Polyamines comprehend a family of biological compounds that are required by all living organisms. At physiological pH, they are positively charged and bind to polyanionic compounds, such as RNA, DNA, proteins, and cell membranes (Michael, 2016). This ability explains their important participation in RNA translation, DNA replication, and membrane stability. The most abundant polyamines in bacteria are the diamine putrescine (Put) and the triamine spermidine (Spd) (Gevrekci, 2017), although polyamines with chemical similarities, such as cadaverine and norspermidine, might be also dominant in some species. In turn, the presence of the tetraamine spermine (which is abundant in mammals and plants) is rather rare.

The intracellular and extracellular contents of these compounds are tightly regulated by their synthesis, catabolism, and transport (Figure 1). Two pathways are used for the formation of Put, which depend on the initial substrate. One of them requires the decarboxylation of the amino acid arginine, whereas an alternative pathway decarboxylates ornithine instead. Decarboxylation of arginine is carried out by SpeA (arginine decarboxylase) to form agmatine, which is then converted into Put by two consecutive enzymatic steps catalyzed by agmatine deiminase (AguA) and N-carbamoylputrescine amidohydrolase (AguB). In turn, ornithine is decarboxylated by SpeC (ornithine decarboxylase) to yield Put as the end product. Then, SpeE (spermidine synthase) converts Put into Spd with the addition of amino propyl groups provided by decarboxylated S-adenosyl methionine, which is the product of the enzymatic step catalyzed by SpeD (S-adenosyl methionine decarboxylase). This anabolic pathway is highly conserved in bacteria, and numerous studies have demonstrated its importance in growth, stress tolerance, and virulence. (Shah and Swiatlo, 2008; Gevrekci, 2017; Igarashi and Kashiwagi, 2018; Chagneau et al., 2019; Banerji et al., 2020, 2021; Guerra et al., 2020; Nanduri and Swiatlo, 2021). Despite the fact that the catabolic and transport pathways have received less attention, there is evidence that they also make great contributions to polyamine homeostasis and cell fitness. (Johnson et al., 2012; Liu et al., 2018; Bolard et al., 2019). Several routes have been described for the degradation of polyamines in bacteria. Thus, they can first be converted into glutamyl derivatives by glutamyl-polyamine synthases and then oxidized to gamma-amino butyric acid (GABA) by glutamyl-polyamine oxidases. This involves the PauA and PauB protein families (as they are known in Pseudomonas aeruginosa), respectively (Schneider and Wendisch, 2011). Put might also be transaminated by SpuC using 2-oxo-glutaric acid or pyruvate as amino receptors to render gamma-amino butanal, which is then converted to GABA. Furthermore, the presence of a Spd dehydrogenase protein has been reported in some species (Hisano et al., 1992; Dasu et al., 2006), but this enzyme is not widely distributed in bacteria. In turn, a variety of transport systems participates in polyamine incorporation and secretion, and bacterial genomes generally exhibit redundancy in cognate genes (Schiller et al., 2000; Shah et al., 2008, 2011; Kurihara and Suzuki, 2015; Pipkins et al., 2017). Among these transporters, PotABCD, PotFGHI, PotE, and SapBCDF are the most studied. PotABCD and PotFGHI are involved in Spd and Put incorporation, respectively, whereas PotE and SapBCDF seem to be involved mostly in polyamine secretion.

The roles of these pathways in bacteria have mostly been studied in the model microbes Escherichia coli and P. aeruginosa (Wright et al., 1986; Xie et al., 1989; Lu et al., 2002; Chattopadhyay et al., 2003, 2009; Dasu et al., 2006; Chou et al., 2008; Kurihara et al., 2010; Yao et al., 2011; Wu et al., 2012; Kurihara and Suzuki, 2015; Sugiyama et al., 2016), and also in other human pathogens, such as Streptococcus pneumoniae (Banerji et al., 2021, 2022; Nanduri and Swiatlo, 2021), Vibrio cholerae (Karatan et al., 2005; Bridges and Bassler, 2021), and Salmonella enterica (Jelsbak et al., 2012; Guerra et al., 2020). We currently have a rather incomplete understanding of the importance of polyamines in the growth and virulence of phytopathogenic bacteria, even though some studies have revealed that they are essential in these organisms as well (Gerlin et al., 2021). For instance, it was reported that Spd is required for the synthesis of the toxin Phevamine A in Pseudomonas syringae, which helps to suppress the oxidative burst elicited in plant tissues following pathogen recognition (O’Neill et al., 2018). In addition, the secretion of Put by phytopathogenic bacteria has been observed in different bacterial species, such as Ralstonia solanacearum, P. syringae, and Dickeya zeae (Lowe‐Power et al., 2018; Vilas et al., 2018; Shi et al., 2019). Based on various reports demonstrating that polyamines can scavenge reactive oxygen species (ROS) and activate the antioxidant machinery (Bors et al., 1989; Das and Misra, 2004; Nayyar and Chander, 2004; Tang and Newton, 2005), it was proposed that Put secretion by bacteria constitutes a mechanism to counteract the oxidative stress imposed by plants at the site of the infection, a function that has yet to be confirmed. Alternatively, it has been postulated that Put secretion by D. zeae is engaged in cell-to-cell communication during the invasion of maize (Shi et al., 2019). More work is needed to reach a better understanding of the roles that polyamines play in such microorganisms.

As the number of molecular studies examining phytopathogenic bacteria is constantly growing, meta-analysis of publicly available transcriptomic data represents a valuable tool to comprehend the functionality of gene networks in pathogenesis (Caldas and Vinga, 2014). Thus, the comparison between these studies can lead to the recognition of common gene expression patterns and the identification of the metabolic pathways linked to cell behavior. Under this premise, we hypothesized that this kind of approach could enable us to discern the signatures characterizing the regulation of the metabolism of polyamines in bacterial pathogens of plants and discover potential connections between polyamine homeostasis and bacterial virulence.

We focused this study on transcriptomic data obtained from the bacterial species Pseudomonas syringae. This plant pathogen is worldwide distributed and more than 50 pathovars have been described, which altogether cause disease in the most important crops (Arnold and Preston, 2018; Xin et al., 2018). In addition, other plant and human pathogens were also considered to test whether identified expression signatures could be generalized to other pathogenic systems. Our study showed that polyamine production is first promoted during plant colonization but later suppressed, implying that these chemicals play an important role in the early stages of infection. In agreement with this, co-expression analysis revealed a close link between the expression of genes from the metabolism of polyamines and genes associated to essential cellular activities, such as gene expression regulation, energy production, transmembrane transport, and virulence. Interestingly, findings showed that the initial induction in synthesis are partially suppressed during the activation of plant defense responses, suggesting that plant immunity induction may affect bacterial polyamine homeostasis. In turn, even though the expression of genes from the catabolism and transport of polyamines varied among studies, we observed that polyamine incorporation is induced in later stages of infection. This probably helps to maintain the supply of polyamines when its synthesis is reduced. Another intriguing observation was that polyamine synthesis is repressed in response to oxidative stress, contrary to the widely held belief that polyamine production is essential for tolerance to these conditions. The implications of such observations are discussed.

The expanding amount of transcriptome data sets provides a valuable resource for identifying key features involved in cellular processes like growth, development, and stress tolerance (Caldas and Vinga, 2014). In the case of pathogenic microbes, these studies not only allow researchers to assess the relative relevance of different pathways but also provides a wealth of information about common mechanisms participating in the activation of virulence. In this work, we explored the importance of the metabolic routes maintaining polyamine homeostasis for disease and oxidative stress response in Pseudomonas species, with a focus on P. syringae.

We first compared polyamine metabolism gene expression levels in cells growing in minimal media in relation to those in rich media. Minimal media are thought to mimic apoplastic conditions leading to the induction of the virulence-associated hrp/hrc operons encoding for the T3SS (Rico and Preston, 2008). The studies selected for our analysis offered a time-lapse analysis spanning 8 h of culture and indicate the existence of a regulatory network suppressing polyamine homeostasis under minimal nutrient environments. These results curtail the importance of polyamines in supporting growth in low-nutrient media (meaning that more preferred metabolites would be synthesized instead under these circumstances) and besides, they also suggest that the mechanisms governing polyamine homeostasis are distinct from those inducing bacterial pathogenicity. Even though this observation contradicts our previous gene expression analysis in Pst using qRT-PCR (where the polyamine biosynthetic gene speC and the catabolic enzymes pauA3 and pauB2 resulted mildly upregulated in minimal medium), it should be considered that in the mentioned work only samples taken 6 hpi were analyzed and that variations in gene expression before or after that time could have been missed (Vilas et al., 2018).

An important point to be considered is that growth of the bacteria in planta generates much broader modifications of the bacterial transcriptomes, and consequently, results obtained from in vitro and in planta conditions are not always mirrored (Vandelle et al., 2021). Thus, we then evaluated then expression data from in planta studies since they should more accurately reflect the alterations of polyamine metabolism and its connection to disease. In line with this, our investigation discovered significant changes with respect to the metabolic behavior showed by cells in vitro, as the synthesis of polyamines was enhanced during the first stages of infection (up to 6 hpi) and repressed subsequently as pathogenesis progresses (48 hpi). If this initial rise in the contents of polyamines through their synthesis is required to support cell fitness at this stage, then mutant strains perturbed in polyamine synthesis should be affected in their virulence. Even though this kind of mutants has not been deeply tested in P. syringae yet, a genome-wide fitness profiling made on Pss by Helmann et al. (2019) showed that deletion of ΔspeE conduces to mild negative fitness scores when growing in green pepper (Helmann et al., 2019). The fact that no other mutant strains affected in genes contributing to polyamine biosynthesis or transport demonstrated negative scores could be explained on the bases of pathway redundancy, which would assure the needed supply of polyamines when these pathways are partially affected. In relation to this, works in Ralstonia solanacearum (which exclusively depends on the SpeC pathway for Put synthesis) demonstrated that a ΔspeC mutant is hypovirulent in tomato (Lowe‐Power et al., 2018).

Then there’s the question of why polyamines are needed for the first stages of plant infection. We could get a glimpse of their functions from our co-expression analysis based on the work made by Nobori et al. (2018, 2020), where most of the samples correspond to cells growing for 6 h both in vitro and in planta. This examination indicated that genes involved in polyamine biosynthesis were positively co-expressed with ribosome components as well as regulators of transcription/translation. On the contrary, polyamine catabolic genes showed a negative co-expression relationship with the same group of genes. This is in agreement with studies demonstrating that polyamines are required to assure correct ribosome ensemble and general transcription efficiency (Winther et al., 2021). We also distinguished the same pattern of co-expression with genes involved in ATP formation, carboxylic acid, and nitrogen metabolism. Thus, polyamine accumulation would be associated to the synthesis of important compounds and a higher rate of energy generation and gene expression.

The fact that polyamine biosynthetic genes were inversely linked with genes involved in flagella production is an interesting finding, since it suggests that bacterial movement could be affected by polyamines in Pseudomonads. In agreement with this, it was recently demonstrated that Put accumulation in P. aeruginosa, which is achieved by disrupting Spd synthesis or Put degradation, promotes cell transition toward the formation of sessile biofilms (Liu et al., 2021). However, this does not seem to be a general rule, as the link between polyamines and cell movement depends on the bacterial species. For instance, even though the lack of speA in Dickeya zeae and Proteus mirabilis decreases swimming and swarming mobilities (Shah and Swiatlo, 2008; Shi et al., 2019), deletion of speA and speC in Yersinia pestis perturbs biofilm formation (Patel et al., 2006).

We also evidenced that pre-induction of plant defense responses leads to a minor induction of polyamine biosynthesis and to the upregulation of polyamine transport genes. This also occurs when the effectorless D36E mutant strain is analyzed, emphasizing the importance of the T3SS function in avoiding the effects of plant defense mechanisms on pathogen’s metabolism. It is conceivable that the increased expression of polyamine transporters under these conditions could be an attempt to incorporate these compounds from the extracellular niche as a result of the reduced de novo production. But why is polyamine synthesis diminished after the elicitation of plant immunity? We hypothesize that the explanation to this phenomenon could be associated to 1) a bacterial response to the deleterious environment imposed by the activation of plant immunity, or 2) that plant defense responses directly target the synthesis of these amines to influence bacterial cell fitness. In connection with this, it is known that one of the main defense mechanisms deployed by plants against endophytic bacteria is the accumulation of reactive oxygen species at the apoplast (Yu et al., 2013; O'Leary et al., 2016), provoking oxidative stress conditions in the bacteria dwelling at this niche. With this is mind, we explored transcription data sets obtained from different bacterial species exposed to oxidative stress and found that, even though at different extents, polyamine biosynthetic genes were downregulated in bacteria. Hence, we speculated that the reduction in polyamine synthesis is more probable a consequence of a bacterial regulatory mechanism acting on polyamine synthesis, even though we cannot rule-out a possible direct effect of plant immunity on the expression of these genes. If lower expression of polyamine synthesis genes is associated with the activation of the antioxidative machinery, then it challenges the accepted paradigm that polyamines are required in bacteria for tolerance to oxidative stress (Chattopadhyay et al., 2003; Jelsbak et al., 2012; Johnson et al., 2012; El-Halfawy and Valvano, 2014). For instance, synthesis and secretion of polyamines by R. solanacearum at the tomato xylem was proposed by Lowe‐Power et al. (2018) as a mechanism that subdue this stress. Importantly, the authors demonstrated that Put is accumulated at this compartment presumably as a product of the activation of bacterial metabolism and that a ΔspeC mutant resulted completely avirulent. In relation to this, we described in a previous work that Pst can also secrete large amounts of Put while growing in apoplastic washing fluids, although we could not find a link between this phenomenon and bacterial fitness/virulence. Our next experiments will try to generate Pst mutants unable to synthesize polyamines to further test their roles in stress tolerance and its link with pathogenesis.

As mentioned above, our study showed that bacterial polyamine biosynthesis is repressed at later stages of infection, which is also associated to the induction of polyamine transporters and catabolic genes. A similar scenario is observed in later stages of pathogenic interactions established by other bacterial species. For instance, Xanthomonas axonopodis pv. glycines reduces the expression of speD but upregulates other polyamine biosynthetic and transport genes after 72 hpi in soybean (Chatnaparat et al., 2016), whereas polyamine synthesis was repressed in Xanthomonas oryzae pv. oryzae after 5 days while polyamine transport was activated (Lee et al., 2017; data not shown). Importantly, our hierarchical clustering analysis of co-expressed genes shows that transcription profiles from samples taken 48 hpi are grouped with those obtained from cells growing in vitro under low nitrogen conditions or in minimal medium, as well as in experiments using primed plants (pre-infiltrated with flg22) or the Pst D36E strain. Thus, it is reasonable to conclude that bacteria are facing more restrictive conditions at the later phase of the plant invasion in terms of nutrient availability. Besides, the induction of a number of polyamine metabolic genes at 48 hpi is correlated with the induction of the T3SS. This observation agrees with the idea that the modulation of specific genes from the metabolism of polyamines, but not all, would be coordinated with the induction of bacterial virulence. More research is needed to fully comprehend the regulatory mechanisms governing polyamine metabolism throughout infection and their impact on pathogenesis.

Altogether, these findings lead to the hypothesis that the synthesis of polyamines in phytopathogenic Pseudomonas species is important during the early periods of plant invasion, a process that is accompanied by the activation of nitrogen and carboxylic acid metabolism, general gene expression, and energy generation (Figure 6). This pathway, however, is repressed at the later stages of infection with the consequent induction of the polyamine transport and catabolic systems, a metabolic shift that would enable the survival of cells in the plant environment. Whether this behavior is extrapolated to other bacterial species should be evaluated with the generation of specific mutant strains perturbed in different branches of the polyamine metabolism.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

LS and AG designed the study. SS, LS, and AG prepared figures. AG wrote the main text. All authors analyzed and reviewed the manuscript.

LS, HR, MP, FR, FR, OR, and AG are members of the Research Staff of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina). This work was partly funded by the project “Universidades Agregando Valor” #8550 from the Secretaría de Políticas Universitarias, Argentina.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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