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The identification and functional characterization of the amino acid 3-methyl-arginine (MeArg) biosynthesis gene cluster from the epiphyte Pss22d is described. The identified MeArg biosynthesis gene cluster might provide the basis for its large-scale biotechnological production to test its potential for control of the soybean pathogen P. syringae pv. glycinea or its potential for pharmacological applications.

Bacillus sp. G2112, an isolate from cucumber plants that inhibited plant pathogens, produces not only surfactins, iturins, and fengycins common to many Bacillus spp., but also a large variety of N -acyl-(depsi)peptides related to A-3302-B and nobilamides. Four known and fourteen previously unreported nobilamide peptides were characterized using high-resolution mass spectrometry, tandem mass spectrometry, and NMR. The stereochemistry of the amino acids of nobilamide peptides was determined using Marfey's method. The diversity of nobilamide peptides from Bacillus sp. G2112 resulted from the incorporation of different acyl groups and amino acids in the sequence. The peptides occur in linear or cyclic form. In addition, a truncated N -acetylpentapeptide was produced. Agar diffusion assays with selected nobilamide peptides against plant pathogens and human pathogens revealed that A-3302-B and its N -acyl homologs, A-3302-A and nobilamide J, exhibited powerful antibiotic activity (at 5 µg/hole) against Lysinibacillus sphaericus that can cause severe sepsis and bacteremia in patients. Moreover, nobilamide peptides from Bacillus sp. G2112 strongly promoted biofilm formation in the Gram-positive Mycobacterium aurum and Gram-negative pseudomonads. Structurally diverse nobilamides from Bacillus sp. G2112, whether linear or cyclic, penta and heptapeptides, induced biofilm formation, suggesting that the common N -acetyl-D-Phe-D-Leu-L-Phe-D-allo-Thr-L-Val amino acid sequence motif is important for the biofilm-inducing activity.

Polyphosphates (polyP) are ubiquitous biomolecules that play a multitude of physiological roles in many cells. We have studied the presence and role of polyP in a unicellular alga, the freshwater diatom Achnanthidium minutissimum. This diatom stores up to 2.0 pg·cell -1 of polyP, with chain lengths ranging from 130 to 500 inorganic phosphate units (P i ). We applied energy dispersive X-ray spectroscopy, Raman/fluorescence microscopy, and biochemical assays to localize and characterize the intracellular polyP granules that were present in large apical vacuoles. We investigated the fate of polyP in axenic A. minutissimum cells grown under phosphorus (P), replete (P (+) ), or P deplete (P (-) ) cultivation conditions and observed that in the absence of exogenous P, A. minutissimum rapidly utilizes their internal polyP reserves, maintaining their intrinsic growth rates for up to 8 days. PolyP-depleted A. minutissimum cells rapidly took up exogenous P a few hours after P i resupply and generated polyP three times faster than cells that were not initially subjected to P limitation. Accordingly, we propose that A. minutissimum deploys a succession of acclimation strategies regarding polyP dynamics where the production or consumption of polyP plays a central role in the homeostasis of the diatom., (© 2024 The Authors. Journal of Phycology published by Wiley Periodicals LLC on behalf of Phycological Society of America.)

Microbial symbionts of plants constitute promising sources of biocontrol organisms to fight plant pathogens. Bacillus sp. G2112 and Pseudomonas sp. G124 isolated from cucumber ( Cucumis sativus ) leaves inhibited the plant pathogens Erwinia and Fusarium . When Bacillus sp. G2112 and Pseudomonas sp. G124 were co-cultivated, a red halo appeared around Bacillus sp. G2112 colonies. Metabolite profiling using liquid chromatography coupled to UV and mass spectrometry revealed that the antibiotic phenazine-1-carboxylic acid (PCA) released by Pseudomonas sp. G124 was transformed by Bacillus sp. G2112 to red pigments. In the presence of PCA (>40 µg/mL), Bacillus sp. G2112 could not grow. However, already-grown Bacillus sp. G2112 (OD 600 > 1.0) survived PCA treatment, converting it to red pigments. These pigments were purified by reverse-phase chromatography, and identified by high-resolution mass spectrometry, NMR, and chemical degradation as unprecedented 5 N -glucosylated phenazine derivatives: 7-imino-5 N -(1'β-D-glucopyranosyl)-5,7-dihydrophenazine-1-carboxylic acid and 3-imino-5 N -(1'β-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid. 3-imino-5 N -(1'β-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid did not inhibit Bacillus sp. G2112, proving that the observed modification constitutes a resistance mechanism. The coexistence of microorganisms-especially under natural/field conditions-calls for such adaptations, such as PCA inactivation, but these can weaken the potential of the producing organism against pathogens and should be considered during the development of biocontrol strategies.

Aphids are a ubiquitous group of pests in agriculture that cause serious losses. For sustainable aphid identification, it is necessary to develop a precise and fast aphid identification tool. A new simple chemotaxonomy approach to rapidly identify aphids was implemented. The method was calibrated in comparison to the established phylogenetic analysis. For chemotaxonomic analysis, aphids were crushed, their headspace compounds were collected through closed-loop stripping (CLS) and analysed using gas chromatography-mass spectrometry (GC-MS). GC-MS data were then subjected to a discriminant analysis using CAP12.exe software, which identified key biomarkers that distinguish aphid species. A dichotomous key taking into account the presence and absence of a set of species-specific biomarkers was derived from the discriminant analysis which enabled rapid and reliable identification of aphid species. As the method overcomes the limits of morphological identification, it works with aphids at all life stages and in both genders. Thus, our method enables entomologists to assign aphids to growth stages and identify the life history of the investigated aphids, i.e., the food plant(s) they fed on. Our experiments clearly showed that the method could be used as a software to automatically identify aphids.

Rosenbergiella bacteria have been previously isolated predominantly from floral nectar and identified in metagenomic screenings as associated with bees. Here, we isolated three Rosenbergiella strains from the robust Australian stingless bee Tetragonula carbonaria sharing over 99.4% sequence similarity with Rosenbergiella strains isolated from floral nectar. The three Rosenbergiella strains (D21B, D08K, D15G) from T. carbonaria exhibited near-identical 16 S rDNA. The genome of strain D21B was sequenced; its draft genome contains 3,294,717 bp, with a GC content of 47.38%. Genome annotation revealed 3236 protein-coding genes. The genome of D21B differs sufficiently from the closest related strain, Rosenbergiella epipactidis 2.1A, to constitute a new species. In contrast to R. epipactidis 2.1A, strain D21B produces the volatile 2-phenylethanol. The D21B genome contains a polyketide/non-ribosomal peptide gene cluster not present in any other Rosenbergiella draft genomes. Moreover, the Rosenbergiella strains isolated from T. carbonaria grew in a minimal medium without thiamine, but R. epipactidis 2.1A was thiamine-dependent. Strain D21B was named R. meliponini D21B, reflecting its origin from stingless bees. Rosenbergiella strains may contribute to the fitness of T. carbonaria.

Background: Environmental contamination from synthetic plastics and their additives is a widespread problem. Phthalate esters are a class of refractory synthetic organic compounds which are widely used in plastics, coatings, and for several industrial applications such as packaging, pharmaceuticals, and/or paints. They are released into the environment during production, use and disposal, and some of them are potential mutagens and carcinogens. Isophthalate (1,3-benzenedicarboxylic acid) is a synthetic chemical that is globally produced at a million-ton scale for industrial applications and is considered a priority pollutant. Here we describe the biochemical characterization of an enzyme involved in anaerobic degradation of isophthalate by the syntrophically fermenting bacterium Syntrophorhabdus aromaticivorans strain UI that activate isophthalate to isophthalyl-CoA followed by its decarboxylation to benzoyl-CoA., Results: Isophthalate:Coenzyme A ligase (IPCL, AMP-forming) that activates isophthalate to isophthalyl-CoA was heterologously expressed in E. coli (49.6 kDa) for biochemical characterization. IPCL is homologous to phenylacetate-CoA ligase that belongs to the family of ligases that form carbon-sulfur bonds. In the presence of coenzyme A, Mg 2+ and ATP, IPCL converts isophthalate to isophthalyl-CoA, AMP and pyrophosphate (PPi). The enzyme was specifically induced after anaerobic growth of S. aromaticivorans in a medium containing isophthalate as the sole carbon source. Therefore, IPCL exhibited high substrate specificity and affinity towards isophthalate. Only substrates that are structurally related to isophthalate, such as glutarate and 3-hydroxybenzoate, could be partially converted to the respective coenzyme A esters. Notably, no activity could be measured with substrates such as phthalate, terephthalate and benzoate. Acetyl-CoA or succinyl-CoA did not serve as CoA donors. The enzyme has a theoretical pI of 6.8 and exhibited optimal activity between pH 7.0 to 7.5. The optimal temperature was between 25 °C and 37 °C. Denaturation temperature (Tm) of IPCL was found to be at about 63 °C. The apparent K M values for isophthalate, CoA, and ATP were 409 μM, 642 μM, and 3580 μM, respectively. Although S. aromaticivorans is a strictly anaerobic bacterium, the enzyme was found to be oxygen-insensitive and catalysed isophthalyl-CoA formation under both anoxic and oxic conditions., Conclusion: We have successfully cloned the ipcl gene, expressed and characterized the corresponding IPCL enzyme, which plays a key role in isophthalate activation that initiates its activation and further degradation by S. aromaticivorans. Its biochemical characterization represents an important step in the elucidation of the complete degradation pathway of isophthalate., (© 2022. The Author(s).)

Actinomycetes, most notably the genus Streptomyces , have great importance due to their role in the discovery of new natural products, especially for finding antimicrobial secondary metabolites that are useful in the medicinal science and biotechnology industries. In the current study, a genome-based evaluation of Streptomyces sp. isolate BR123 was analyzed to determine its biosynthetic potential, based on its in vitro antimicrobial activity against a broad range of microbial pathogens, including gram-positive and gram-negative bacteria and fungi. A draft genome sequence of 8.15 Mb of Streptomyces sp. isolate BR123 was attained, containing a GC content of 72.63% and 8103 protein coding genes. Many antimicrobial, antiparasitic, and anticancerous compounds were detected by the presence of multiple biosynthetic gene clusters, which was predicted by in silico analysis. A novel metabolite with a molecular mass of 1271.7773 in positive ion mode was detected through a high-performance liquid chromatography linked with mass spectrometry (HPLC-MS) analysis. In addition, another compound, meridamycin, was also identified through a HPLC-MS analysis. The current study reveals the biosynthetic potential of Streptomyces sp. isolate BR123, with respect to the synthesis of bioactive secondary metabolites through genomic and spectrometric analysis. Moreover, the comparative genome study compared the isolate BR123 with other Streptomyces strains, which may expand the knowledge concerning the mechanism involved in novel antimicrobial metabolite synthesis.

Leaf-cutting ants live in mutualistic symbiosis with their garden fungus Leucoagaricus gongylophorus that can be attacked by the specialized pathogenic fungus Escovopsis . Actinomyces symbionts from Acromyrmex leaf-cutting ants contribute to protect L. gongylophorus against pathogens. The symbiont Streptomyces sp. Av25_4 exhibited strong activity against Escovopsis weberi in co-cultivation assays. Experiments physically separating E. weberi and Streptomyces sp. Av25_4 allowing only exchange of volatiles revealed that Streptomyces sp. Av25_4 produces a volatile antifungal. Volatile compounds from Streptomyces sp. Av25_4 were collected by closed loop stripping. Analysis by NMR revealed that Streptomyces sp. Av25_4 overproduces ammonia (up to 8 mM) which completely inhibited the growth of E. weberi due to its strong basic pH. Additionally, other symbionts from different Acromyrmex ants inhibited E. weberi by production of ammonia. The waste of ca. one third of Acomyrmex and Atta leaf-cutting ant colonies was strongly basic due to ammonia (up to ca. 8 mM) suggesting its role in nest hygiene. Not only complex and metabolically costly secondary metabolites, such as polyketides, but simple ammonia released by symbionts of leaf-cutting ants can contribute to control the growth of Escovopsis that is sensitive to ammonia in contrast to the garden fungus L. gongylophorus .

Background: Degradation of acetone by aerobic and nitrate-reducing bacteria can proceed via carboxylation to acetoacetate and subsequent thiolytic cleavage to two acetyl residues. A different strategy was identified in the sulfate-reducing bacterium Desulfococcus biacutus that involves formylation of acetone to 2-hydroxyisobutyryl-CoA., Results: Utilization of short-chain ketones (acetone, butanone, 2-pentanone and 3-pentanone) and isopropanol by the sulfate reducer Desulfosarcina cetonica was investigated by differential proteome analyses and enzyme assays. Two-dimensional protein gel electrophoresis indicated that D. cetonica during growth with acetone expresses enzymes homologous to those described for Desulfococcus biacutus: a thiamine diphosphate (TDP)-requiring enzyme, two subunits of a B 12 -dependent mutase, and a NAD + -dependent dehydrogenase. Total proteomics of cell-free extracts confirmed these results and identified several additional ketone-inducible proteins. Acetone is activated, most likely mediated by the TDP-dependent enzyme, to a branched-chain CoA-ester, 2-hydroxyisobutyryl-CoA. This compound is linearized to 3-hydroxybutyryl-CoA by a coenzyme B 12 -dependent mutase followed by oxidation to acetoacetyl-CoA by a dehydrogenase. Proteomic analysis of isopropanol- and butanone-grown cells revealed the expression of a set of enzymes identical to that expressed during growth with acetone. Enzyme assays with cell-free extract of isopropanol- and butanone-grown cells support a B 12 -dependent isomerization. After growth with 2-pentanone or 3-pentanone, similar protein patterns were observed in cell-free extracts as those found after growth with acetone., Conclusions: According to these results, butanone and isopropanol, as well as the two pentanone isomers, are degraded by the same enzymes that are used also in acetone degradation. Our results indicate that the degradation of several short-chain ketones appears to be initiated by TDP-dependent formylation in sulfate-reducing bacteria.

When Streptomyces violaceoruber grows together with Streptomyces sp. MG7-G1, it reacts with strongly induced droplet production on its aerial mycelium. Initially the metabolite profile of droplets from S. violaceoruber in co-culture with Streptomyces sp. MG7-G1 was compared to samples from S. violaceoruber in single-culture by using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Then, the exudate from agar plates of co-cultures and single cultures (after freezing and thawing) was also analysed. Several compounds were only observed when S. violaceoruber was grown in co-culture. Based on their high-resolution ESI mass spectra and their comparable retention times to the calcium-dependent antibiotics (CDAs) produced by S. violaceoruber, the new compounds were suspected to be deacylated calcium-dependent antibiotics (daCDAs), lacking the 2,3-epoxyhexanoyl residue of CDAs. This was verified by detailed analysis of the MS/MS spectra of the daCDAs in comparison to the CDAs. The major CDA compounds present in calcium ion-supplemented agar medium of co-cultures were daCDAs, thus suggesting that Streptomyces sp. MG7-G1 expresses a deacylase that degrades CDAs., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The anaerobic degradation of aniline was studied in the sulfate-reducing bacterium Desulfatiglans anilini . Our aim was to identify the genes and their proteins that are required for the initial activation of aniline as well as to characterize intermediates of this reaction. Aniline-induced genes were revealed by comparison of the proteomes of D. anilini grown with different substrates (aniline, 4-aminobenzoate, phenol, and benzoate). Most genes encoding proteins that were highly abundant in aniline- or 4-aminobenzoate-grown D. anilini cells but not in phenol- or benzoate-grown cells were located in the putative gene clusters ani (aniline degradation), hcr (4-hydroxybenzoyl-CoA reductase) and phe (phenol degradation). Of these putative gene clusters, only the phe gene cluster has been studied previously. Based on the differential proteome analysis, four candidate genes coding for kinase subunits and carboxylase subunits were suspected to be responsible for the initial conversion of aniline to 4-aminobenzoate. These genes were cloned and overproduced in E. coli . The recombinant proteins were obtained in inclusion bodies but could be refolded successfully. Two subunits of phenylphosphoamidate synthase and two carboxylase subunits converted aniline to 4-aminobenzoate with phenylphosphoamidate as intermediate under consumption of ATP. Only when both carboxylase subunits, one from gene cluster ani and the other from gene cluster phe , were combined, phenylphosphoamidate was converted to 4-aminobenzoate in vitro, with Mn 2+ , K + , and FMN as co-factors. Thus, aniline is degraded by the anaerobic bacterium D. anilini only by recruiting genes for the enzymatic machinery from different gene clusters. We conclude, that D. anilini carboxylates aniline to 4-aminobenzoate via phenylphosphoamidate as an energy rich intermediate analogous to the degradation of phenol to 4-hydroxybenzoate via phenylphosphate., (Copyright © 2020 Xie, Spiteller, Huhn, Schink and Müller.)

Bacterial degradation of the sugar sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) produced by plants, algae, and cyanobacteria, is an important component of the biogeochemical carbon and sulfur cycles. Here, we reveal a third biochemical pathway for primary SQ degradation in an aerobic Bacillus aryabhattai strain. An isomerase converts SQ to 6-deoxy-6-sulfofructose (SF). A novel transaldolase enzyme cleaves the SF to 3-sulfolactaldehyde (SLA), while the non-sulfonated C 3 -(glycerone)-moiety is transferred to an acceptor molecule, glyceraldehyde phosphate (GAP), yielding fructose-6-phosphate (F6P). Intestinal anaerobic bacteria such as Enterococcus gilvus , Clostridium symbiosum , and Eubacterium rectale strains also express transaldolase pathway gene clusters during fermentative growth with SQ. The now three known biochemical strategies for SQ catabolism reflect adaptations to the aerobic or anaerobic lifestyle of the different bacteria. The occurrence of these pathways in intestinal (family) Enterobacteriaceae and (phylum) Firmicutes strains further highlights a potential importance of metabolism of green-diet SQ by gut microbial communities to, ultimately, hydrogen sulfide., Competing Interests: The authors declare they have no competing interests., (© 2020 The Author(s).)

Syntrophorhabdus aromaticivorans is a syntrophically fermenting bacterium that can degrade isophthalate (3-carboxybenzoate). It is a xenobiotic compound which has accumulated in the environment for more than 50 years due to its global industrial usage and can cause negative effects on the environment. Isophthalate degradation by the strictly anaerobic S. aromaticivorans was investigated to advance our understanding of the degradation of xenobiotics introduced into nature, and to identify enzymes that might have ecological significance for bioremediation. Differential proteome analysis of isophthalate- vs benzoate-grown cells revealed over 400 differentially expressed proteins of which only four were unique to isophthalate-grown cells. The isophthalate-induced proteins include a phenylacetate:CoA ligase, a UbiD-like decarboxylase, a UbiX-like flavin prenyltransferase, and a hypothetical protein. These proteins are encoded by genes forming a single gene cluster that putatively codes for anaerobic conversion of isophthalate to benzoyl-CoA. Subsequently, benzoyl-CoA is metabolized by the enzymes of the anaerobic benzoate degradation pathway that were identified in the proteomic analysis. In vitro enzyme assays with cell-free extracts of isophthalate-grown cells indicated that isophthalate is activated to isophthalyl-CoA by an ATP-dependent isophthalate:CoA ligase (IPCL), and subsequently decarboxylated to benzoyl-CoA by a UbiD family isophthalyl-CoA decarboxylase (IPCD) that requires a prenylated flavin mononucleotide (prFMN) cofactor supplied by UbiX to effect decarboxylation. Phylogenetic analysis revealed that IPCD is a novel member of the functionally diverse UbiD family (de)carboxylases. Homologs of the IPCD encoding genes are found in several other bacteria, such as aromatic compound-degrading denitrifiers, marine sulfate-reducers, and methanogenic communities in a terephthalate-degrading reactor. These results suggest that metabolic strategies adapted for degradation of isophthalate and other phthalate are conserved between microorganisms that are involved in the anaerobic degradation of environmentally relevant aromatic compounds.

Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is produced by plants and other phototrophs and its biodegradation is a relevant component of the biogeochemical carbon and sulfur cycles. SQ is known to be degraded by aerobic bacterial consortia in two tiers via C 3 -organosulfonates as transient intermediates to CO 2 , water and sulfate. In this study, we present a first laboratory model for anaerobic degradation of SQ by bacterial consortia in two tiers to acetate and hydrogen sulfide (H 2 S). For the first tier, SQ-degrading Escherichia coli K-12 was used. It catalyzes the fermentation of SQ to 2,3-dihydroxypropane-1-sulfonate (DHPS), succinate, acetate and formate, thus, a novel type of mixed-acid fermentation. It employs the characterized SQ Embden-Meyerhof-Parnas pathway, as confirmed by mutational and proteomic analyses. For the second tier, a DHPS-degrading Desulfovibrio sp. isolate from anaerobic sewage sludge was used, strain DF1. It catalyzes another novel fermentation, of the DHPS to acetate and H 2 S. Its DHPS desulfonation pathway was identified by differential proteomics and demonstrated by heterologously produced enzymes: DHPS is oxidized via 3-sulfolactaldehyde to 3-sulfolactate (SL) by two NAD + -dependent dehydrogenases (DhpA, SlaB); the SL is cleaved by an SL sulfite-lyase known from aerobic bacteria (SuyAB) to pyruvate and sulfite. The pyruvate is oxidized to acetate, while the sulfite is used as electron acceptor in respiration and reduced to H 2 S. In conclusion, anaerobic sulfidogenic SQ degradation was demonstrated as a novel link in the biogeochemical sulfur cycle. SQ is also a constituent of the green-vegetable diet of herbivores and omnivores and H 2 S production in the intestinal microbiome has many recognized and potential contributions to human health and disease. Hence, it is important to examine bacterial SQ degradation also in the human intestinal microbiome, in relation to H 2 S production, dietary conditions and human health.

Degradation of acetone by the sulfate-reducing bacterium Desulfococcus biacutus involves an acetone-activation reaction different from that used by aerobic or nitrate-reducing bacteria, because the small energy budget of sulfate-reducing bacteria does not allow for major expenditures into ATP-consuming carboxylation reactions. In the present study, an inducible coenzyme B 12 -dependent conversion of 2-hydroxyisobutyryl-CoA to 3-hydroxybutyryl-CoA was demonstrated in cell-free extracts of acetone-grown D. biacutus cells, together with a NAD + -dependent oxidation of 3-hydroxybutyryl-CoA to acetoacetyl-CoA. Genes encoding two mutase subunits and a dehydrogenase, which were found previously to be strongly induced during growth with acetone, were heterologously expressed in E. coli. The activities of the purified recombinant proteins matched with the inducible activities observed in cell-free extracts of acetone-grown D. biacutus: proteins (IMG locus tags) DebiaDRAFT_04573 and 04574 constituted a B 12 -dependent 2-hydroxyisobutyryl-CoA/3-hydroxybutyryl-CoA mutase, and DebiaDRAFT_04571 was a 3-hydroxybutyryl-CoA dehydrogenase. Hence, these enzymes play key roles in the degradation of acetone and define an involvement of CoA esters in the pathway. Further, the involvement of 2-hydroxyisobutyryl-CoA strongly indicates that the carbonyl-C 2 of acetone is added, most likely, to formyl-CoA through a TDP-dependent enzyme that is co-induced in acetone-grown cells and is encoded in the same gene cluster as the identified mutase and dehydrogenase., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The specialized, fungal pathogen Escovopsis weberi threatens the mutualistic symbiosis between leaf-cutting ants and their garden fungus (Leucoagaricus gongylophorus). Because E. weberi can overwhelm L. gongylophorus without direct contact, it was suspected to secrete toxins. Using NMR and mass spectrometry, we identified several secondary metabolites produced by E. weberi. E. weberi produces five shearinine-type indole triterpenoids including two novel derivatives, shearinine L and shearinine M, as well as the polyketides, emodin and cycloarthropsone. Cycloarthropsone and emodin strongly inhibited the growth of the garden fungus L. gongylophorous at 0.8 and 0.7 μmol, respectively. Emodin was also active against Streptomyces microbial symbionts (0.3 μmol) of leaf-cutting ants. Shearinine L instead did not affect the growth of L. gongylophorus in agar diffusion assays. However, in dual choice behavioral assays Acromyrmex octospinosus ants clearly avoided substrate treated with shearinine L for the garden fungus after a 2 d learning period, indicating that the ants quickly learn to avoid shearinine L., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Streptomyces violaceoruber grown in co-culture with Streptomyces aburaviensis produces an about 17-fold higher volume of droplets on its aerial mycelium than in single-culture. Physical separation of the Streptomyces strains by either a plastic barrier or by a dialysis membrane, which allowed communication only by the exchange of volatile compounds or diffusible compounds in the medium, respectively, still resulted in enhanced droplet formation. The application of molecular sieves to bioassays resulted in the attenuation of the droplet-inducing effect of S. aburaviensis indicating the absorption of the compound. 1 H-NMR analysis of molecular-sieve extracts and the selective indophenol-blue reaction revealed that the volatile droplet-inducing compound is ammonia. The external supply of ammonia in biologically relevant concentrations of ≥8 mM enhanced droplet formation in S. violaceoruber in a similar way to S. aburaviensis. Ammonia appears to trigger droplet production in many Streptomyces strains because four out of six Streptomyces strains exposed to ammonia exhibited induced droplet production.

The pathway of anaerobic degradation of o-phthalate was studied in the nitrate-reducing bacterium Azoarcus sp. strain PA01. Differential two-dimensional protein gel profiling allowed the identification of specifically induced proteins in o-phthalate-grown compared to benzoate-grown cells. The genes encoding o-phthalate-induced proteins were found in a 9.9 kb gene cluster in the genome of Azoarcus sp. strain PA01. The o-phthalate-induced gene cluster codes for proteins homologous to a dicarboxylic acid transporter, putative CoA-transferases and a UbiD-like decarboxylase that were assigned to be specifically involved in the initial steps of anaerobic o-phthalate degradation. We propose that o-phthalate is first activated to o-phthalyl-CoA by a putative succinyl-CoA-dependent succinyl-CoA:o-phthalate CoA-transferase, and o-phthalyl-CoA is subsequently decarboxylated to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase. Results from in vitro enzyme assays with cell-free extracts of o-phthalate-grown cells demonstrated the formation of o-phthalyl-CoA from o-phthalate and succinyl-CoA as CoA donor, and its subsequent decarboxylation to benzoyl-CoA. The putative succinyl-CoA:o-phthalate CoA-transferase showed high substrate specificity for o-phthalate and did not accept isophthalate, terephthalate or 3-fluoro-o-phthalate whereas the putative o-phthalyl-CoA decarboxylase converted fluoro-o-phthalyl-CoA to fluoro-benzoyl-CoA. No decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA. Both enzyme activities were oxygen-insensitive and inducible only after growth with o-phthalate. Further degradation of benzoyl-CoA proceeds analogous to the well-established anaerobic benzoyl-CoA degradation pathway of nitrate-reducing bacteria., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Bacteria were isolated from necrotic apple and pear tree tissue and from dead wood in Germany and Austria as well as from pear tree exudate in China. They were selected for growth at 37 °C, screened for levan production and then characterized as Gram-negative, facultatively anaerobic rods. Nucleotide sequences from 16S rRNA genes, the housekeeping genes dnaJ, gyrB, recA and rpoB alignments, BLAST searches and phenotypic data confirmed by MALDI-TOF analysis showed that these bacteria belong to the genus Gibbsiella and resembled strains isolated from diseased oaks in Britain and Spain. Gibbsiella-specific PCR primers were designed from the proline isomerase and the levansucrase genes. Acid secretion was investigated by screening for halo formation on calcium carbonate agar and the compound identified by NMR as acetic acid. Its production by Gibbsiella spp. strains was also determined in culture supernatants by GC/MS analysis after derivatization with pentafluorobenzyl bromide. Some strains were differentiated by the PFGE patterns of SpeI digests and by sequence analyses of the lsc and the ppiD genes, and the Chinese Gibbsiella strain was most divergent. The newly investigated bacteria as well as Gibbsiella querinecans, Gibbsiella dentisursi and Gibbsiella papilionis, isolated in Britain, Spain, Korea and Japan, are taxonomically related Enterobacteriaceae, tolerate and secrete acetic acid. We therefore propose to unify them in the species Gibbsiella acetica sp. nov., (Copyright © 2015. Published by Elsevier GmbH.)

Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid SQ-diacylglycerol, and SQ comprises a major proportion of the organosulfur in nature, where it is degraded by bacteria. A first degradation pathway for SQ has been demonstrated recently, a "sulfoglycolytic" pathway, in addition to the classical glycolytic (Embden-Meyerhof) pathway in Escherichia coli K-12; half of the carbon of SQ is abstracted as dihydroxyacetonephosphate (DHAP) and used for growth, whereas a C3-organosulfonate, 2,3-dihydroxypropane sulfonate (DHPS), is excreted. The environmental isolate Pseudomonas putida SQ1 is also able to use SQ for growth, and excretes a different C3-organosulfonate, 3-sulfolactate (SL). In this study, we revealed the catabolic pathway for SQ in P. putida SQ1 through differential proteomics and transcriptional analyses, by in vitro reconstitution of the complete pathway by five heterologously produced enzymes, and by identification of all four organosulfonate intermediates. The pathway follows a reaction sequence analogous to the Entner-Doudoroff pathway for glucose-6-phosphate: It involves an NAD(+)-dependent SQ dehydrogenase, 6-deoxy-6-sulfogluconolactone (SGL) lactonase, 6-deoxy-6-sulfogluconate (SG) dehydratase, and 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase. The aldolase reaction yields pyruvate, which supports growth of P. putida, and 3-sulfolactaldehyde (SLA), which is oxidized to SL by an NAD(P)(+)-dependent SLA dehydrogenase. All five enzymes are encoded in a single gene cluster that includes, for example, genes for transport and regulation. Homologous gene clusters were found in genomes of other P. putida strains, in other gamma-Proteobacteria, and in beta- and alpha-Proteobacteria, for example, in genomes of Enterobacteria, Vibrio, and Halomonas species, and in typical soil bacteria, such as Burkholderia, Herbaspirillum, and Rhizobium.

The bacterium Streptomyces coelicolor M145 reacts to transition-metal-ion stress with myriad growth responses, leading to different phenotypes. In particular, in the presence of Co(2+) ions (0.7 mM) S. coelicolor consistently produced a red phenotype. This phenotype, when compared to the wild type, differed strongly in its production of volatile compounds as well as high molecular weight secondary metabolites. LC-MS analysis revealed that in the red phenotype the production of the prodigiosins, undecylprodigiosin and streptorubin B, was strongly induced and, in addition, several intense signals appeared in the LC-MS chromatogram. Using LC-MS/MS and NMR spectroscopy, two new prodigiosin derivatives were identified, that is, coeligiosin A and B, which contained an additional undecylpyrrolyl side chain attached to the central carbon of the tripyrrole ring system of undecylprodigiosin or streptorubin B. This example demonstrates that environmental factors such as heavy metal ion stress can not only induce the production of otherwise not observed metabolites from so called sleeping genes but alter the products from well-studied biosynthetic pathways., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sulphoquinovose (SQ, 6-deoxy-6-sulphoglucose) has been known for 50 years as the polar headgroup of the plant sulpholipid in the photosynthetic membranes of all higher plants, mosses, ferns, algae and most photosynthetic bacteria. It is also found in some non-photosynthetic bacteria, and SQ is part of the surface layer of some Archaea. The estimated annual production of SQ is 10,000,000,000 tonnes (10 petagrams), thus it comprises a major portion of the organo-sulphur in nature, where SQ is degraded by bacteria. However, despite evidence for at least three different degradative pathways in bacteria, no enzymic reaction or gene in any pathway has been defined, although a sulphoglycolytic pathway has been proposed. Here we show that Escherichia coli K-12, the most widely studied prokaryotic model organism, performs sulphoglycolysis, in addition to standard glycolysis. SQ is catabolised through four newly discovered reactions that we established using purified, heterologously expressed enzymes: SQ isomerase, 6-deoxy-6-sulphofructose (SF) kinase, 6-deoxy-6-sulphofructose-1-phosphate (SFP) aldolase, and 3-sulpholactaldehyde (SLA) reductase. The enzymes are encoded in a ten-gene cluster, which probably also encodes regulation, transport and degradation of the whole sulpholipid; the gene cluster is present in almost all (>91%) available E. coli genomes, and is widespread in Enterobacteriaceae. The pathway yields dihydroxyacetone phosphate (DHAP), which powers energy conservation and growth of E. coli, and the sulphonate product 2,3-dihydroxypropane-1-sulphonate (DHPS), which is excreted. DHPS is mineralized by other bacteria, thus closing the sulphur cycle within a bacterial community.

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones.

Ralfuranones are aryl-substituted furanone secondary metabolites of the Gram-negative plant pathogen Ralstonia solanacearum. New sulfur-containing ralfuranone derivatives were identified, including the methyl thioether-containing ralfuranone D. Isotopic labeling in vivo, as well as headspace analyses of volatiles from R. solanacearum liquid cultures, established a mechanism for the transfer of an intact methylthio group from L-methionine or α-keto-γ-methylthiobutyric acid. The methylthio acceptor molecule ralfuranone I, a previously postulated biosynthetic intermediate in ralfuranone biosynthesis, was isolated and characterized by NMR. The highly reactive Michael acceptor system of this intermediate readily reacts with various thiols, including glutathione., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The epiphyte Pantoea agglomerans 48b/90 (Pa48b) is a promising biocontrol strain against economically important bacterial pathogens such as Erwinia amylovora. Strain Pa48b produces the broad-spectrum antibiotic 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine (APV) in a temperature-dependent manner. An APV-negative mutant still suppressed the E. amylovora population and fire blight disease symptoms in apple blossom experiments under greenhouse conditions, but was inferior to the Pa48b wild-type indicating the influence of APV in the antagonism. In plant experiments with the soybean pathogen Pseudomonas syringae pv. glycinea both, Pa48b and the APV-negative mutant, successfully suppressed the pathogen. Our results demonstrate that the P. agglomerans strain Pa48b is an efficient biocontrol organism against plant pathogens, and we prove its ability for fast colonization of plant surfaces over a wide temperature range., (© 2012 The Authors. Published by Blackwell Publishing Ltd.)

The volatiles released by several streptomycetes were collected by using a closed-loop stripping apparatus (CLSA) and analysed by GC-MS. The obtained headspace extracts of various species contained blastmycinone, a known degradation product of the fungicidal antibiotic, antimycin A(3b), and several unknown derivatives. The suggested structures of these compounds, based on their mass spectra and GC retention indices, were confirmed by comparison to synthetic reference samples. Additional compounds found in the headspace extracts were butenolides formed from the blastmycinones by elimination of the carboxylic acid moiety. Analysis of a gene knockout mutant in the antimycin biosynthetic gene cluster demonstrated that all blastmycinones and butenolides are formed via the antimycin biosynthetic pathway. The structural variation of the blastmycinones identified here is much larger than within the known antimycins, thus suggesting that several antimycin derivatives remain to be discovered., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)