Homospermidine Synthase Research Articles

Pyrrolizwilline, a Unique Bacterial Alkaloid Assembled by a Nonribosomal Peptide Synthetase and non-Enzymatic Dimerization.

Pyrrolizidine alkaloids (PAs) are a structurally diverse group of heterocyclic specialized metabolites characterized by a core structure comprising a hexahydro-1H-pyrrolizine. PAs are synthesized through two main pathways. In plants, assembly occurs via a homospermidine synthase, and in bacteria, through combined action of a nonribosomal peptide synthetase and a Baeyer-Villiger monooxygenase. While the toxic properties of plant-derived PAs and their prevalence in animal and human foods have been extensively studied, the biological roles and biosynthesis of more complex bacterial PAs are not well understood. Here, we report the identification and characterization of a bacterial biosynthetic gene cluster from Xenorhabdus hominickii, xhpA-G, which is responsible for producing the PA pseudo-dimer pyrrolizwilline. Analysis of X. hominickii promoter exchange mutants together with heterologous expression of xhpA-G in E. coli, revealed a set of pathway intermediates, two of which were chemically synthesized, as well as multiple derivatives. This information was leveraged to propose a detailed biosynthetic pathway to pyrrolizwilline. Furthermore, we have characterized the hydrolase XhpG, the key enzyme in the conversion of the pathway intermediate pyrrolizixenamide to pyrrolizwilline, using X-ray crystallography and small-angle X-ray scattering (SAXS).

Seeing double: two different homospermidine oxidases are involved in pyrrolizidine alkaloid biosynthesis in different organs of comfrey (Symphytum officinale).

Pyrrolizidine alkaloids (PAs) are toxic specialized metabolites produced in several plant species and frequently contaminate herbal teas or livestock feed. In comfrey (Symphytum officinale, Boraginaceae), they are produced in two different organs of the plant, the root and young leaves. In this study, we demonstrate that homospermidine oxidase (HSO), a copper-containing amine oxidase (CuAO) responsible for catalyzing the formation of the distinctive pyrrolizidine ring in PAs, is encoded by two individual genes. Specifically, SoCuAO1 is expressed in young leaves, while SoCuAO5 is expressed in roots. CRISPR/Cas9-mediated knockout of socuao5 resulted in hairy roots (HRs) unable to produce PAs, supporting its function as HSO in roots. Plants regenerated from socuao5 knockout HRs remained completely PA-free until the plants began to develop inflorescences, indicating the presence of another HSO that is expressed only during flower development. Stable expression of SoCuAO1 in socuao5 knockout HRs rescued the ability to produce PAs. In vitro assays of both enzymes transiently expressed in Nicotiana benthamiana confirmed their HSO activity and revealed the ability of HSO to control the stereospecific cyclization of the pyrrolizidine backbone. The observation that the first specific step of PA biosynthesis catalyzed by homospermidine synthase requires only one gene copy, while two independent paralogs are recruited for the subsequent homospermidine oxidation in different tissues of the plant, suggests a complex regulation of the pathway. This adds a new level of complexity to PA biosynthesis, a system already characterized by species-specific, tight spatio-temporal regulation, and independent evolutionary origins in multiple plant lineages.

Rational engineering of homospermidine synthase for enhanced catalytic efficiency toward spermidine synthesis

Spermidine is a naturally occurring polyamine widely utilized in the prevention and treatment of various diseases. Current spermidine biosynthetic methods have problems such as low efficiency and complex multi-enzyme catalysis. Based on sequence-structure-function relationships, we engineered the widely studied homospermidine synthase from Blastochloris viridis (BvHSS) and obtained mutants that could catalyze the production of spermidine from 1,3-diaminopropane and putrescine. The specific activities of BvHSS and the mutants D361E and E232D + D361E (E232D-D) were 8.72, 46.04 and 48.30 U/mg, respectively. The optimal pH for both mutants was 9.0, and the optimal temperature was 50 °C. Molecular docking and dynamics simulations revealed that mutating aspartic acid at position 361 to glutamic acid narrowed the substrate binding pocket, promoting stable spermidine production. Conversely, mutating glutamic acid at position 232 to aspartic acid enlarged the substrate channel entrance, facilitating substrate entry into the active pocket and enhancing spermidine generation. In whole-cell catalysis lasting 6 h, D361E and E232D-D synthesized 725.3 and 933.5 mg/L of spermidine, respectively. This study offers a practical approach for single-enzyme catalyzed spermidine synthesis and sheds light on the crucial residues influencing homospermidine synthase catalytic activity in spermidine production.

Caldomycin, a new guanidopolyamine produced by a novel agmatine homocoupling enzyme involved in homospermidine biosynthesis

An extreme thermophilic bacterium, Thermus thermophilus produces more than 20 unusual polyamines, but their biosynthetic pathways, including homospermidine, are not yet fully understood. Two types of homospermidine synthases have been identified in plants and bacteria, which use spermidine and putrescine or two molecules of putrescine as substrates. However, homospermidine synthases with such substrate specificity have not been identified in T. thermophilus. Here we identified a novel agmatine homocoupling enzyme that is involved in homospermidine biosynthesis in T. thermophilus. The reaction mechanism is different from that of a previously described homospermidine synthase, and involves conjugation of two molecules of agmatine, which produces a diamidino derivative of homospermidine (caldomycin) as an immediate precursor of homospermidine. We conclude that there is a homospermidine biosynthetic pathway from agmatine via caldomycin synthase followed by ureohydrolase in T. thermophilus. Furthermore, it is shown that caldomycin is a novel compound existing in nature.

Pyrrolizidine alkaloids are synthesized and accumulated in flower of Myosotis scorpioides.

Pyrrolizidine alkaloids (PAs) are specialized metabolites that are produced by various plant families that act as defense compounds against herbivores. On the other hand, certain lepidopteran insects uptake and utilize these PAs as defense compounds against their predators and as precursors of their sex pheromones. Adult males of Parantica sita, a danaine butterfly, convert PAs into their sex pheromones. In early summer, P. sita swarms over the flowers of Myosotis scorpioides, which belongs to the family Boraginaceae. M. scorpioides produces PAs, but the organs in which PAs are produced and whether P. sita utilizes PAs in M. scorpioides are largely unknown. In the present study, we clarified that M. scorpioides accumulates retronecine-core PAs in N-oxide form in all organs, including flowers. We also identified two M. scorpioides genes encoding homospermidine synthase (HSS), a key enzyme in the PA biosynthetic pathway, and clarified that these genes are expressed in all organs where PAs accumulate. Phylogenetic analysis suggested that these two HSS genes were originated from gene duplication of deoxyhypusine synthase gene like other HSS genes in PA-producing plants. These results suggest that PAs are synthesized and accumulated in the flower of M. scorpioides and provide a possibility for a PA-mediated interaction between P. sita and M. scorpioides.

Insights into the Unusual Activity of a Novel Homospermidine Synthase with a Promising Application to Produce Spermidine.

Spermidine is a naturally occurring polyamine with multiple biological activities and potential food and agricultural applications. However, sustainable and scalable spermidine production has not yet been attained. In this study, a homospermidine synthase (HSS) from Pseudomonas frederiksbergensis (PfHSS) capable of catalyzing the synthesis of spermidine from 1,3-diaminopropane and putrescine was identified based on multiple sequence alignment using Blastochloris viridis HSS (BvHSS) as a template. The optimal reaction pH and temperature for purified PfHSS were determined to be 8.5 and 45 °C, respectively, and K+ was able to promote the enzyme activity. Further analysis of the structural and functional relationships through molecular docking and molecular dynamics simulation indicates that glutamic acid at position 359 is the essential residue for the enzyme-catalyzed synthesis of spermidine. The whole-cell catalytic reaction yielded 1321.4 mg/L spermidine and 678.2 mg/L of homospermidine. This study presents a novel, promising, and sustainable biological method for producing spermidine.

Functional polyamine metabolic enzymes and pathways encoded by the virosphere

Viruses produce more viruses by manipulating the metabolic and replication systems of their host cells. Many have acquired metabolic genes from ancestral hosts and use the encoded enzymes to subvert host metabolism. The polyamine spermidine is required for bacteriophage and eukaryotic virus replication, and herein, we have identified and functionally characterized diverse phage- and virus-encoded polyamine metabolic enzymes and pathways. These include pyridoxal 5'-phosphate (PLP)-dependent ornithine decarboxylase (ODC), pyruvoyl-dependent ODC and arginine decarboxylase (ADC), arginase, S-adenosylmethionine decarboxylase (AdoMetDC/speD), spermidine synthase, homospermidine synthase, spermidine N-acetyltransferase, and N-acetylspermidine amidohydrolase. We identified homologs of the spermidine-modified translation factor eIF5a encoded by giant viruses of the Imitervirales. Although AdoMetDC/speD is prevalent among marine phages, some homologs have lost AdoMetDC activity and have evolved into pyruvoyl-dependent ADC or ODC. The pelagiphages that encode the pyruvoyl-dependent ADCs infect the abundant ocean bacterium Candidatus Pelagibacter ubique, which we have found encodes a PLP-dependent ODC homolog that has evolved into an ADC, indicating that infected cells would contain both PLP- and pyruvoyl-dependent ADCs. Complete or partial spermidine or homospermidine biosynthetic pathways are found encoded in the giant viruses of the Algavirales and Imitervirales, and in addition, some viruses of the Imitervirales can release spermidine from the inactive N-acetylspermidine. In contrast, diverse phages encode spermidine N-acetyltransferase that can sequester spermidine into its inactive N-acetyl form. Together, the virome-encoded enzymes and pathways for biosynthesis and release or biochemical sequestration of spermidine or its structural analog homospermidine consolidate and expand evidence supporting an important and global role of spermidine in virus biology.

The long road of functional recruitment-The evolution of a gene duplicate to pyrrolizidine alkaloid biosynthesis in the morning glories (Convolvulaceae).

In plants, homospermidine synthase (HSS) is a pathway‐specific enzyme initiating the biosynthesis of pyrrolizidine alkaloids (PAs), which function as a chemical defense against herbivores. In PA‐producing Convolvulaceae (“morning glories”), HSS originated from deoxyhypusine synthase at least >50 to 75 million years ago via a gene duplication event and subsequent functional diversification. To study the recruitment of this ancient gene duplicate to PA biosynthesis, the presence of putative hss gene copies in 11 Convolvulaceae species was analyzed. Additionally, various plant parts from seven of these species were screened for the presence of PAs. Although all of these species possess a putative hss copy, PAs could only be detected in roots of Ipomoea neei (Spreng.) O'Donell and Distimake quinquefolius (L.) A.R.Simões & Staples in this study. A precursor of PAs was detected in roots of Ipomoea alba L. Thus, despite sharing high sequence identities, the presence of an hss gene copy does not correlate with PA accumulation in particular species of Convolvulaceae. In vitro activity assays of the encoded enzymes revealed a broad spectrum of enzyme activity, further emphasizing a functional diversity of the hss gene copies. A recently identified HSS specific amino acid motif seems to be important for the loss of the ancestral protein function—the activation of the eukaryotic initiation factor 5A (eIF5A). Thus, the motif might be indicative for a change of function but allows not to predict the new function. This emphasizes the challenges in annotating functions for duplicates, even for duplicates from closely related species.

Structure-Based Engineering of Substrate Specificity for Blastochloris Viridis Homospermidine Synthase

Structure-Based Engineering of Substrate Specificity for Blastochloris Viridis Homospermidine Synthase

Structural and catalytic characterization of Blastochloris viridis and Pseudomonas aeruginosa homospermidine synthases supports the essential role of cation-π interaction.

Polyamines influence medically relevant processes in the opportunistic pathogen Pseudomonas aeruginosa, including virulence, biofilm formation and susceptibility to antibiotics. Although homospermidine synthase (HSS) is part of the polyamine metabolism in various strains of P. aeruginosa, neither its role nor its structure has been examined so far. The reaction mechanism of the nicotinamide adenine dinucleotide (NAD+)-dependent bacterial HSS has previously been characterized based on crystal structures of Blastochloris viridis HSS (BvHSS). This study presents the crystal structure of P. aeruginosa HSS (PaHSS) in complex with its substrate putrescine. A high structural similarity between PaHSS and BvHSS with conservation of the catalytically relevant residues is demonstrated, qualifying BvHSS as a model for mechanistic studies of PaHSS. Following this strategy, crystal structures of single-residue variants of BvHSS are presented together with activity assays of PaHSS, BvHSS and BvHSS variants. For efficient homospermidine production, acidic residues are required at the entrance to the binding pocket (`ionic slide') and near the active site (`inner amino site') to attract and bind the substrate putrescine via salt bridges. The tryptophan residue at the active site stabilizes cationic reaction components by cation-π interaction, as inferred from the interaction geometry between putrescine and the indole ring plane. Exchange of this tryptophan for other amino acids suggests a distinct catalytic requirement for an aromatic interaction partner with a highly negative electrostatic potential. These findings substantiate the structural and mechanistic knowledge on bacterial HSS, a potential target for antibiotic design.

The Orphan Crop Crassocephalum crepidioides Accumulates the Pyrrolizidine Alkaloid Jacobine in Response to Nitrogen Starvation.

Crassocephalum crepidioides is an African orphan crop that is used as a leafy vegetable and medicinal plant. Although it is of high regional importance in Sub-Saharan Africa, the plant is still mainly collected from the wild and therefore efforts are made to promote its domestication. However, in addition to beneficial properties, there was first evidence that C. crepidioides can accumulate the highly toxic pyrrolizidine alkaloid (PA) jacobine and here it was investigated, how jacobine production is controlled. Using ecotypes from Africa and Asia that were characterized in terms of their PA profiles, it is shown that the tetraploid C. crepidioides forms jacobine, an ability that its diploid close relative Crassocephalum rubens appears to lack. Evidence is provided that nitrogen (N) deficiency strongly increases jacobine in the leaves of C. crepidioides, that this capacity depends more strongly on the shoot than the root system, and that homospermidine synthase (HSS) activity is not rate-limiting for this reaction. A characterization of HSS gene representation and transcription showed that C. crepidioides and C. rubens possess two functional versions, one of which is conserved, that the HSS transcript is mainly present in roots and that its abundance is not controlled by N deficiency. In summary, this work improves our understanding of how environmental cues impact PA biosynthesis in plants and provides a basis for the development of PA-free C. crepidioides cultivars, which will aid its domestication and safe use.

CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids.

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.

Gene products and processes contributing to lanthanide homeostasis and methanol metabolism in Methylorubrum extorquens AM1

Lanthanide elements have been recently recognized as “new life metals” yet much remains unknown regarding lanthanide acquisition and homeostasis. In Methylorubrum extorquens AM1, the periplasmic lanthanide-dependent methanol dehydrogenase XoxF1 produces formaldehyde, which is lethal if allowed to accumulate. This property enabled a transposon mutagenesis study and growth studies to confirm novel gene products required for XoxF1 function. The identified genes encode an MxaD homolog, an ABC-type transporter, an aminopeptidase, a putative homospermidine synthase, and two genes of unknown function annotated as orf6 and orf7. Lanthanide transport and trafficking genes were also identified. Growth and lanthanide uptake were measured using strains lacking individual lanthanide transport cluster genes, and transmission electron microscopy was used to visualize lanthanide localization. We corroborated previous reports that a TonB-ABC transport system is required for lanthanide incorporation to the cytoplasm. However, cells were able to acclimate over time and bypass the requirement for the TonB outer membrane transporter to allow expression of xoxF1 and growth. Transcriptional reporter fusions show that excess lanthanides repress the gene encoding the TonB-receptor. Using growth studies along with energy dispersive X-ray spectroscopy and transmission electron microscopy, we demonstrate that lanthanides are stored as cytoplasmic inclusions that resemble polyphosphate granules.

Reduction of Pyrrolizidine Alkaloid Levels in Comfrey (Symphytum officinale) Hairy Roots by RNAi Silencing of Homospermidine Synthase.

Comfrey is a medicinal plant, extracts of which are traditionally used for the treatment of painful inflammatory muscle and joint problems, because the plant contains allantoin and rosmarinic acid. However, its medicinal use is limited because of its toxic pyrrolizidine alkaloid (PA) content. PAs encompass more than 400 different compounds that have been identified from various plant lineages. To date, only the first pathway-specific enzyme, homospermidine synthase (HSS), has been characterized. HSS catalyzes the formation of homospermidine, which is exclusively incorporated into PAs. HSS has been recruited several times independently in various plant lineages during evolution by duplication of the gene encoding deoxyhypusine synthase (DHS), an enzyme of primary metabolism. Here, we describe the establishment of RNAi knockdown hairy root mutants of HSS in Symphytum officinale. A knockdown of HSS by 60 - 80% resulted in a significant reduction of homospermidine by ~ 86% and of the major PA components 7-acetylintermedine N-oxide and 3-acetylmyoscorpine N-oxide by approximately 60%. The correlation of reduced transcript levels of HSS with reduced levels of homospermidine and PAs provides in planta support for HSS being the central enzyme in PA biosynthesis. Furthermore, the generation of PA-depleted hairy roots might be a cost-efficient way for reducing toxic by-products that limit the medicinal applicability of S. officinale extracts.

Pyrrolizidine Alkaloids: Biosynthesis, Biological Activities and Occurrence in Crop Plants.

Pyrrolizidine alkaloids (PAs) are heterocyclic secondary metabolites with a typical pyrrolizidine motif predominantly produced by plants as defense chemicals against herbivores. They display a wide structural diversity and occur in a vast number of species with novel structures and occurrences continuously being discovered. These alkaloids exhibit strong hepatotoxic, genotoxic, cytotoxic, tumorigenic, and neurotoxic activities, and thereby pose a serious threat to the health of humans since they are known contaminants of foods including grain, milk, honey, and eggs, as well as plant derived pharmaceuticals and food supplements. Livestock and fodder can be affected due to PA-containing plants on pastures and fields. Despite their importance as toxic contaminants of agricultural products, there is limited knowledge about their biosynthesis. While the intermediates were well defined by feeding experiments, only one enzyme involved in PA biosynthesis has been characterized so far, the homospermidine synthase catalyzing the first committed step in PA biosynthesis. This review gives an overview about structural diversity of PAs, biosynthetic pathways of necine base, and necic acid formation and how PA accumulation is regulated. Furthermore, we discuss their role in plant ecology and their modes of toxicity towards humans and animals. Finally, several examples of PA-producing crop plants are discussed.

Low pyrrolizidine alkaloid levels in perennial ryegrass is associated with the absence of a homospermidine synthase gene

BackgroundPyrrolizidine alkaloids (PAs) are a class of secondary metabolites that function as feeding deterrents in a range of different plant species. In perennial ryegrass (Lolium perenne L.) the only PAs that have been identified are the thesinine-rhamnoside group, which displays significant genetic variation. Homospermidine synthase (HSS) has evolved from deoxyhypusine synthase (DHS) and catalyses the first step in the PA pathway, making it a key candidate for the investigation of genes influencing observed PA trait variation.ResultsDuring PCR amplification and sequence analysis of DHS we identified two putative HSS genes in perennial ryegrass. One of the genes (LpHSS1) was absent in some perennial ryegrass plants. Thesinine-rhamnoside levels were measured using liquid chromatography coupled with mass spectrometry in a diverse association mapping population, consisting of 693 plants free of fungal endophytic symbionts. Association tests that accounted for population structure identified a significant association of absence of the LpHSS1 gene with lower levels of thesinine-rhamnoside PAs. HSS-like gene sequences were identified for other grass species of the Poaceae, including tall fescue, wheat, maize and sorghum.ConclusionHSS is situated at the crucial first step in the PA pathway making it an important candidate gene for investigation of involvement in PA phenotypic variation. In this study, PA level in perennial ryegrass was strongly associated with the presence or absence of the LpHSS1 gene. A genetic marker, developed for the presence/absence of LpHSS1, may be used for marker-assisted breeding to either lower or increase PAs in breeding populations of perennial or Italian ryegrass to investigate a potential role in the deterrence of herbivore pests. The presence of HSS-like genes in several other Poaceae species suggests that PA biosynthesis may occur in plant family members beyond perennial ryegrass and tall fescue and identifies a potential route for manipulating PA levels.

Evolution of pyrrolizidine alkaloid biosynthesis in Apocynaceae: revisiting the defence de-escalation hypothesis.

Summary Plants produce specialized metabolites for their defence. However, specialist herbivores adapt to these compounds and use them for their own benefit. Plants attacked predominantly by specialists may be under selection to reduce or eliminate production of co‐opted chemicals: the defence de‐escalation hypothesis.We studied the evolution of pyrrolizidine alkaloids (PAs) in Apocynaceae, larval host plants for PA‐adapted butterflies (Danainae, milkweed and clearwing butterflies), to test if the evolutionary pattern is consistent with de‐escalation.We used the first PA biosynthesis specific enzyme (homospermidine synthase, HSS) as tool for reconstructing PA evolution. We found hss orthologues in diverse Apocynaceae species, not all of them known to produce PAs. The phylogenetic analysis showed a monophyletic origin of the putative hss sequences early in the evolution of one Apocynaceae lineage (the APSA clade). We found an hss pseudogene in Asclepias syriaca, a species known to produce cardiac glycosides but no PAs, and four losses of an HSS amino acid motif. APSA clade species are significantly more likely to be Danainae larval host plants than expected if all Apocynaceae species were equally likely to be exploited.Our findings are consistent with PA de‐escalation as an adaptive response to specialist attack.

Identification of a Second Site of Pyrrolizidine Alkaloid Biosynthesis in Comfrey to Boost Plant Defense in Floral Stage.

Pyrrolizidine alkaloids (PAs) are toxic secondary metabolites that are found in several distantly related families of the angiosperms. The first specific step in PA biosynthesis is catalyzed by homospermidine synthase (HSS), which has been recruited several times independently by duplication of the gene encoding deoxyhypusine synthase, an enzyme involved in the posttranslational activation of the eukaryotic initiation factor 5A. HSS shows highly diverse spatiotemporal gene expression in various PA-producing species. In comfrey (Symphytum officinale; Boraginaceae), PAs are reported to be synthesized in the roots, with HSS being localized in cells of the root endodermis. Here, we show that comfrey plants activate a second site of HSS expression when inflorescences start to develop. HSS has been localized in the bundle sheath cells of specific leaves. Tracer feeding experiments have confirmed that these young leaves express not only HSS but the whole PA biosynthetic route. This second site of PA biosynthesis results in drastically increased PA levels within the inflorescences. The boost of PA biosynthesis is proposed to guarantee optimal protection especially of the reproductive structures.

Spermidine Inversely Influences Surface Interactions and Planktonic Growth in Agrobacterium tumefaciens.

In bacteria, the functions of polyamines, small linear polycations, are poorly defined, but these metabolites can influence biofilm formation in several systems. Transposon insertions in an ornithine decarboxylase (odc) gene in Agrobacterium tumefaciens, predicted to direct synthesis of the polyamine putrescine from ornithine, resulted in elevated cellulose. Null mutants for odc grew somewhat slowly in a polyamine-free medium but exhibited increased biofilm formation that was dependent on cellulose production. Spermidine is an essential metabolite in A. tumefaciens and is synthesized from putrescine in A. tumefaciens via the stepwise actions of carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase (CASDC). Exogenous addition of either putrescine or spermidine to the odc mutant returned biofilm formation to wild-type levels. Low levels of exogenous spermidine restored growth to CASDH and CASDC mutants, facilitating weak biofilm formation, but this was dampened with increasing concentrations. Norspermidine rescued growth for the odc, CASDH, and CASDC mutants but did not significantly affect their biofilm phenotypes, whereas in the wild type, it stimulated biofilm formation and depressed spermidine levels. The odc mutant produced elevated levels of cyclic diguanylate monophosphate (c-di-GMP), exogenous polyamines modulated these levels, and expression of a c-di-GMP phosphodiesterase reversed the enhanced biofilm formation. Prior work revealed accumulation of the precursors putrescine and carboxyspermidine in the CASDH and CASDC mutants, respectively, but unexpectedly, both mutants accumulated homospermidine; here, we show that this requires a homospermidine synthase (hss) homologue. Polyamines are small, positively charged metabolites that are nearly ubiquitous in cellular life. They are often essential in eukaryotes and more variably in bacteria. Polyamines have been reported to influence the surface-attached biofilm formation of several bacteria. In Agrobacterium tumefaciens, mutants with diminished levels of the polyamine spermidine are stimulated for biofilm formation, and exogenous provision of spermidine decreases biofilm formation. Spermidine is also essential for A. tumefaciens growth, but the related polyamine norspermidine exogenously rescues growth and does not diminish biofilm formation, revealing that the growth requirement and biofilm control are separable. Polyamine control of biofilm formation appears to function via effects on the cellular second messenger cyclic diguanylate monophosphate, regulating the transition from a free-living to a surface-attached lifestyle.

Homospermidine synthase contributes to salt tolerance in free-living Rhizobium tropici and in symbiosis with Phaseolus vulgaris

Background and aims Homospermidine is known to be the most abundant polyamine in root nodules of Phaseolus vulgaris induced by Rhizobium tropici. In addition, homospermidine is involved in the stress tolerance of fast-growing rhizobia and in the bacteroid protection from environmental changes. For that reason, in this work, we have investigated the role of the rhizobial homospermidine synthase in the response of P. vulgaris root nodules to salinity.