Using the structural diversity of siderophore biosynthesis genes for intra-and interspecific differentiation of pathogenic yersinia

The paper analyzes the siderophore biosynthesis genes that are located in the ysu and ynp loci of only Y. pestis and Y. pseudotuberculosis, have variable structure between different strains of both species and contain previously unknown variable number tandem repeats (VNTR). The purpose of the study was to assess the possibility of using these VNTR as genetic markers for intra-and interspecific differentiation of pathogenic Yersinia. Based on the novel VNTR-markers, three pairs of primers (ysu-interF/R, ilp1F/R и ilp2F/R) were designed and used for the in silico and in vitro PCR analysis of various Y.pestis and Y. pseudotuberculosis strains. All studied Y. pestis strains of the main subspecies (ssp pestis), unlike the strains of non-main subspecies and Y. pseudotuberculosis, did not give amplicon with ilp1F/R primers, since the area between them contains an IS100 element. To identify the strains of the main subspecies, the fourth pair of primers ilp1F-is100R was designed, allowing the most dangerous ssp pestis strains to be distinguished from the not dangerous non-main ssp strains. Y. pseudotuberculosis strains were characterized by a significant variety of amplicons with three pairs of primers, and which made it possible to carry out intraspecies strain genotyping. At the same time, for those strains whose serotype is known, the correlation between the serogroup and the genotype of the strains was observed. Analysis of the 1 serotype strains representing most sequenced Y. pseudotuberculosis strains allowed us to separate two gene groups differing from the rest of 1 serotype gene groups. The first one included the serotype 1a strains isolated from people in Europe, which are known to have the greatest pathogenetic potential. The other one was formed by serotype 1b strains isolated from people in Siberia and Primorye, which are characterized by the high epidemic potential. Thus, four pairs of primers designed in this study can be used to develop additional tests for the identification and differential diagnostics of the most dangerous Y. pestis ssp pestis and Y. pseudotuberculosis serotype 1a and 1b strains.

AB569, a non-toxic combination of acidified nitrite and EDTA, is effective at killing the notorious Iraq/Afghanistan combat wound pathogens, multi-drug resistant Acinetobacter baumannii and Acinetobacter spp.

Multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Acinetobacter spp. present monumental global health challenges. These organisms represent model Gram-negative pathogens with known antibiotic resistance and biofilm-forming properties. Herein, a novel, nontoxic biocide, AB569, consisting of acidified nitrite (A-NO2-) and ethylenediaminetetraacetic acid (EDTA), demonstrated bactericidal activity against all Ab and Acinetobacter spp. strains, respectively. Average fractional inhibitory concentrations (FICs) of 0.25 mM EDTA plus 4 mM A-NO2- were observed across several clinical reference and multiple combat wound isolates from the Iraq/Afghanistan wars. Importantly, toxicity testing on human dermal fibroblasts (HDFa) revealed an upper toxicity limit of 3 mM EDTA plus 64 mM A-NO2-, and thus are in the therapeutic range for effective Ab and Acinetobacter spp. treatment. Following treatment of Ab strain ATCC 19606 with AB569, quantitative PCR analysis of selected genes products to be responsive to AB569 revealed up-regulation of iron regulated genes involved in siderophore production, siderophore biosynthesis non-ribosomal peptide synthetase module (SBNRPSM), and siderophore biosynthesis protein monooxygenase (SBPM) when compared to untreated organisms. Taken together, treating Ab infections with AB569 at inhibitory concentrations reveals the potential clinical application of preventing Ab from gaining an early growth advantage during infection followed by extensive bactericidal activity upon subsequent exposures.

Draft Genome Sequence of Pseudomonas sp. Strain FEN, Isolated from the Fe- and Organic Matter-Rich Schlöppnerbrunnen Fen

ABSTRACT Here, we report the draft genome sequence of Pseudomonas sp. strain FEN, a nonfluorescent siderophore producer that was isolated from the Schlöppnerbrunnen fen, which is characterized by high concentrations of Fe, dissolved organic matter (DOM), and Fe-DOM complexes. This draft genome sequence provides insight into the mechanisms of siderophore biosynthesis and siderophore-mediated iron uptake by this bacterium.

A Pseudoalteromonas clade with remarkable biosynthetic potential

Pseudoalteromonas species produce a diverse range of biologically active compounds, including those biosynthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here we report the biochemical and genomic analysis of Pseudoalteromonas sp. HM-SA03, isolated from the blue-ringed octopus, Hapalochalaena sp. Genome mining for secondary metabolite pathways revealed seven putative NRPS/PKS biosynthesis gene clusters, including those for the biosynthesis of alterochromides, pseudoalterobactins, alteramides and four hitherto novel compounds. Among these was a novel siderophore biosynthesis gene cluster with unprecedented architecture (NRPS-PKS-NRPS-PKS-NRPS-PKS-NRPS). Alterochromide production in HM-SA03 was also confirmed by liquid chromatography-mass spectrometry. An investigation of the biosynthetic potential of 42 publicly available Pseudoalteromonas genomes indicated that some of these gene clusters are distributed throughout the genus. Through phylogenetic analysis, a particular subset of strains formed a clade with extraordinary biosynthetic potential, with an average density of ten biosynthesis gene clusters per genome. In contrast, the majority of Pseudoalteromonas strains outside this clade contained an average of three clusters encoding complex biosynthesis. These results highlight the under-explored potential of Pseudoalteromonas as a source of new natural products. Importance This study demonstrates that the Pseudoalteromonas strain, HM-SA03, isolated from the venomous blue-ringed octopus, Hapalochalaena sp., is a biosynthetically talented organism, capable of producing alterochromides and potentially six other specialized metabolites. We have identified a pseudoalterobactin biosynthesis gene cluster and proposed a pathway for the production of the associated siderophore. A novel siderophore biosynthesis gene cluster with unprecedented architecture was also identified in the HM-SA03 genome. Finally, we have demonstrated that HM-SA03 belongs to a phylogenetic clade of strains with extraordinary biosynthetic potential. While our results do not support a role of HM-SA03 in Hapalochalaena sp. venom (tetrodotoxin) production, they emphasize the untapped potential of Pseudoalteromonas as a source of novel natural products.

Hierarchical routing in carbon metabolism favors iron-scavenging strategy in iron-deficient soilPseudomonasspecies

High-affinity iron (Fe) scavenging compounds, or siderophores, are widely employed by soil bacteria to survive scarcity in bioavailable Fe. Siderophore biosynthesis relies on cellular carbon metabolism, despite reported decrease in both carbon uptake and Fe-containing metabolic proteins in Fe-deficient cells. Given this paradox, the metabolic network required to sustain the Fe-scavenging strategy is poorly understood. Here, through multiple13C-metabolomics experiments with Fe-replete and Fe-limited cells, we uncover how soilPseudomonasspecies reprogram their metabolic pathways to prioritize siderophore biosynthesis. Across the three species investigated (Pseudomonas putidaKT2440,Pseudomonas protegensPf-5, andPseudomonas putidaS12), siderophore secretion is higher during growth on gluconeogenic substrates than during growth on glycolytic substrates. In response to Fe limitation, we capture decreased flux toward the tricarboxylic acid (TCA) cycle during the metabolism of glycolytic substrates but, due to carbon recycling to the TCA cycle via enhanced anaplerosis, the metabolism of gluconeogenic substrates results in an increase in both siderophore secretion (up to threefold) and Fe extraction (up to sixfold) from soil minerals. During simultaneous feeding on the different substrate types, Fe deficiency triggers a hierarchy in substrate utilization, which is facilitated by changes in protein abundances for substrate uptake and initial catabolism. Rerouted metabolism further promotes favorable fluxes in the TCA cycle and the gluconeogenesis–anaplerosis nodes, despite decrease in several proteins in these pathways, to meet carbon and energy demands for siderophore precursors in accordance with increased proteins for siderophore biosynthesis. Hierarchical carbon metabolism thus serves as a critical survival strategy during the metal nutrient deficiency.

Biochemical and Molecular Characterization, and Bioprospecting of Drought Tolerant Actinomycetes from Maize Rhizosphere Soil

Drought is a major limitation to maize cultivation around the globe. Seven actinomycetes strains were isolated from maize rhizosphere soils in Mahikeng, North-West Province, South Africa. The isolates were biochemically characterized and identified with 16S rRNA gene sequence analysis. Isolates were also screened in vitro for abiotic stress tolerance to different concentrations of NaCl, pH, and polyethylene glycol (PEG 8000), as well as for biosynthesis of drought tolerance genes namely Glutathione peroxidase (GPX), Glycine-rich RNA binding protein (GRP), Desiccation protectant protein (DSP), Guanosine triphosphate binding protein (GTP) and plant growth-promoting genes:1-aminocyclopropane-1-carboxylate deaminase (accd) and siderophore biosynthesis (Sid). About 71.43% of isolates were of the genus Streptomyces (99-100% similarity), while 14.29% belong to the genus Arthrobacter (R15) and 14.29% to the genus Microbacterium (S11) respectively (99% similarity). Five isolates had their optimum growth at 35°C. Arthrobacter arilaitensis (R15) grew and tolerated 5%, 10%, and 20% PEG at 120 h. Root length increased by 110.53% in PEG treated maize seeds (−0.30 MPa) inoculated with Streptomyces pseudovenezuelae (S20) compared to the un-inoculated control. Likewise, germination percentage and vigor index increased by 37.53% and 194.81% respectively in PEG treated seeds inoculated with S20 than the un-inoculated PEG treated seeds. ACC deaminase gene was amplified in all the isolates, while the gene for siderophore biosynthesis was amplified in 85.71% of the isolates. Genes for the synthesis of GPX, GRP, DSP and GTP were amplified in Arthrobacter arilaitensis (R15) and Streptomyces pseudovenezuelae (S20) which lacked GTP. The amplification of drought-tolerant and plant growth-promoting primers indicates the possible presence of these genes in the isolates. These isolates have the potential for use as bio-inoculants, not only to improve drought tolerance in maize but also to be utilized as biofertilizers and biocontrol agents to facilitate growth promotion.

Arginine Auxotrophy Affects Siderophore Biosynthesis and Attenuates Virulence of Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic human pathogen mainly infecting immunocompromised patients. The aim of this study was to characterize the role of arginine biosynthesis in virulence of A. fumigatus via genetic inactivation of two key arginine biosynthetic enzymes, the bifunctional acetylglutamate synthase/ornithine acetyltransferase (argJ/AFUA_5G08120) and the ornithine carbamoyltransferase (argB/AFUA_4G07190). Arginine biosynthesis is intimately linked to the biosynthesis of ornithine, a precursor for siderophore production that has previously been shown to be essential for virulence in A. fumigatus. ArgJ is of particular interest as it is the only arginine biosynthetic enzyme lacking mammalian homologs. Inactivation of either ArgJ or ArgB resulted in arginine auxotrophy. Lack of ArgJ, which is essential for mitochondrial ornithine biosynthesis, significantly decreased siderophore production during limited arginine supply with glutamine as nitrogen source, but not with arginine as sole nitrogen source. In contrast, siderophore production reached wild-type levels under both growth conditions in ArgB null strains. These data indicate that siderophore biosynthesis is mainly fueled by mitochondrial ornithine production during limited arginine availability, but by cytosolic ornithine production during high arginine availability via cytosolic arginine hydrolysis. Lack of ArgJ or ArgB attenuated virulence of A. fumigatus in the insect model Galleria mellonella and in murine models for invasive aspergillosis, indicating limited arginine availability in the investigated host niches.