Manfred Nimtz

Biosynthetic Pathway of Pseudomonas aeruginosa 4-Hydroxy-2-Alkylquinolines

The role of intercellular communication in the regulation of bacterial multicellular behavior has received widespread attention, and a variety of signal molecules involved in bacterial communication have been discovered. In addition to the N-acyl-homoserine lactones, 4-hydroxy-2-alkylquinolines (HAQs), including the Pseudomonas quinolone signal, have been shown to function as signal molecules in Pseudomonas aeruginosa. In this study we unraveled the biosynthetic pathway of HAQs using feeding experiments with isotope-labeled precursors and analysis of extracted HAQs by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. Our results show that the biosynthesis of various HAQ metabolites is directed via a common metabolic pathway involving a head-to-head condensation of anthranilic acid and beta-keto fatty acids. Moreover, we provide evidence that the beta-keto-(do)decanoic acids, crucial for the biosynthesis of the heptyl and nonyl derivatives of the 4-hydroxyquinolines in P. aeruginosa, are at least in part derived from a common pool of beta-hydroxy(do)decanoic acids involved in rhamnolipid biosynthesis.

Cross-feeding and interkingdom communication in dual-species biofilms of Streptococcus mutans and Candida albicans.

Polymicrobial biofilms are of large medical importance, but relatively little is known about the role of interspecies interactions for their physiology and virulence. Here, we studied two human pathogens co-occuring in the oral cavity, the opportunistic fungus Candida albicans and the caries-promoting bacterium Streptococcus mutans. Dual-species biofilms reached higher biomass and cell numbers than mono-species biofilms, and the production of extracellular polymeric substances (EPSs) by S. mutans was strongly suppressed, which was confirmed by scanning electron microscopy, gas chromatography–mass spectrometry and transcriptome analysis. To detect interkingdom communication, C. albicans was co-cultivated with a strain of S. mutans carrying a transcriptional fusion between a green fluorescent protein-encoding gene and the promoter for sigX, the alternative sigma factor of S. mutans, which is induced by quorum sensing signals. Strong induction of sigX was observed in dual-species biofilms, but not in single-species biofilms. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion of comS encoding the synthesis of the sigX-inducing peptide precursor abolished this activity, whereas deletion of comC encoding the competence-stimulating peptide precursor had no effect. Transcriptome analysis of S. mutans confirmed induction of comS, sigX, bacteriocins and the downstream late competence genes, including fratricins, in dual-species biofilms. We show here for the first time the stimulation of the complete quorum sensing system of S. mutans by a species from another kingdom, namely the fungus C. albicans, resulting in fundamentally changed virulence properties of the caries pathogen.

Adaptation of Pseudomonas aeruginosa to various conditions includes tRNA-dependent formation of alanyl-phosphatidylglycerol

The opportunistic bacterium Pseudomonas aeruginosa synthesizes significant amounts of an additional phospholipid, identified as 2′ alanyl‐phosphatidylglycerol (A‐PG), when exposed to acidic growth conditions. At pHu20035.3 A‐PG contributed up to 6% to the overall lipid content of the bacterium. Sequence analysis of P.u2003aeruginosa revealed open reading frame PA0920 showing 34% sequence identity to a protein from Staphylococcus aureus involved in tRNA‐dependent formation of lysyl‐phosphatidylglycerol. The P.u2003aeruginosa deletion mutant ΔPA0920 failed to synthesize A‐PG. Heterologous overproduction of PA0920 in Escherichia coli resulted in the formation of significant amounts of A‐PG, otherwise not synthesized by E.u2003coli. Consequently, the protein encoded by PA0920 was named A‐PG synthase. The enzyme was identified as an integral component of the inner membrane. The protein was partially purified by detergent solubilization and subjected to an in vitro activity assay. tRNAAla‐dependent catalysis was demonstrated. Transcriptional analysis of the corresponding gene in P.u2003aeruginosa using lacZ reporter gene fusion under various pH conditions indicated a 4.4‐fold acid‐activated transcription. A phenotype microarray analysis was used to identify further conditions for A‐PG function.

Convergent Synthesis of Columnar Twins and Tetramers from Triphenylene Building Blocks – The First Example of a Columnar Spiro-Twin

The novel spiro-twins 3a–f were prepared in three steps by one-pot oxidative coupling of guaiacol (9) and 1,2-dialkoxybenzenes 10a–f, followed by demethylation of the triphenylene 11 and subsequent etherification with the tetrabromide 6. A related strategy gave the tetramers 7a–f in two steps. Whereas derivatives 3a,b and 7a,b with pentyloxy and hexyloxy chains showed isotropic melting behavior, compounds 3c–f and 7c–f with longer alkyl chains displayed columnar mesophases. DSC, polarizing microscopy, X-ray diffraction and molecular modeling studies were used to further characterize the type of mesophase. For the spiro-twin 3 either a rectangular or a hexagonal columnar phase were conceivable. In case of 7f a hexagonal columnar mesophase was assigned.

An esterase from the basidiomycete Pleurotus sapidus hydrolyzes feruloylated saccharides

Investigating the secretion of esterases by the basidiomycetous fungus Pleurotus sapidus in a Tween 80-rich nutrient medium, an enzyme was discovered that hydrolyzed the ester bond of feruloylated saccharides. The enzyme was purified by ion exchange and size exclusion chromatography. Polyacrylamide gel electrophoresis analysis showed a monomeric protein of about 55xa0kDa. The complete coding sequence with an open reading frame of 1,665xa0bp encoded a protein (Est1) consisting of 554 amino acids. The enzyme showed no significant homology to any published feruloyl esterase sequences, but possessed putative conserved domains of the lipase/esterase superfamily. Substrate specificity studies classified the new enzyme as type-A feruloyl esterase, hydrolyzing methyl ferulate, methyl sinapate, and methyl p-coumarate but no methyl caffeate. The enzyme had a pH optimum of 6 and a temperature optimum at 50xa0°C. Ferulic acid was efficiently released from ferulated saccharides, and the feruloyl esterase exhibited moderate stability in biphasic systems (50xa0% toluene or tert-butylmethyl ether).

Lyngbyazothrins A−D, Antimicrobial Cyclic Undecapeptides from the Cultured Cyanobacterium Lyngbya sp.

Four novel cyclic undecapeptides, lyngbyazothrins A (1), B (2), C (3), and D (4), were isolated from the cultured Lyngbya sp. 36.91 as binary mixtures (1/2 and 3/4). Their structures were elucidated by analysis of 1D (1H and 13C) and 2D (COSY, TOCSY, ROESY, NOESY, HMQC, and HMBC) NMR spectra, ESIMSMS, ESITOFMS, and amino acid analyses. Three unusual amino acids were present and identified as 4-methoxyhomophenylalanine in 1 and 3, homophenylalanine in 2 and 4, and 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Aound) in all compounds, while 3 and 4 have an additional N-acetyl-N-methyltyrosine unit. The mixture of lyngbyazothrins A (1) and B (2) shows only low antimicrobial activity against Micrococcus flavus, whereas the mixture of lyngbyazothrins C (3) and D (4) was active against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens. It seems that the acyl residue at C-5 of the Aound unit plays an important role in antimicrobial activity.

Production and structural analysis of the polysaccharide secreted by Trametes (Coriolus) versicolor ATCC 200801.

Trametes versicolor ATCC 200801 secretes 4.1xa0g L−1 of exopolysaccharide (EPS) when synthetic minimal medium and low-shear bioreactor cultivation technique are used. Structural and compositional analyses by thin layer chromatography, gas chromatography–mass spectrometry, electrospray ionization tandem mass spectrometry, and nuclear magnetic resonance spectroscopy yielded predominantly glucose and small amounts of galactose, mannose, arabinose, and xylose. The main EPS is composed of β-1,3/β-1,6-linked d-glucose molecules which is identical with Schizophyllan but does not possess a triple helical arrangement as secondary structure. Two molar mass fractions were detected by size exclusion chromatography yielding weight-average molecular weights of 4,100 and 2.6xa0kDa. Protein content varies between 2–3.6% (w/w). The exopolysaccharide is different in the nature of the glycosidic linkage, composition of monosaccharides, protein content, and weight-average molecular weight compared to the well-known polysaccharopeptide (PSP) and polysaccharopeptide Krestin (PSK).

Resistance Phenotypes Mediated by Aminoacyl-Phosphatidylglycerol Synthases

The specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine or with lysine catalyzed by aminoacyl-phosphatidylglycerol synthases (aaPGS) was shown to render various organisms less susceptible to antibacterial agents. This study makes use of Pseudomonas aeruginosa chimeric mutant strains producing lysyl-phosphatidylglycerol (L-PG) instead of the naturally occurring alanyl-phosphatidylglycerol (A-PG) to study the resulting impact on bacterial resistance. Consequences of such artificial phospholipid composition were studied in the presence of an overall of seven antimicrobials (β-lactams, a lipopeptide antibiotic, cationic antimicrobial peptides [CAMPs]) to quantitatively assess the effect of A-PG substitution (with L-PG, L-PG and A-PG, increased A-PG levels). For the employed Gram-negative P. aeruginosa model system, an exclusive charge repulsion mechanism does not explain the attenuated antimicrobial susceptibility due to PG modification. Additionally, the specificity of nine orthologous aaPGS enzymes was experimentally determined. The newly characterized protein sequences allowed for the establishment of a significant group of A-PG synthase sequences which were bioinformatically compared to the related group of L-PG synthesizing enzymes. The analysis revealed a diverse origin for the evolution of A-PG and L-PG synthases, as the specificity of an individual enzyme is not reflected in terms of a characteristic sequence motif. This finding is relevant for future development of potential aaPGS inhibitors.

Characterization of an antifungal compound produced by Bacillus sp. strain A5F that inhibits Sclerotinia sclerotiorum

A potential antagonist, Bacillus sp. strain A5F was isolated from soybean rhizosphere following in vitro dual plate screening. The bacterium displayed strong inhibitory activity in vitro against soybean stem rot pathogen, Sclerotinia sclerotiorum. The culture supernatant of strain A5F completely suppressed the mycelial growth of the pathogen, indicating that suppression was due to the presence of antifungal compounds in the culture filtrate. The culture filtrate also suppressed other phytopathogenic fungi including Fusarium oxysporum and Macrophomina phaseolina, in vitro suggesting a broad spectrum antagonistic activity against fungal pathogens. Chemical extraction followed by chromatographic analysis resulted in two antifungal fractions. The high resolution‐electron spin ionization‐mass spectrometry (HR‐ESI‐MS) and Nuclear Magnetic Resonance (1D and 2D1H) spectra of these antifungal fractions revealed the presence of antifungal compounds, one of which showed similarity to bacillomycin D. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Identification and localization of a lipase-like acyltransferase in phenylpropanoid metabolism of tomato (Solanum lycopersicum).

We have isolated an enzyme classified as chlorogenate: glucarate caffeoyltransferase (CGT) from seedlings of tomato (Solanum lycopersicum) that catalyzes the formation of caffeoylglucarate and caffeoylgalactarate using chlorogenate (5-O-caffeoylquinate) as acyl donor. Peptide sequences obtained by trypsin digestion and spectrometric sequencing were used to isolate the SlCGT cDNA encoding a protein of 380 amino acids with a putative targeting signal of 24 amino acids indicating an entry of the SlCGT into the secretory pathway. Immunogold electron microscopy revealed the localization of the enzyme in the apoplastic space of tomato leaves. Southern blot analysis of genomic cDNA suggests that SlCGT is encoded by a single-copy gene. The SlCGT cDNA was functionally expressed in Nicotiana benthamiana leaves and proved to confer chlorogenate-dependent caffeoyltransferase activity in the presence of glucarate. Sequence comparison of the deduced amino acid sequence identified the protein unexpectedly as a GDSL lipase-like protein, representing a new member of the SGNH protein superfamily. Lipases of this family employ a catalytic triad of Ser-Asp-His with Ser as nucleophile of the GDSL motif. Site-directed mutagenesis of each residue of the assumed respective SlCGT catalytic triad, however, indicated that the catalytic triad of the GDSL lipase is not essential for SlCGT enzymatic activity. SlCGT is therefore the first example of a GDSL lipase-like protein that lost hydrolytic activity and has acquired a completely new function in plant metabolism, functioning in secondary metabolism as acyltransferase in synthesis of hydroxycinnamate esters by employing amino acid residues different from the lipase catalytic triad.