like protease from Streptomyces albogriseolus. Data show that Corynebacterium glutamicum secretes two proteins that can be exploited for industrial-scale production.

To the Editor: New Delhi metallo-[beta]-lactamase 1 (NDM-1) and OXA-48-group [beta]-lactamase have been increasingly reported as carbapenemases responsible for carbapenem resistance in Enterobacteriaceae worldwide (1). However, in the United States, [...]

Research demonstrates that a major pilin subunit of the Pap pilus, PapA, is the natural substrate of the DegP protein, which is a multifunctional chaperone and protease from Escherichia coli. A 7-mer peptide of PapA is preferentially cleaved by DegP, which occurs between paired hydrophobic amino acid residues.

Research describes the occurrence of two different gene clusters, cat1 and cat2, for catechol degradation in Burkholderia sp. strain TH2. The gene sets code for catechol dehydrogenases, CatA1 and CatA2, but produced under different conditions. The inducer of CatA1 is benzoate, whereas that of CatA2 is cis,cis-muconate.

Research reveals that human large intestine harbors distinct groups of butyrate-producing bacteria of different functionality. Data show that strains of Roseburia sp. and Faecalibacterium prausnitzii and strain L2-50 of Coprococcus sp. possess different functional enzyme systems.

Research has been conducted on the fungal cutinase and yeast esterase. The role of these enzymes in butyl benzyl phthalate degradation has been investigated and the details are reported.

Research has been conducted on the erythritol production by Torula corallina. The mechanism of Cu (super)2+ enhancement of this production has been investigated and the results are reported.

Research has been conducted on the glycogen debranching enzyme from Saccharomyces cerevisiae. The role of this enzyme in amylose and its ability to produce cycloamylose have been examined and the details are reported.

Research has been conducted on Streptomyces glaucescens FabH. Results demonstrate that the presence of FabH in S. glaucescens during tetracenomycin C production does not affect acetyl-CoA biosynthesis initiation process.

Research has been conducted on the cysteines' heme binding domain. Results indicate that deletion of this domain in the CXXC motif leads to the redox sensitivity loss in the cystathionine beta-synthase.

Research has been conducted on the dihydrolipoyl acyltransferase's mutant peripheral subunit-binding domain. The use of surface plasmon resonance and microcalorimetry in investigating interactions between this domain and dihydrolipoyl dehydrogenase is discussed.

Research has been conducted on Trichomonas vaginalis purine nucleoside phosphorylase. The identification and characterization of this phosphorylase have been carried out and the results suggest that it will favor the anabolic reaction for the purine nucleoside synthesis.

Results demonstrate that Glu160 residue in Thermoanaerobacterium saccharolyticum beta-xylosidase prticipates in acid/base catalysis either singly or in cooperation with a second carboxylic acid residue. The enzyme activity proceeds using acid catalysis, which involves active site residue reverse protonation.