Different biosynthetic pathways to fosfomycin in Pseudomonas syringae and Streptomyces species.
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ABSTRACT:
SUBMITTER: Kim SY
PROVIDER: S-EPMC3421606 | biostudies-literature | 2012 Aug
REPOSITORIES: biostudies-literature
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Project description:The final step in the biosynthesis of fosfomycin in Streptomyces wedmorensis is catalyzed by ( S)-2-hydroxypropylphosphonic acid (HPP) epoxidase ( Sw-HppE). A homologous enzyme from Pseudomonas syringae whose encoding gene ( orf3) shares a relatively low degree of sequence homology with the corresponding Sw-HppE gene has recently been isolated. This purified P. syringae protein was determined to catalyze the epoxidation of ( S)-HPP to fosfomycin and the oxidation of ( R)-HPP to 2-oxopropylphosphonic acid under the same conditions as Sw-HppE. Therefore, this protein is indeed a true HPP epoxidase and is termed Ps-HppE. Like Sw-HppE, Ps-HppE was determined to be post-translationally modified by the hydroxylation of a putative active site tyrosine (Tyr95). Analysis of the Fe(II) center by EPR spectroscopy using NO as a spin probe and molecular oxygen surrogate reveals that Ps-HppE's metal center is similar, but not identical, to that of Sw-HppE. The identity of the rate-determining step for the ( S)-HPP and ( R)-HPP reactions was determined by measuring primary deuterium kinetic effects, and the outcome of these results was correlated with density functional theory calculations. Interestingly, the reaction using the nonphysiological substrate ( R)-HPP was 1.9 times faster than that with ( S)-HPP for both Ps-HppE and Sw-HppE. This is likely due to the difference in bond dissociation energy of the abstracted hydrogen atom for each respective reaction. Thus, despite the low level of amino acid sequence identity, Ps-HppE is a close mimic of Sw-HppE, representing a second example of a non-heme iron-dependent enzyme capable of catalyzing dehydrogenation of a secondary alcohol to form a new C-O bond.
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Project description:Streptomyces bacteria are renowned for their ability to produce bioactive secondary metabolites. Recently, synthetic biology has enabled the production of intermediates and shunt products, which may have altered biological activities compared to the end products of the pathways. Here, we have evaluated the potential of recently isolated alnumycins and other closely related pyranonaphthoquinone (PNQ) polyketides against Staphylococcus aureus biofilms. The antimicrobial potency of the compounds against planktonic cells and biofilms was determined by redox dye-based viability staining, and the antibiofilm efficacy of the compounds was confirmed by viable counting. A novel antistaphylococcal polyketide, alnumycin D, was identified. Unexpectedly, the C-ribosylated pathway shunt product alnumycin D was more active against planktonic and biofilm cells than the pathway end product alnumycin A, where a ribose unit has been converted into a dioxane moiety. The evaluation of the antibiofilm potential of other alnumycins revealed that the presence of the ribose moiety in pyranose form is essential for high activity against preformed biofilms. Furthermore, the antibiofilm potential of other closely related PNQ polyketides was examined. Based on their previously reported activity against planktonic S. aureus cells, granaticin B, kalafungin, and medermycin were also selected for testing, and among them, granaticin B was found to be the most potent against preformed biofilms. The most active antibiofilm PNQs, alnumycin D and granaticin B, share several structural features that may be important for their antibiofilm activity. They are uncharged, glycosylated, and also contain a similar oxygenation pattern of the lateral naphthoquinone ring. These findings highlight the potential of antibiotic biosynthetic pathways as a source of effective antibiofilm compounds.
| S-EPMC4576117 | biostudies-literature
Project description:Two methionine biosynthetic genes in Pseudomonas syringae pv. syringae, metX and metW, were isolated, sequenced, and evaluated for their roles in methionine biosynthesis and bacterial fitness on leaf surfaces. The metXW locus was isolated on a 1.8-kb DNA fragment that was required for both methionine prototrophy and wild-type epiphytic fitness. Sequence analysis identified two consecutive open reading frames (ORFs), and in vitro transcription-translation experiments provided strong evidence that the ORFs encode proteins with the predicted molecular masses of 39 and 22.5 kDa. The predicted amino acid sequence of MetX (39 kDa) showed homology to several known and putative homoserine O-acetyltransferases. This enzyme is the first enzyme in the methionine biosynthetic pathway of fungi, gram-negative bacteria of the genus Leptospira, and several gram-positive bacterial genera. Both metX and metW were required for methionine biosynthesis, and transcription from both genes was not repressed by methionine. MetW (22.5 kDa) did not show significant homology to any known protein, including prokaryotic and eukaryotic methionine biosynthetic enzymes. Several classes of methionine auxotrophs, including metX and metW mutants, exhibit reduced fitness on leaf surfaces, indicating a requirement for methionine prototrophy in wild-type epiphytic fitness. This requirement is enhanced under environmentally stressful conditions, suggesting a role for methionine prototrophy in bacterial stress tolerance.
| S-EPMC107460 | biostudies-literature
Project description:Pseudomonas viridiflava is a gram-negative pseudomonad that is phylogenetically placed within the Pseudomonas syringae species complex. P. viridiflava has a wide host range and causes a variety of symptoms in different plant parts, including stems, leaves, and blossoms. Outside of its role as a pathogen, P. viridiflava also exists as an endophyte, epiphyte, and saprophyte. Increased reports of P. viridiflava causing disease on new hosts in recent years coincide with increased research on its genetic variability, virulence, phylogenetics, and phenotypes. There is high variation in its core genome, virulence factors, and phenotypic characteristics. The main virulence factors of this pathogen include the enzyme pectate lyase and virulence genes encoded within one or two pathogenicity islands. The delineation of P. viridiflava in the P. syringae complex has been investigated using several molecular approaches. P. viridiflava comprises its own species, within the complex. While seemingly an outsider to the complex as a whole due to differences in the core genome and virulence genes, low average nucleotide identity to other of P. syringae complex members, and some phenotypic traits, it remains as part of the complex. Defining phylogenetic, phenotypic, and genomic characteristics of P. viridiflava in comparison to other P. syringae members is important to understanding this pathogen and for the development of disease resistance and management practices.TaxonomyKingdom Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Family Pseudomonadaceae; Genus Pseudomonas; Species Pseudomonas syringae species complex, Genomospecies 6, Phylogroup 7 and 8.Microbiological propertiesGram-negative, fluorescent, aerobic, motile, rod-shaped, oxidase negative, arginine dihydrolase negative, levan production negative (or positive), potato rot positive (or negative), tobacco hypersensitivity positive.GenomeThere are two complete genomes, five chromosome-level genomes, and 1,540 genomes composed of multiple scaffolds of P. viridiflava available in the National Center for Biotechnology Information Genome database. The median total length of these assemblies is 5,975,050 bp, the median number of protein coding genes is 5,208, and the median G + C content is 59.3%.Disease symptomsP. viridiflava causes a variety of disease symptoms, including spots, streaks, necrosis, rots, and more in above- and below-ground plant parts on at least 50 hosts.EpidemiologyThere have been several significant disease outbreaks on field and horticultural crops caused by P. viridiflava since the turn of the century. P. viridiflava has been reported as a pathogen, epiphyte, endophyte, and saprophyte. This species has been isolated from a variety of environmental sources, including asymptomatic wild plants, snow, epilithic biofilms, and icepacks.
| S-EPMC8659605 | biostudies-literature
Project description:A complex programme of regulation governs gene expression during development of the morphologically and biochemically complex eubacterial genus Streptomyces. Earlier work has suggested a model in which 'higher level' pleiotropic regulators activate 'pathway-specific' regulators located within chromosomal gene clusters encoding biosynthesis of individual antibiotics. We used mutational analysis and adventitious overexpression of key Streptomyces coelicolor regulators to investigate functional interactions among them. We report here that cluster-situated regulators (CSRs) thought to be pathway-specific can also control other antibiotic biosynthetic gene clusters, and thus have pleiotropic actions. Surprisingly, we also find that CSRs exhibit growth-phase-dependent control over afsR2/afsS, a 'higher level' pleiotropic regulatory locus not located within any of the chromosomal gene clusters it targets, and further demonstrate that cross-regulation by CSRs is modulated globally and differentially during the S. coelicolor growth cycle by the RNaseIII homologue AbsB. Our results, which reveal a network of functional interactions among regulators that govern production of antibiotics and other secondary metabolites in S. coelicolor, suggest that revision of the currently prevalent view of higher-level versus pathway-specific regulation of secondary metabolism in Streptomyces species is warranted. Groups of assays that are related as part of a time series. Keywords: time_series_design
2006-02-02 | GSE4138 | GEO
Project description:The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (K(m), 7 mM; k(cat), 85 min(-1)). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.
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Project description:We propose Pseudomonas coronafaciens sp. nov. as a new species in genus Pseudomonas, which is diverse from P. syringae. We also classified strains from onions which are responsible for yellow bud (YB) disease as P. coronafaciens. Sequencing of 16S rRNA gene and multi-locus sequence analysis (MLSA) of housekeeping genes (gyrB, rpoD, gltA and gap1 genes) for the P. syringae pv. coronafaciens strains along with other strains of P. syringae pathovars resulted in a distinct cluster separate from other P. syringae pathovars. Based on DNA-DNA relatedness, pathotype strain of P. syringae pv. coronafaciens (CFBP 2216PT) exhibited ≤35.5% similarity with the pathotype strains of P. syringae pv. syringae (CFBP 1392PT, 4702T) but exhibited ≥90.6% with the YB strains (YB 12-1, YB 12-4, YB 09-1). Also, the YB strains (YB 12-1, YB 12-4, YB 09-1) were able to infect only onion but not oat, rye and Italian ryegrass (common hosts for P. syrinage pv. coronafaciens). Contrastingly, P. syringae pv. coronafaciens strains (NCPPB 600PT, ATCC 19608, Pcf 83-300) produced typical halo blight symptoms on oat, rye and Italian rye grass but did not produce any symptoms on onion. These results provide evidence that P. syringae pv. coronafaciens should be elevated to a species level and the new YB strains may potentially be a novel pathovar of hereto proposed P. coronafaciens species.
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Project description:Pseudomonas syringe PB-5123, a producer of fosfomycin, is resistant to high concentrations of the antibiotic. Two possible mechanisms of resistance have been detected: (i) impermeability to exogenous fosfomycin, even in the presence of sugar phosphate uptake inducers, and (ii) antibiotic phosphorylation. The gene responsible for this last activity, fosC, encodes a ca. 19,000-Da protein and is immediately followed by a second open reading frame, which shows sequence similarities to glutathione S-transferases. FosC uses ATP as a cosubstrate in an inactivation reaction that can be reversed with alkaline phosphatase. Other nucleotide triphosphates cannot be substituted for ATP in this reaction. No relationship between fosC and the previously described genes of fosfomycin resistance was found.
| S-EPMC162783 | biostudies-other
Project description:Interactions between epiphytic bacteria and herbivorous insects are ubiquitous on plants, but little is known about their ecological implications. Aphids are devastating crop pests worldwide, so understanding how epiphytic bacteria impact aphid populations is critically important. Recent evidence demonstrates that plant-associated bacteria, such as Pseudomonas syringae, can be highly virulent to one species of aphid, the pea aphid (Acyrthosiphon pisum). Currently, we have no knowledge on how broad this phenomenon is across diverse aphid species that are of high agricultural concern. In controlled experiments using oral exposure in an artificial diet, we challenged five aphid species of agricultural importance with three strains of P. syringae that vary in virulence to the pea aphid. These strains also vary in epiphytic ability and comprise two phytopathogens and one non-plant-pathogenic strain. In general, differences in virulence to aphids remained relatively constant across strains regardless of the aphid species, except for the bird cherry-oat aphid (Rhopalosiphum padi), which is significantly less susceptible to two P. syringae strains. We demonstrate that lower infection incidence likely plays a role in the reduced susceptibility. Importantly, these data support previous results showing that interactions with epiphytic bacteria are important for aphids and may play a large, but underappreciated, role in insect population dynamics. Our study illustrates a potential role of epiphytic bacteria in the biological control of aphid pests broadly but suggests the need for more research encompassing a greater diversity of pest species.IMPORTANCE Sap-sucking aphids are insects of huge agricultural concern, not only because of direct damage caused by feeding but also because of their ability to transmit various plant pathogens. Some bacteria that grow on leaf surfaces, such as Pseudomonas syringae, can infect and kill aphids, making them potentially useful in the biological control of pest aphids. However, only one aphid species, the pea aphid (Acyrthosiphon pisum), has been tested for infection by P. syringae Here, we challenged five aphid species of agricultural importance with three strains of P. syringae that vary in virulence to the pea aphid. We found that four of these aphid species were susceptible to infection and death, suggesting that these bacteria are broadly useful for biological control. However, one aphid species was much more resistant to infection, indicating that more testing on diverse aphid species is needed.
| S-EPMC8208145 | biostudies-literature
Project description:A complex programme of regulation governs gene expression during development of the morphologically and biochemically complex eubacterial genus Streptomyces. Earlier work has suggested a model in which 'higher level' pleiotropic regulators activate 'pathway-specific' regulators located within chromosomal gene clusters encoding biosynthesis of individual antibiotics. We used mutational analysis and adventitious overexpression of key Streptomyces coelicolor regulators to investigate functional interactions among them. We report here that cluster-situated regulators (CSRs) thought to be pathway-specific can also control other antibiotic biosynthetic gene clusters, and thus have pleiotropic actions. Surprisingly, we also find that CSRs exhibit growth-phase-dependent control over afsR2/afsS, a 'higher level' pleiotropic regulatory locus not located within any of the chromosomal gene clusters it targets, and further demonstrate that cross-regulation by CSRs is modulated globally and differentially during the S. coelicolor growth cycle by the RNaseIII homologue AbsB. Our results, which reveal a network of functional interactions among regulators that govern production of antibiotics and other secondary metabolites in S. coelicolor, suggest that revision of the currently prevalent view of higher-level versus pathway-specific regulation of secondary metabolism in Streptomyces species is warranted. Groups of assays that are related as part of a time series. Computed
2006-02-01 | E-GEOD-4138 | biostudies-arrayexpress