Alternative pathways utilize or circumvent putrescine for biosynthesis of putrescine-containing rhizoferrin

The siderophore rhizoferrin (N1,N4-dicitrylputrescine) is produced in fungi and bacteria to scavenge iron. Putrescine-producing bacterium Ralstonia pickettii synthesizes rhizoferrin and encodes a single nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. From biosynthetic logic, we hypothesized that this single enzyme is sufficient for rhizoferrin biosynthesis. We confirmed this by expression of R. pickettii NIS synthetase in E. coli, resulting in rhizoferrin production. This was further confirmed in vitro using the recombinant NIS synthetase, synthesizing rhizoferrin from putrescine and citrate. Heterologous expression of homologous lbtA from Legionella pneumophila, required for rhizoferrin biosynthesis in that species, produced siderophore activity in E. coli. Rhizoferrin is also synthesized by Francisella tularensis and F. novicida, but unlike R. pickettii or L. pneumophila, Francisella species lack putrescine biosynthetic pathways due to genomic decay. Francisella encodes a NIS synthetase FslA/FigA and an ornithine decarboxylase (ODC) homologue FslC/FigC, required for rhizoferrin biosynthesis. ODC produces putrescine from ornithine but we show here in vitro that FigA synthesizes N-citrylornithine, and FigC is an N-citrylornithine decarboxylase that together synthesize rhizoferrin without using putrescine. We co-expressed F. novicida figA and figC in E. coli, and produced rhizoferrin. A 2.1Å X-ray crystal structure of the FigC N-citrylornithine decarboxylase reveals how the larger substrate is accommodated and how active site residues have changed to recognize N-citrylornithine. FigC belongs to a new subfamily of alanine racemase-fold PLP-dependent decarboxylases that are not involved in polyamine biosynthesis. These data reveal a natural product biosynthetic workaround that evolved to bypass a missing precursor and re-establish it in the final structure.

Rice rhizospheric microbes confer limited Arsenic protection under high Arsenic conditions

Rice (Oryza sativa) is a staple food crop worldwide and plays a critical role in ensuring food security as the global population continues to expand exponentially. Groundwater contamination with Arsenite [As(III)], a naturally occurring inorganic form of arsenic (As), leads to uptake and accumulation within rice plants. As a result, grain yield is lowered, the overall plant health is diminished, and there is a risk of arsenic toxicity from grain consumption. It was previously shown that a novel bacterial strain from the rice rhizosphere may reduce As accumulation in rice plants exposed to low levels of environmental As. We hypothesized that different rice varieties may exhibit varying responses to high As levels, resulting in differences in As uptake and toxicity. Utilizing the natural rice rhizospheric microbes, we initiated a set of hydroponic experiments with two rice varieties, Nipponbare (As tolerant) and IR66 (As susceptible). Rice varieties exposed to high As(III) concentration (50 μM) showed changes in both aboveground and belowground traits. As-tolerant Nipponbare varieties show grain production at high As(III) concentrations compared to the As-susceptible IR66 variety. Supplementation of natural rice rhizospheric microbes as single inoculums showed varied responses in both As-tolerant and As-susceptible varieties. Three natural rice rhizospheric microbes Pantoea sps (EA106), Pseudomonas corrugata (EA104), and Arthrobacter oxydans (EA201) were selected based on previously reported high Iron (Fe)-siderophore activity and were used for the hydroponic experiments as well as a non-rice rhizospheric strain, Bacillus subtilis UD1022. Interestingly, treatment with two strains (EA104 and EA201) led to reduction in As(III) uptake in shoots, roots, and grains and the degree of reduction of As(III) was pronounced in As-susceptible IR66 varieties. Non-rice rhizospheric UD1022 showed subtle protection against high As toxicity. High As(III) treatment led to lack or delay of flowering and seed setting in the As-susceptible IR66 variety. The data presented here may further the understanding of how beneficial microbes in the rhizosphere may help rice plants cope with high concentrations of As in the soil or groundwater.

Phylogenomic Analyses of Non-Dikarya Fungi Supports Horizontal Gene Transfer Driving Diversification of Secondary Metabolism in the Amphibian Gastrointestinal Symbiont, Basidiobolus

Research into secondary metabolism (SM) production by fungi has resulted in the discovery of diverse, biologically active compounds with significant medicinal applications. The fungi rich in SM production are taxonomically concentrated in the subkingdom Dikarya, which comprises the phyla Ascomycota and Basidiomycota. Here, we explore the potential for SM production in Mucoromycota and Zoopagomycota, two phyla of nonflagellated fungi that are not members of Dikarya, by predicting and identifying core genes and gene clusters involved in SM. The majority of non-Dikarya have few genes and gene clusters involved in SM production except for the amphibian gut symbionts in the genus Basidiobolus. Basidiobolus genomes exhibit an enrichment of SM genes involved in siderophore, surfactin-like, and terpene cyclase production, all these with evidence of constitutive gene expression. Gene expression and chemical assays also confirm that Basidiobolus has significant siderophore activity. The expansion of SMs in Basidiobolus are partially due to horizontal gene transfer from bacteria, likely as a consequence of its ecology as an amphibian gut endosymbiont.

Transporter genes in biosynthetic gene clusters predict metabolite characteristics and siderophore activity

Biosynthetic gene clusters (BGCs) are operonic sets of microbial genes that synthesize specialized metabolites with diverse functions, including siderophores and antibiotics, which often require export to the extracellular environment. For this reason, genes for transport across cellular membranes are essential for the production of specialized metabolites, and are often genomically co-localized with BGCs. Here we conducted a comprehensive computational analysis of transporters associated with characterized BGCs. In addition to known exporters, in BGCs we found many importer-specific transmembrane domains that co-occur with substrate binding proteins possibly for uptake of siderophores or metabolic precursors. Machine learning models using transporter gene frequencies were predictive of known siderophore activity, molecular weights, and a measure of lipophilicity (log P) for corresponding BGC-synthesized metabolites. Transporter genes associated with BGCs were often equally or more predictive of metabolite features than biosynthetic genes. Given the importance of siderophores as pathogenicity factors, we used transporters specific for siderophore BGCs to identify both known and uncharacterized siderophore-like BGCs in genomes from metagenomes from the infant and adult gut microbiome. We find that 23% of microbial genomes from the infant gut have siderophore-like BGCs, but only 3% of those assembled from adult gut microbiomes do. While siderophore-like BGCs from the infant gut are predominantly associated with Enterobactericaee and Staphylococcus, siderophore-like BGCs can be identified from taxa in the adult gut microbiome that have rarely been recognized for siderophore production. Taken together, these results show that consideration of BGC-associated transporter genes can inform predictions of specialized metabolite structure and function.

Phylogenomic analyses of non-Dikarya fungi supports horizontal gene transfer driving diversification of secondary metabolism in the amphibian gastrointestinal symbiont, Basidiobolus

Research into secondary metabolism (SM) production by fungi has resulted in the discovery of diverse, biologically active compounds with significant medicinal applications. However, the fungi rich in SM production are taxonomically restricted to Dikarya, two phyla of Kingdom Fungi, Ascomycota and Basidiomycota. Here, we explore the potential for SM production in Mucoromycota and Zoopagomycota, two phyla of nonflagellated fungi that are not members of Dikarya, by predicting and identifying core genes and gene clusters involved in SM. The majority of non-Dikarya have few genes and gene clusters involved in SM production except for the amphibian gut symbionts in the genus Basidiobolus. Basidiobolus genomes exhibit an enrichment of SM genes involved in siderophore, surfactin-like, and terpene cyclase production, all these with evidence of constitutive gene expression. Gene expression and chemical assays confirm that Basidiobolus has significant siderophore activity. The expansion of SMs in Basidiobolus are partially due to horizontal gene transfer from bacteria, likely as a consequence of its ecology as an amphibian gut endosymbiont.

Loss of O-Linked Protein Glycosylation in Burkholderia cenocepacia Impairs Biofilm Formation and Siderophore Activity and Alters Transcriptional Regulators

ABSTRACT O-linked protein glycosylation is a conserved feature of the Burkholderia genus. The addition of the trisaccharide β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc to membrane exported proteins in Burkholderia cenocepacia is required for bacterial fitness and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. Using proteomics, luciferase reporter assays, and DNA cross-linking, we demonstrate the loss of glycosylation leads to changes in transcriptional regulation of multiple proteins, including the repression of the master quorum CepR/I. These proteomic and transcriptional alterations lead to the abolition of biofilm formation and defects in siderophore activity. Surprisingly, the abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants, except for BCAL1086 and BCAL2974, which were found in reduced amounts, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, biofilm formation, or siderophore activity. Together, our results show that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the transcriptional regulatory systems, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins. IMPORTANCE Protein glycosylation is increasingly recognized as a common posttranslational protein modification in bacterial species. Despite this commonality, our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption of O-linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular, and phenotypic assays. We find that in contrast to recent findings on the N-linked glycosylation systems of Campylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that loss of glycosylation in B. cenocepacia strains leads to global proteome and transcriptional changes, including the repression of the quorum-sensing regulator cepR (BCAM1868) gene. These alterations lead to dramatic phenotypic changes in glycosylation-null strains, which are paralleled by both global proteomic and transcriptional alterations, which do not appear to directly result from the loss of glycosylation per se. This research unravels the pleiotropic effects of O-linked glycosylation in B. cenocepacia, demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome.

KARAKTERISASI RIZOBAKTERI YANG BERPOTENSI MENGENDALIKAN BAKTERI Xanthomonas oryzae pv. oryzae DAN MENINGKATKAN PERTUMBUHAN TANAMAN PADI

Characterization of rhizobacteri having potential to control Xanthomonas oryzae pv. oryzae and increase plant growth of rice. Rhizobacteria which are isolated from root could produce HCN, siderophore, and plant growth regulator, induce systemic resistance, and are increase uptake of plant nutrition such as phosphate. The objective of this research was to characterize rhizobacteri as controling agent for Xanthomonas oryzae pv.oryzae (Xoo) and as plant growth promoter. The results show that the isolates of P. diminuta A6, P. aeruginosa A54, B.subtilis 11 /C, and B. subtilis 5/B inhibited the growth of Xoo. B. subtilis 5/B isolate produced the highest siderophore activity, followed by of P. aeruginosa A54, P. diminuta A6 and B. subtilis 11/C. Only P. diminuta A6 isolate produced HCN. The results also showed that all rhizobacteri produced IAA i.e. B.subtilis 5/B (22.10 µg/ml), B. subtilis 11/C (19.05 µg/ml), P. diminuta A6 (8.68 ug/ml), and P. aeruginosa A54 (2.95 µg/ml). The content of phosphatase enzyme was as folows B.subtilis 5/B (2.78 units/ ml), B.subtilis 11/C (5.7 units/ml), P. diminuta A6 (2.25 units/ml), and P. aeruginosa A54 (5.71 units / ml). Content of peroxidase enzymes in plants that were treated by using isolates was as follows B.subtilis 5/B (1.30 x 10-3 units/mg protein), P. aeruginosa A6 (1.20 x 10-3 units/mg protein), B.subtilis 11/C (1.15 x 10-3 units/mg protein), and P. aeruginosa A54 (1.05 x 10-3 units/mg protein).